Datasets:
heegyu
/
elsevier-oa-cc-by

Dataset card Data Studio Files
xet
Community
title
stringlengths
7
332
abstract
stringlengths
0
6.99k
subjareas
listlengths
1
6
keywords
listlengths
0
47
asjc
listlengths
1
8
body_text
listlengths
21
5.39k
author_highlights
listlengths
0
21
Automated extraction of free surface topography using SfM-MVS photogrammetry
This paper describes a spatial measurement technique to measure the free surface of natural fluid flows in laboratory applications. This effective solution is based on “Structure-from- Motion/Multi-view Stereo” (SfM-MVS) photogrammetry and is capable of reconstructing water surface morphology, both at an instant and with a high spatial resolution. The efficiency and accuracy of the method is dependent upon the acquisition of high quality imagery (i.e. sharply focussed, no motion blur) with appropriate multi-frame camera coverage and configuration, and data processing must utilise appropriate camera calibration data. The potential of the technique for developing hydraulic understanding is demonstrated using two contrasting approaches. First, the water surface behind a living vegetation element is analysed along a single transect. Second, the full three-dimensional characteristics of the captured water surfaces are examined using statistical methods which demonstrate surface dissimilarity between vegetated and non-vegetated cases. The technique is transferable to real-world field sites.
[ "COMP", "ENGI", "MATH", "PHYS" ]
[ "Eco-hydraulics", "Free surface measurement", "Laboratory studies", "SfM-MVS photogrammetry" ]
[ "1706", "2208", "2611", "3105" ]
[ "The need for accurate water depth readings has always been a basic requirement in river engineering practice.", "Consequently, a wide range of approaches aiming to gauge water surface elevations in-situ or in the laboratory have been applied traditionally, ranging from rudimentary techniques involving graduated cables or rods, to more sophisticated methods such as ground penetrating radar or ultrasonic sounders [3].", "In more recent times, researchers working on fluvial hydraulics have extended these measurements, notably in terms of potential applications and spatial description.", "For example: Han and Endreny [20] investigated intra-meander hyporheic fluxes based on head loss measurements determined with a point gauge; Biron et al. [2], analysed the water surface topography at an asymmetrical confluence by means of a total station; Cochard and Ancey [9], employed an image projection approach to trace the free surface associated to a dam-break; Felder and Chanson [14], studied the free surface profiles over broad-crested weirs using sidewall photography; Dabiri and Gharib [13], proposed a system combining the free surface gradient detector technique with digital particle image velocimetry to synchronously appraise the free surface distortion and the underlying turbulence in a free shear facility; Nichols et al. [28], fabricated an acoustic wave probe to monitor the free surface over a fixed gravel bed flume; Tsubaki and Fujita [37], explored a stereoscopic method to reproduce the free surface spawned over an asymmetric trench; and, Fujita et al. [18], obtained the water surface profile induced by hemisphere particles by applying an image processing protocol to laser light sheet based binary images.", "Common practice in fluvial hydraulics research is still however typically characterised by a sparse water elevation or topography (depending whether datum coincides or not with the bed) spatial resolution (see e.g. [42]).", "Hence, the work described in this paper provides water surface descriptions at a very high spatial resolution and at an instant through the application of Structure-from-Motion/Multi-view Stereo (SfM-MVS) photogrammetry.", "As the term implies, SfM-MVS photogrammetry has its roots in a well-established spatial measurement method known as photogrammetry.", "Many excellent introductory texts explain the basic principles of photogrammetry.", "Among others, Fryer et al. [17] provides a useful textbook which includes a chapter on earth science applications, comprehending for example a flume bed evolution study [6], and Luhmann et al. [24] offers a more extensive source of reference of photogrammetry science.", "Strictly, the phrase “Structure-from-Motion” evolved from the machine vision community, specifically for tracking points across sequences of images occupied from different positions (see e.g. [31]).", "SfM-MVS implements mathematical models developed many years ago in photogrammetry, including: coplanarity and collinearity, and particularly the self-calibrating bundle adjustment [22].", "The SfM-MVS approach has been explained by a range of authors (e.g. [21,25,30]), but in essence it involves acquiring digital photographs from a number of positions relative to the object of interest.", "A scale invariant feature transform identifies distinctive features appearing upon multiple images and establishes the spatial relationships between the original camera positions in an arbitrary and unscaled coordinated system.", "A self-calibrating bundle adjustment is then used to calibrate the cameras used and derive a sparse set of coordinates to represent the object.", "This is then intensified using MVS (Multi-View Stereo) techniques, to generate a very high resolution point cloud, which is colour-coded using the original image data.", "External geometric constraints can be then optionally applied, to transform the 3-D data to a desired coordinate system.", "Despite the unanimous agreement on the significance of free surface activity regarding, e.g., gas transfer mechanisms [37], or as an “echo” of the spatial/time development of the flow [8], few studies, of which Cochard and Ancey [9], Dabiri and Gharib [13] and Tsubaki and Fujita [37] are notable examples, have quantified the spatial variation of free surface elevation at large spatial scales (i.e. in the three-dimensional space) and at a single instant in time.", "The methodological development reported in this paper provides a way of quantifying free surface heterogeneities produced by vegetation elements at large space scales, with a high spatial resolution and instantaneously.", "The approach exploits the ability of three synchronised cameras to freeze motion, combined with SfM-MVS photogrammetry.", "Besides the obvious advantages over one and two-dimensional sensors, the technology introduced in this paper improves certain aspects and overcomes some limitations of past work.", "Major advances regarding the pioneering study by Tsubaki and Fujita [37] can be summarized in three points.", "First, cameras pose and scene geometry are here automatically ascertained due to the high number of conjugate points measured, due to the superior performance of the scale invariant feature transform algorithm.", "Second, linear and non-linear errors arising due to inadequate ground control or insufficiencies with kernel based approaches ([37] use a cross-correlation technique) are suppressed, enhancing the reliability of derived data [15].", "Third, the need to stain the water with a colourant and project a pattern to the free surface is obviated, making the workflow presented in this paper practicable for field applications.", "Finally, contrary to Dabiri and Gharib [13], SfM-MVS photogrammetry is able to cope with small and steep free surface slopes at competitive data acquisition and processing times since expensive phase unwrapping algorithms are avoided [9].", "The paper is organized as follows: the experimental set-up is described; then, results are presented and further processed using two complementary approaches, before some conclusions are finally drawn.", "Experiments were conducted in a 5.24 m long and 91.5 cm wide straight open-channel flume in the Sir Frank Gibb Civil and Building Laboratories of Loughborough University.", "Water was supplied to the test area by means of an upstream tank with plastic tube bundles for straightening the incoming flow.", "Previous experiments have shown that close to the inlet, the spanwise and vertical time-averaged velocity components were much lower than the magnitude of the principal velocity component, thus indicating that entrance effects were reduced by this apparatus.", "At the outlet, a manually operated tail gate was manipulated to fix the desired water depth.", "Although our goal was to develop a method to remotely measure free surface heterogeneities in vegetated flows, it was considered pertinent to capture imagery both with and without vegetation for comparative purposes.", "The discharge and the tail gate level were kept constant across these two tests (some branches of the vegetation were pruned to make the vegetation height coincident with the water depth).", "For the latter, a localized hole was created on a smooth bed for vegetation insertion, with the vegetation being placed at approximately 2.9 m from the flume inlet and at its centreline, i.e., free surface readings were taken in the second half of the flume length.", "The chosen species, a conifer tree (Fig. 1a), was used while fresh and exhibited a static mode of reconfiguration under stressed conditions [1].", "Our work included the measurement of flow discharge Q, specimen frontal projected area (in dry conditions) Ap and wet mass wmass, and the free surface topography (Section 3).", "The values obtained were: Q=0.133 m3 s-1, Ap=0.053±0.003 m2 and wmass=65±2 gr.", "As free surface irregularities were minor in our experiments, and the flume bed gradient was rather small (0.0014), the SfM-MVS data was not de-trended in this work.", "A multi-camera system comprising three Nikon D80 digital SLR cameras, each equipped with a 24 mm fixed focus lens, was used for water surface measurement.", "Cameras were mounted on a foam pad fastened to a wood stand (positioned two meters above the free surface, each camera separated by 23 cm, Fig. 1b).", "Image synchronization is critical when measuring dynamic entities as asynchronism may cause the surface model extraction algorithm to fail.", "Synchronisation of up to 1/1000 of a second was ensured by adapting an existing electronically controlled triggering box to accommodate a third D80 camera.", "Image matching can only be successful if some visible surface texture is captured, which is challenging in naturally dark laboratory conditions, and particularly involving a rapidly moving and dynamic water surface.", "For our experimental arrangement appropriate texture was created using artificial “seeds” (Section 2.3), necessitating the following exposure settings for the three cameras: manual mode, aperture f-2.8, shutter speed 1/400 second and ISO 1600.", "It is now fully recognized that consumer grade digital cameras are adequate for accurate photogrammetric measurement, provided camera calibration is considered [5].", "Hence, similarly to Wackrow et al. [39], calibration data was obtained offline and for each individual camera.", "Additionally, external lighting sources had to be used to provide appropriate illumination necessary to capture sharp imagery free of motion blur.", "Direct light generated numerous specular reflections which would compromise matching strategies.", "Therefore, indirect and more diffuse illumination was achieved by directing the light sources to a projector screen (Fig. 1b) located above the flume.", "Scene reconstruction requires adding floating marks or “seeds” to the free surface and the selection of a suitable material was challenging.", "Although in practice they worked as flow tracers, these particles were here solely used to provide suitable image texture to guarantee SfM-MVS effectiveness.", "Following Weitbrecht et al. [40] and Chandler et al. [7], the guiding criteria for selection were: density, diameter, durability and cost.", "The authors identified 1 mm cork particles (http://www.ccmoore.com/).", "Preliminary tests revealed that these particles indeed float, are well perceived by the cameras at the prescribed object to camera distance, offer a good contrast with the free surface and can be reused after drying (typically within a few days if no artificial drying is used).", "Seeds were spread manually with the aid of a wood container with two (superimposed and mismatched) metal sieves in the bottom.", "By this way, we could adjust the “dispenser effective diameter” and were able to seed the water at a rate compatible with the free surface velocity.", "The particles were collected near the tail gate via a small floating wooden bar, which guided the floating particles to a collecting net.", "Photogrammetric control was established to define a local scaled coordinate system (with its origin at the flume bed), but also provided the opportunity to assess the accuracy of the experimental layout (Fig. 1b).", "Ringed Automatically Detected (RAD) coded targets [29] were printed at a diameter of 7 mm and coordinated using professional survey grade instrumentation, a Trimble M3 Total Station [36].", "While evaluating the quality of spatial data it is important to distinguish three components, notably precision, accuracy and reliability, and relate these elements to random, systematic and gross error sources, respectively [11].", "Random errors are unavoidable, are subject to change and can be estimated by statistics.", "Systematic errors arise from inexact functional models and improperly calibrated equipment.", "Finally, gross errors are genuine mistakes or “blunders”, but can be normally isolated if there are redundant data to provide checks.", "Ideally, these three aspects are assessed by precision, accuracy and reliability metrics.", "This section presents some simple test cases that give an insight into the magnitude of these descriptors for the present arrangement.", "Measures of precision can be estimated by assessing the covariance matrix, which is a bi-product of the internal bundle adjustment.", "This least-squares estimation procedure is used to determine the position and orientation of the cameras and position of points measured in the images [11].", "These values are uniquely dependent on “endogenous factors” such as image geometry, the precision of the image coordinates and the number of images that each point appears upon.", "Thus, such precision estimates can be directly retrieved from PhotoModeler Scanner (64 bit) [29].", "Conventional photogrammetric practice suggests that “control” or “reference” points should be widely and uniformly distributed around the periphery of the “volume of interest” (i.e. over the area covered by the images) to improve points positioning accuracy [24].", "This requisite was difficult to achieve in our flume study (e.g. it is impracticable to distribute markers beneath the water surface).", "Having this in mind, linear targeted arrays were located on either side of the flume, plus four extra targets just above the water surface (due to the drawing perspective, two of these four extra targets are omitted in Fig. 1b).", "The merit of this configuration was initially gauged by simulating the water surface through an array of photogrammetric targets placed on a flat board, temporarily placed at the same plane as the expected water surface.", "A photographic sequence was recorded and processed using PhotoModeler..", "This demonstrated that the point precision for a conjugate point located close to the water surface was ±0.8 mm, ±0.8 mm and ±1.2 mm in the x (longitudinal), y (lateral) and z (vertical) directions, respectively.", "The lower precision in the vertical direction arises from the fact that cameras were confined to a single zone above the free surface.", "The hypothetical adoption of additional cameras at different levels would augment such precisions by creating stronger image geometry.", "Accuracies can only be assessed by comparing estimated data with some form of external truth or accepted standard [11].", "In this work, accuracy was evaluated by three methods, with the first and second involving the board mentioned above.", "Previous work [38,5] has shown that this provides a rigorous way to assess accuracy and identifying unresolved systematic errors in the measurement process.", "Typically, these are manifested by an error surface or “dome”, which arise due to minor inaccuracies in the parameters used to model lens distortion.", "By assuming board planarity and ignoring surface irregularities, it was possible to estimate discrepancies from this plane surface, with the mean error being found to be 1.1 mm.", "Complementary to this, four of these RAD targets were surveyed using the Trimble M3 Total Station and their readings compared against PhotoModeler estimates.", "Likewise, average height differences of 1.1 mm were determined.", "The third test was executed in the course of the experiments reported in Section 3, and involved a single surface estimate by reading a “survey staff strip” placed towards the side of the flume (Figs. 3a and 3b).", "This allowed us to confirm a general agreement between the SfM-MVS measurement system and the water depth marker (difference of 2 mm in both cases).", "It should be noted that this prediction was obtained using the closest point of the digital elevation model (DEM) (i.e. the cork particle) relative to the water depth marker.", "Due to the analysis performed in Section 3, this accuracy (and precision, see above) were considered appropriate for the present context.", "However, it should be stated that future studies focussing on other hydraulic processes may well be more demanding in terms of the required accuracy and precision.", "Cooper and Cross [12] defined reliability as a “measure of the ease with which outliers may be detected” and proposed a broadly accepted indicator (the τ factor), which is difficult to quantify when studying deformable surfaces (such as a free surface).", "In the spirit of Cooper and Cross [12], the authors of the present work consider that visualisation is a valuable tool that can replace numerical metrics when ascertaining reliability since, as pointed out by Wood and Fisher [41], “it (visualisation) can easily convey the significance of data uncertainty”.", "Accordingly, SfM-MVS photogrammetry aptitude to “replicate the free surface relief” of an undulating free surface was appraised by manually agitating (with a wooden piece) still water that was seeded with cork particles.", "As shown in Fig. 2 (and in http://geomaticsjc.lboro.ac.uk/ven/WaveSurface-Control.gif), SfM-MVS photogrammetry has successfully reconstructed the mechanically generated wave, thus confirming the reliability of SfM-MVS photogrammetry to derive meaningful free surfaces.", "Digital photogrammetry systems (DPSs) incorporate a series of user definable strategy parameters which, when inappropriately assigned, deteriorate the efficiency of image correlation algorithms and, consequently, the quality of the surface representation.", "The number and precise value of the strategy parameters varies with the choice of DPS and particularly the illumination and texture captured in the imagery [19,23].", "Therefore, the writers carried out a simple test to guide parameter selection.", "A small rectangular plastic water storage tank was filled with water and seeded with cork particles, under similar seeding and lighting conditions as the flume.", "Images were gathered and processed, keeping the default strategy parameters currently recommended and utilised by PhotoModeler SfM-MVS software.", "A patch consisting of 55734 points was arbitrarily picked from the DEM and its standard deviation (σ) computed.", "The calculated value (0.5 mm) suggested that the main feature of this surface (i.e. horizontal planarity) was being accurately detected, thus validating the use of the default strategy parameters in our main tests.", "Fig. 3 shows original imagery captured for both the un-vegetated (Fig. 3a) and vegetated (Fig. 3b) flows.", "Fig. 3c is a visualisation of the dense colour-coded point cloud representing the vegetated case.", "It should be emphasized that this point cloud was created in less than 5 min on a personal computer with an Intel Core i7-3770 3.4 GHz processor and 8 GB of RAM.", "Prior to the analysis described below, points of the free surface lying outside our region of interest (before x=0 where the conifer tree was eventually positioned) were removed and dubious point elevations were filtered using a semiautomatic local variance based filter inspired in Lane et al. [23].", "Based on an iterative process, a conservative filter threshold was set so that only free surface points were preserved.", "At the end of this stage, 122604 (over an area of approximately 1.5 m2) and 151383 (over an area of approximately 1.6 m2) free surface points were retained for the reference flow (no conifer tree) and perturbed flow (with the conifer tree), respectively.", "This high number of points ensured the representativeness of the studied water surfaces, a sine qua non condition for the analysis performed in Sections 3.1 and 3.2.", "To portray the spatial variability of the water surface, two sampling formats were tried: (1) a single transect in the middle of the flume containing 825 points taken at 2 mm intervals (Fig. 4 and Section 3.1, only applied to the vegetated perturbed case) and (2) considering the full DEMs 3D coordinates (Section 3.2).", "The major advantage of the second approach is the provision of three-dimensional empirical information, thus enabling researchers to embrace the concept of the random-field approach (see e.g. [10]) to study the free surface shape/structure and ultimately, its signature on different flow zones [33].", "Thus far, this view has been limited to the analysis of flow-bed interactions through the use of spectra, structure functions and statistics.", "We advocate that SfM-MVS photogrammetry is a promising solution to extend this approach to the free surface.", "In this study, eminently focused on the methodological traits of SfM-MVS photogrammetry, we start by considered two specific snapshots from a broader dataset to better illustrate some outputs that can be derived from SfM-MVS photogrammetry.", "Hence, for the first time, we will look at the following basic questions: What type of waves are induced by these flexible vegetation elements?", "Are the water heights normally distributed in this case?", "Fig. 4 provides a plan view representation of the captured water surface with variations in grey representing absolute elevation above the flume bed (White: 0.295 m and Black: 0.280 m), illustrating an elevation range of approximately 13 mm.", "In the course of the vegetated trial, small scale free surface disturbances were originally detected on the wake of the vegetation, with these disturbances being progressively dissipated downstream (Fig. 4).", "Hence, it was attempted, in a first instance, to fit a damping wave equation to the profile data (i.e. along the blue line in Fig. 4) through a non-linear regression.", "Any real water wave is the manifestation of many waves.", "However, it would have some practical significance if the waves past the conifer tree could be described by a simple analytical form (which will only eventually occur if sine-like wave modes prevail).", "At a fixed time, a damped sinusoidal wave is given by the product of a decaying exponential signal and a pure sine wave: z= e-bxAcos(kx +φ), where b is a damping coefficient, A is the wave amplitude, k is the wave number, φ is the phase and z and x have the same meaning as before.", "Fig. 5 compares the measured water surface profile with the damping equation.", "Overall, and excluding the first few centimetres immediately after the conifer tree, two main regions can be distinguished.", "Before x≈0.75 m, the water depth (initially) increases and the damping wave equation consistently underestimates the wave peaks; after this instance, the measured water wave is well described by the damping equation.", "Further statistical analysis utilised filtered water surface elevation points extracted immediately downstream of the conifer tree until x≈0.23 m.", "This approach used all elevation points within this area, rather than just points extracted in a downstream sectional plane.", "As the inner flow region was not measured, this area was delineated from visual inspection as a tentative to isolate possible side wall effects.", "This area is therefore assumed to be mainly affected by the wake of the conifer tree.", "As expected, the first moment (μ) and the standard deviation (σ) of the water depth slightly increased from the non-vegetated reference flow (μ=0.284 m and σ=0.001 m) to the vegetated perturbed flow (μ=0.289 m and σ=0.002 m).", "It is unlikely to retrieve a pure normal distribution from experimental data and Totton and White [35] review distinct methods to judge data normality.", "However, Montgomery and Runger [26] state that normality tests are fruitless when extensive datasets (as in our case) are exploited.", "An alternative is to generate normal probability plots to examine water elevation distributions, as shown by Nichols & Shepherd [27].", "Basically, this approach involves plotting the experimental observations zi (i=1:n with n being the total number of samples) versus the standardized normal scores (SNS) (Fig. 6a).", "Fig. 6a suggests that free surface heights are non-normally distributed in the vicinity of flexible isolated (or highly sparse) vegetation elements.", "This non-normal behaviour has been earlier observed on steep ocean waves [32].", "Both curves display an “S-like shape” but exhibit noticeable differences between their tails.", "Note that in the non-vegetated reference flow case, the vast majority of points lie in a straight line (comparing to the vegetated plot) and the tails are below the auxiliary line on the left and above that same line on the right, which is typical of light tailed distributions., In contrast, the vegetated perturbed flow has a more “intermittent” or irregular free surface, thus demonstrating a heavy tailed distribution..", "This departure from normality is reinforced in Fig. 6b, where it seems apparent that a logistic distribution works better than a normal one..", "At present we are acquiring more data and in a follow-up study intend to more systematically characterize the free surface pattern and its potential link with vegetation properties.", "The existence of such a connection can have ground-breaking implications for the creation of new theories in fluvial hydraulics.", "This work has presented a method to yield high resolution DEMs of water surfaces using SfM-MVS photogrammetry.", "A description of the method was accompanied by an examination of the quality of the data extracted, suggesting that accuracies of 1–2 mm can be achieved.", "Furthermore, two distinct analyses have demonstrated how a dense numerical description of high resolution surface morphology captured at an instant, can answer fundamental questions linked to the nature of the free surface.", "The authors believe that SfM-MVS photogrammetry might open new avenues for researchers working on free surface dynamics/structure, especially for those trying to infer subsurface properties from the free surface pattern [4].", "In summary, this work is seen as an important step to improve our understanding of flow-free surface interactions, a necessity recently claimed by Sukhodolov [34].", "More tenuous progresses in this area are in part a manifestation of technological constraints, specifically an inability to adequately measure the free surface topography, a task that this work has proved to be suited for SfM-MVS photogrammetry.", "This technique can also be extremely useful in other domains, particularly the validation of computational models, among others.", "Due to the innovative character of this work, future applications will increase the cumulative experience with SfM-MVS photogrammetry and dictate eventual operational and methodological refinements (e.g. increasing the sampling rate beyond the 3 Hz current achievable, among others) needed in laboratory and field conditions.", "In general, SfM-MVS photogrammetry is anticipated to be similarly applicable to other free surface formats, for example above rough beds or at confluences.", "The objective of each study should dictate the quality of data required, but it is clear that higher accuracies and precisions can be attained using SfM-MVS photogrammetry through, for example, enlarging the number of overlapping photos (i.e. by using more cameras at different locations), decreasing the cameras to object distance or increasing the focal length.", "Recent preliminary work conducted by the authors (results not shown here), indicates that SfM-MVS photogrammetry is also viable in field environments and that under certain circumstances, e.g. in white water flows, image texture is “naturally supplied” by the free surface features.", "Alternatively, provided weather conditions (illumination and wind) are favourable, bio-degradable seeding particles can be used with the aid of seeding distribution mechanisms.", "Finally, in large scale field experiments, low altitude aerial photography may be used instead of more traditional acquisition platforms (e.g. bridges).", "This work was supported by the Engineering and Physical Sciences Research Council (EPSRC) [Grant: EP/K004891/1]." ]
[ "New experimental tools are needed to better quantify the free surface topography.", "A remote and non-intrusive approach based on SfM-MVS photogrammetry is presented.", "Statistical analysis shows differences between vegetated and non-vegetated flows." ]
An investigation into per- and polyfluoroalkyl substances (PFAS) in nineteen Australian wastewater treatment plants (WWTPs)
Quantifying the emissions of per- and polyfluoroalkyl substances (PFAS) from Australian wastewater treatment plants (WWTP) is of high importance due to potential impacts on receiving aquatic ecosystems. The new Australian PFAS National Environmental Management Plan recommends 0.23 ng L−1 of PFOS as the guideline value for 99% species protection for aquatic systems. In this study, 21 PFAS from four classes were measured in WWTP solid and aqueous samples from 19 Australian WWTPs. The mean ∑21PFAS was 110 ng L−1 (median: 80 ng L−1; range: 9.3–520 ng L−1) in aqueous samples and 34 ng g−1 dw (median: 12 ng g−1 dw; range: 2.0–130 ng g−1 dw) in WWTP solids. Similar to WWTPs worldwide, perfluorocarboxylic acids were generally higher in effluent, compared to influent. Partitioning to solids within WWTPs increased with increasing fluoroalkyl chain length from 0.05 to 1.22 log units. Many PFAS were highly correlated, and PCA analysis showed strong associations between two groups: odd chained PFCAs, PFHxA and PFSAs; and 6:2 FTS with daily inflow volume and the proportion of trade waste accepted by WWTPs (as % of typical dry inflow). The compounds PFPeA, PFHxA, PFHpA, PFOA, PFNA, and PFDA increased significantly between influent and final effluent. The compounds 6:2 FTS and 8:2 FTS were quantified and F–53B detected and reported in Australian WWTP matrices. The compound 6:2 FTS was an important contributor to PFAS emissions in the studied Australian WWTPs, supporting the need for future research on its sources (including precursor degradation), environmental fate and impact in Australian aquatic environments receiving WWTP effluent.
[ "MULT" ]
[ "Chemistry", "Chromatography", "Environmental analysis", "Environmental assessment", "Environmental chemistry", "Environmental hazard", "Environmental pollution", "Environmental science", "FTS", "PFAS", "PFOA", "PFOS", "Sludge", "Waste", "Wastewater", "Water pollution F–53B" ]
[ "1000" ]
[ "Per- and polyfluoroalkyl substances (PFAS) are a group of man-made pollutants that pose an emerging risk to the water sector, challenging established practices such as recycling and environmental discharges.", "They are omnipresent in water, air, food, wildlife, and humans, are resistant to typical environmental degradation processes, and can have negative impacts on exposed organisms [1, 2, 3, 4, 5, 6, 7, 8, 9, 10].", "Most PFAS are recalcitrant through conventional water treatment processes and, therefore, wastewater effluents can contain PFAS that has originated from domestic and industrial sources [11, 12].", "Understanding the sources of PFAS to the environment is of high importance in Australia due to the recently recommended perfluorooctane sulfonic acid (PFOS) guideline value for 99% species protection of 0.23 ng L−1 in aquatic ecosystems in the PFAS National Environmental Management Plan [13].", "The unique and useful chemical and physical properties of PFAS have resulted in many commercial applications, such as stain-resistant coatings, water-resistant fabrics, metal plating paints, pesticides, fluoropolymers, greaseproof paper, and aqueous film-forming foams (AFFF) used in firefighting, amongst others [3, 4, 7, 9].", "Although PFAS are a broad class of compounds comprising over 4700 known PFAS [10], many studies have focused on a small number of perfluoroalkyl acids (PFAAs), specifically the perfluorocarboxylic acids (PFCAs; CF3(CF2)nCOOH) and perfluorosulfonic acids (PFSAs; CF3(CF2)nS(=O)OH) [9].", "Despite being manufactured since the 1950s, it wasn't until 2001 that the extent of PFAS global contamination was first demonstrated for perfluorooctane sulfonate (PFOS; C8F17SO3H) and perfluorooctanoic acid (PFOA; C7F15COOH) [1].", "Since then, PFAS have been detected in almost every wildlife sample measured [14], ubiquitously in humans throughout the world [15], and in most environmental compartments, including pristine locations [7].", "The perfluoroalkyl substances contain at least one fully fluorinated alkyl chain bonded to a functional group, whereas polyfluoroalkyl substances contain a partially fluorinated alkyl chain with a range of functional groups.", "In general, the sorption potential of PFAS is determined by functional group, chemical structure, and fluorinated chain length; however, for many newer PFAS, this information is not yet available.", "In environmental aquatic systems, the different partitioning behavior will typically result in short-chain compounds (PFCAs: ≤ C6, and PFSAs: ≤ C5) partitioning to the aqueous phase and long-chain compounds adsorbed to the solid compartments [16, 17].", "Furthermore, some PFAS (viz. fluorotelomer alcohols, phosphate esters, etc.) are precursor compounds and will transform in the environment, forming many intermediate transformation products with PFAAs such as PFOA as terminal products [18].", "The growing understanding of the risks of many legacy PFAS has led to the phase-out of production of PFOS (and related compounds) and PFOA in North America (in 2000 and 2002, respectively) and an increased use of less problematic alternative compounds (such as short-chain and fluorotelomer based chemistries) [4].", "An example of two PFOS alternatives used as mist suppressants in metal plating are 6:2 fluorotelomer sulfonate (6:2 FTS) and the chlorinated perfluoroether sulfonate F–53B [4, 19, 20].", "In some regions, 6:2 FTS is not used as a PFOS substitute in metal plating as it cannot match the low surface tension of PFOS and approximately three to ten times the quantity is required [21].", "However, 6:2 FTS has found further uses as a PFOS substitute in AFFF, oil production and primarily occurs as an intermediate degradant of complex fluorotelomer-based substances [4].", "In initial testing by Dupont scientists, 6:2 FTS was found to show low risk to aquatic ecosystems making it a desirable substitute, however, studies on the environmental fate and effects were still needed [22].", "Alternatively, F-53 (6:2 PFESA) then the chlorine substituted F–53B (6:2 Cl-PFESA), have been used almost exclusively in China since the 1970s with little PFOS ever used in metal plating [19].", "As investigations into the fate and toxicity of F–53B progresses, it is now becoming apparent that it shows similar recalcitrance, toxicity and physiochemical properties to PFOS and is becoming widely distributed in the environment making it a less desirable substitute for PFOS [20, 23, 24, 25].", "Wastewater treatment plants (WWTPs) can act as a conduit for many recalcitrant anthropogenic compounds, such as PFAS, to the environment through effluent discharges and the land application of biosolids [26].", "PFAS have been detected in WWTP influent, effluent and solids worldwide [11].", "Similar to other environmental compartments, hydrophobic partitioning in WWTPs is the dominant sorption mechanism, which results in long-chain PFAAs partitioning to WWTP solid matrices [27, 28, 29, 30].", "Typical wastewater treatment processes are unable to remove PFAS from the final effluent.", "In some studies, concentrations of compounds such as perfluorocarboxylic acids (PFCA) and perfluorosulfonic acids (PFSA) have increased from influent to final effluent [11, 27, 31].", "The increase of PFAAs has been attributed to the degradation of the PFAS precursor compounds [32, 33], fluorotelomer sulfonoates (FTS) and fluorotelomer alcohols (FOTH), that have been shown to transform to stable PFAAs in WWTP sludge [34, 35].", "The awareness of PFAS environmental contamination associated with AFFF application on government military sites, and evidence of widespread distribution in the Australian environment [36, 37, 38, 39, 40], have led to the development of the Australian PFAS National Environmental Management Plan (NEMP) [13].", "Within the NEMP, the recommended freshwater and marine guideline values (water concentrations) for 99% species protection are 0.23 and 1900 ng L−1 PFOS and PFOA, respectively (HEPA 2018).", "As a result, there is strong interest from water industry professionals and regulators to understand the quantities of PFAS released into the environment through treated effluent, and the potential impact these emissions may have upon Australian aquatic environments.", "Initial studies on PFAS emissions in Australian WWTPs have focused on the removal efficiency in two reclaimed water plants (18 PFAS measured ranging from 1.1 to 38.6 ng L−1) [41] and one WWTP (8 PFAS measured ranging from 3 to 82 ng L−1) [42].", "An Australian-wide study measuring nine PFAS in WWTP effluent (range from n. d. to 240 ng L−1) and biosolids, sampled in 2016, estimated that Australian WWTPs have discharged an estimated 33 kg PFOS and 67 kg PFOA, annually [37].", "More recently, PFAS levels in influent over a four year period at two large Australian WWTPs (mean ∑11PFAS levels 57 ± 3.3–94 ± 17 ng L−1 at WWTP A; and 31 ± 6.1–142 ± 73 ng L−1 at WWTP B) were determined to have: 1) no significant difference in daily PFAS mass load between weekdays and weekends (composite samples over 7 consecutive days), 2) very few significant seasonal differences of ∑11PFAS (with most significant differences linked to a pulse release of PFOS at both WWTPs), and, 3) only one significantly different annual mean mass load in WWTP B over the entire four year period (linked to the same PFOS pulse event of October 2017) [43].", "Australian WWTPs represent a unique case as there is no reported PFAS manufacture and low rates of PFAS are imported for direct use in industries such as car manufacture, chrome plating, leather treatment, medical imaging, firefighting and in goods already impregnated (carpets, furniture, etc.) or in products containing PFAS as impurities [13, 44].", "Furthermore, unlike many parts of the world, in Australian cities, sewer systems are closed, with separate stormwater sewers and low infiltration rates, this means rainfall has limited effect on influent PFAS composition as opposed to pulse events from industrial effluent discharge.", "It is, however, becoming apparent that many PFAS, including PFOS and PFOA, are present in Australian WWTP effluents and are being discharged to the aquatic environment.", "The aims of this study were to measure the mass loading of PFAS (including PFAAs, FTSs, and F–53B) within solid and liquid matrices from 19 Australian WWTPs of varying size, capacity, localities and treatment types.", "Samples were taken from various stages within the treatment train from a range of WWTPs to determine the trends in the mass flux and partitioning of PFAS within the sampled WWTPs.", "Finally, the data were compared to recent work estimating the Australian annual PFAS discharge, providing important data for ongoing assessments of the potential impact of PFAS on aquatic environments.", "Field sampling kits including field blanks were prepared at RMIT University laboratories and shipped overnight to each WWTP.", "Three replicate aqueous and solid samples were collected from each of nineteen Australian WWTPs throughout 2017 (Table 1).", "Aqueous samples (influent, primary effluent, secondary effluent, final effluent, recycled water) consisting of either triplicate sub-samples from a single 24 h composite or three replicate grab samples were collected in 250 mL polypropylene bottles pre-rinsed with ultrapure water, methanol, and site water.", "Solid samples (primary sludge, secondary sludge, lagoon sludge, and one lagoon sludge dredge pile) were collected in 50 mL polypropylene centrifuge tubes.", "On receipt, samples were sterilized (aqueous samples with sodium azide ∼1g L−1 and solid samples with 2 % w/w sodium azide solution) and refrigerated until extraction.", "The compounds quantified in this study were the perfluorocarboxylic acids (PFCAs): PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUdA, PFDoA, PFTrA & PFTeA; the perfluorosulfonic acids (PFSAs): PFBS, PFPeS, PFHxS, PFHpS, PFOS, PFDS; the fluorotelomer sulfonates 6:2 FTS, 8:2 FTS, and the chlorinated perfluoroether sulfonic acids (components of the commercial product F–53B): 6:2 Cl-PFESA (F–53B) and the F–53B impurity 8:2 Cl-PFESA (full compound details and MS/MS transitions listed in Table S1).", "These compounds were selected as PFCAs and PFSAs have previously been demonstrated to be present in Australian WWTPs and need further baseline data [37, 41, 42].", "The FTSs were selected as 6:2 FTS has been demonstrated as present in AFFF formulations impacting WWTPs [33], used as a PFOS replacement [4] and there is little current published Australian data on FTSs.", "Furthermore, the F–53B components are an emerging contaminant in China due to substitution for PFOS in chrome plating [20].", "As Australian is part of the Asia Pacific region, and Cl-PFESAs have been detected in WWTPs in China [19, 24] it was included in this study to determine if there is an emerging risk in Australia.", "Analytical standards and isotopically labeled analogues of PFAS were purchased from Wellington Laboratories (Ontario, Canada) as solutions of 50 μg mL−1 in methanol.", "Stock solutions of 100 ng mL−1 for native PFAS and 100 ng mL−1 for surrogate PFAS were prepared gravimetrically in methanol for spiking.", "The solvents methanol (LC-MS grade, Honeywell, USA and LiChrosolv hypergrade, Merck Millipore, Australia) and ultrapure water (pH 8, Merck Millipore, Australia) were tested for PFAS contamination prior to use.", "Ammonium hydroxide solution (28% in H2O, ≥ 99.99%), sodium acetate, glacial acetic acid and ammonium acetate (≥99.99%) were purchased from Sigma-Aldrich (Australia).", "The dispersive solid-phase extraction sorbents (d-SPE), sorbents C18, and primary secondary amine (PSA) were purchased in bulk from Agilent Technologies (USA).", "Aqueous samples were extracted using similar methods outlined in Szabo, Coggan [40], Hepburn, Madden [45] and Coggan, Anumol [46].", "Briefly, samples were filtered using 1 μm glass fibre filters (Merck Millipore, Australia), spiked with 5 ng of isotopically labelled PFAS, followed by solid-phase extraction (SPE) using Oasis weak anion exchange (6 mL, 150 mg WAX) cartridges with 15 mL polypropylene centrifuge vials used as collection vessels.", "Cartridges were conditioned sequentially with 4 mL 0.1% (v/v) ammonium hydroxide in methanol, 4 mL methanol, and 4 mL ultrapure water.", "The entire sample was passed through the cartridge under vacuum at approximately one drop per second, then washed with 4 mL of a pH 4 buffer (sodium acetate/acetic acid) and dried under vacuum for 10 min.", "SPE cartridges were eluted using 2 mL of methanol that was used to rinse the sample bottle, followed by 4 mL of 0.1% (v/v) ammonium hydroxide in methanol.", "Extracts were evaporated to 500 μL under a gentle stream of nitrogen (at 25 °C) and reconstituted to 1 mL in methanol and transferred to a polypropylene chromatography vial with polyethylene lid for analysis.", "Freeze-dried sludge samples (0.5–1 g) were spiked with 25 ng of isotopically labelled PFAS before adding 4.65 mL of 10 mM NaOH in methanol.", "Samples were sonicated for 30 min and shaken overnight for 12 h. Extracts were neutralized with 100 μL of glacial acetic acid and cooled on ice.", "Five mL of extract was then transferred to a 15 mL polypropylene (PP) tube before adding 100 mg of C18 and 50 mg primary secondary amine (PSA) to remove interfering compounds.", "Extracts were agitated for approximately 1 min and centrifuged (10,000 rpm, 10 °C, 10 min), with this process repeated twice.", "Finally, extracts were filtered using a 0.45 μm PES syringe filter (pre-rinsed with LC-MS grade methanol) into a propylene chromatography vial with polyethylene lid for analysis.", "The analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on an Agilent 6495B mass spectrometer coupled with an Agilent 1290 II Infinity liquid chromatograph optimised for PFAS analysis.", "Twenty-one PFAS compounds were quantified using isotope dilution.", "A surrogate compound for each PFAS was set as a mass-labeled compound from a similar class and/or close elution time.", "For compounds where two or more transition ions were present, the transition with the highest response was set as the quantifier, with others set as qualifier ions.", "The branched plus linear isomers of PFPeS, PFHxS, PFHpS, and PFOS were quantified using linear-only calibration standards and reported as a combined branched plus linear concentration.", "The twenty-one PFAS quantified in the analytical method are listed in the supplementary information (Table S1).", "The method employed dynamic multiple reaction monitoring (dMRM) and a 2 μL injection, in negative ESI mode.", "MS parameters were: gas temperature 250 °C, gas flow 11 L min−1, Nebulizer 25 psi, sheath gas temp 375 °C, sheath gas flow 11 L min−1, capillary voltage 2500 V, high pressure ion funnel RF 90 V and low pressure ion funnel RF 60 V. Separation was achieved using a Zorbax eclipse plus RRHD C18 column (3.0 × 50 mm, 1.8 μm, Agilent Technologies, USA) with a guard column attached.", "Gradient elution with the solvents 5 mM ammonium acetate in ultrapure water (A) and methanol (B) at 400 μL min−1 was performed, and the first 1.5 min was diverted to waste (t0 = 10% B; t0.5 = 10% B; t2.5 = 55% B; t9 = 90% B; t9.5 = 100% B; t11.5 = 100% B; t11.6 = 10% B; t14 = 10% B).", "A delay column (Zorbax Eclipse Plus C18 RRHD, 4.6 × 50 mm, 3.5 μm, Agilent Technologies, USA) was installed between the solvent mixer and injector module to delay instrument PFAS contamination.", "Linear calibration curves were prepared by gravimetric dilution of a mixed PFAS standard solution (100 ng mL−1 in methanol) with methanol to 9 levels with r2 > 0.99.", "Limit of quantification (LOQ) was set as the lowest calibration point multiplied by four and ranged from 0.1 to 0.6 ng L−1 depending on the compound type and gravimetric dilution.", "Limit of detection (LOD) was set as the instrument detection limit (IDL), varied on a compound-by-compound basis, and ranged from 0.01 to 0.1 ng L−1.", "IDL and instrument variability was determined using similar methods to Coggan, Anumol [46] on the same instrument and using the same instrument configuration.", "A field blank was prepared with every kit, transferred to a clean bottle on-site and then extracted concurrently with samples.", "Field blanks were extracted within the same batches as samples and matched with corresponding WWTPs.", "Only one compound (PFBA) was detected above LOD in field blanks from treatment plants WWTP-7 and WWTP-17; due to this, PFBA results for these two treatment plants were set as < LOD.", "Aqueous samples were extracted in batches containing two method blanks and a laboratory control sample (LCS).", "Laboratory control samples consisted of ultrapure water spiked with a native PFAS mixture containing all measured compounds at a mass of 5 ng, 1 ng or 0.25 ng.", "Mean recovery of all compounds in LCS samples ranged from 80 to 120% with s.d. < 15%, except for PFDS (72%, s.d. 13%), 8:2 Cl-PFESA (73%, s.d. 7%), PFTrA (70%, s.d. 6%) and PFTeA (76%, s.d. 6%) (Table S2).", "Solid LCS samples consisted of acid-washed sand spiked with 10 ng of PFAS and extracted alongside batches of 12 samples.", "Mean recovery of all compounds in LCS samples ranged from 80 to 120% with s.d. < 15%, except for 6:2 FTS (61% s.d. 8%).", "Method blanks returned less than the limit of detection (<LOD) for all batches.", "The use of ultrapure water and acid-washed sand as laboratory control samples may not adequately represent WWTP matrices (and the associated interferences) and present some uncertainty with analytical results.", "However, similar methods have been successfully employed in WWTP matrices in other studies [32, 46, 47] and overall we considered the QA/QC results provided an acceptable assurance of the quality of the data set for this study.", "Quantitation was carried out using MassHunter QQQ quantitative analysis software (version 08.00, Agilent Technologies, USA).", "Descriptive statistics were computed using pooled data from all samples.", "Statistical analysis was carried out using R [48].", "Data visualizations were also produced in R [48] using the packages reshape2 [49] and ggplot2 [50].", "To compare distribution coefficients to those previously published in Eriksson, Haglund [32] and Sun, Zhang [51], similar estimation methods were employed.", "This calculation method is only an approximation as it assumed that the concentration of PFAS in solids and liquids at the sampled location were in equilibrium and does not consider the differences in effluent and sludge retention times.", "Distribution coefficients (log Kd) were calculated for the compounds PFHxA, PFHxS, 6:2 FTS, PFOA, PFNA, PFOS, and PFDA.", "Distribution coefficients were only calculated for these compounds at sample locations where both aqueous and solid samples were above the limit of quantitation.", "Due to non-normal distributions, data for 11 compounds were first log10-transformed.", "Pearson correlation coefficients were computed using the transformed, pooled, influent and pooled final effluent data.", "Linear mixed-effects analysis was performed on the transformed data using the R package lme4 [52].", "P-values were obtained by likelihood ratio tests of the full model with treatment stage (influent and final effluent) included against the model without the effect in question.", "PCA analysis was performed on the untransformed data for the 11 compounds plus percentage trade waste and daily inflow using the correlation matrix (standardised) and visualised in R using the package factoextra [53] and ggplot2 [50].", "Twenty-one PFAS from four classes (PFCA, PFSA, FTS, Cl-PFESA) were measured in aqueous (n = 201) and solid (n = 51) samples from the 19 Australian WWTPs.", "PFAS were detected in all samples from all matrices.", "The summary statistics are presented in Table 2 and the data is further provided in the Supplementary Information, Table S3, and Table S4.", "The mean ∑21PFAS in aqueous samples was 110 ng L−1 (median: 80 ng L−1; range: 9.3–520 ng L−1) (Table 2).", "The highest concentration measured in aqueous matrices for any compound was 370 ng L−1 for PFBA in final effluent at WWTP-12.", "PFBA has been used as a short-chain PFAS substitute for some PFCAs [54].", "The high concentration of PFBA in final effluent and distribution within the sampled WWTPs may reflect current PFBA use.", "Eleven of the 21 compounds were detected in >90% of samples (PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFBS, PFHxS, PFOS, 6:2 FTS), and these were used in the subsequent statistical analysis of influent and final effluent.", "Mean concentrations in aqueous samples followed the trend: PFHxA > PFOA > PFOS > PFHxS > PFBA > PFPeA>6:2 FTS > PFHpA > PFBS > PFDA > PFPeS > PFNA > PFHpS>8:2 FTS > PFTeA > PFDS.", "Mean concentrations of PFAS in aqueous samples were similar to concentrations previously reported for Australian WWTP aqueous samples [37, 41, 43].", "To date concentrations of PFBA and 6:2 FTS have not been widely reported in Australian WWTPs.", "PFBA and 6:2 FTS have been used as C8 substitutes and are end-stage and intermediate metabolites (respectively) of many PFAS, their prevalence in the studied WWTPs may be an indicator of changing PFAS use trends in Australia.", "WWTP-2 had the highest ∑21PFAS in influent and final effluent, with concentrations of 410 and 520 ng L−1, respectively.", "Major contributors to ∑21PFAS loading at WWTP-2 were PFHxS (influent 130 ng L−1, effluent 190 ng L−1) and PFOS (influent 120 ng L−1, effluent 130 ng L−1) (Fig. 1).", "The WWTP operator reported that approximately 45% of the inflow at WWTP-2 is attributed to baseflows, and largely a result of groundwater infiltration.", "Furthermore, WWTP-2 is within a highly industrialised catchment which may be causing elevated ∑21PFAS levels in both groundwater and influent.", "Final effluent from WWTP-2 is mixed with reverse osmosis (RO) reject water.", "The RO process is an effective long-chain PFAA treatment, removing them from effluent then partitioning them to the RO reject water [41].", "Therefore, the re-introduction of RO reject water at this WWTP is likely contributing to the elevated PFAS concentrations observed in the final effluent.", "PFAS were detected in all WWTP solid samples, and the mean ∑21PFAS in solid samples was 34 ng g−1 dw (median: 12 ng g−1 dw; range: 2.0–130 ng g−1 dw) (Table 2).", "Mean concentrations of PFAS in solids followed the trend PFOS > PFDA > PFDoA > PFOA > PFHxA > PFHxS > PFBS > PFDS>8:2 FTS > PFTeA > PFBA > PFHpA > PFHpS>6:2 FTS.", "The compounds PFOS, PFDoA, and PFTeA were detected in >90% of samples, while the compounds PFOA, PFDA, PFHxA, and PFTrA were detected in 70–90% of solid samples.", "Six of the seven compounds with detection frequencies above 70% had a carbon chain length of eight or higher.", "The increased partitioning of PFAS to the solid phase within WWTPs has been associated with increasing fluoroalkyl chain length [11, 51, 55].", "The calculated mean partitioning coefficients from this study reflected this trend, increasing with increased fluoroalkyl chain length, and were higher in PFSAs compared to PFCAs of similar carbon chain length; except for 6:2 FTS which displayed the lowest mean partitioning coefficient, being primarily partitioned to the aqueous phase (Table S5).", "The highest mean concentration found in WWTP solids was for PFOS (mean: 14; median: 4.7; range < LOD - 90 ng g−1 dw).", "The lagoon-based treatment plant sludge and AS primary sludge displayed low PFAS concentrations compared to AS secondary sludge.", "The process of concentrating and recycling AS secondary sludge through the treatment process, combined with the aeration/agitation provided, likely facilitates increased secondary sludge PFAS concentrations.", "6:2 FTS was detected in 99% of aqueous samples (mean 7.3 ng L−1) and 26% of solid samples (mean 0.26 ng g−1 dw), At three of the larger WWTPs (-9, 330 ML day−1; -18, 143 ML day−1; and -19, 498 ML day−1), elevated total PFAS loading was a result of increased 6:2 FTS in influent (56, 23 and 38 ng L−1, respectively).", "6:2 FTS has been employed in aqueous film-forming firefighting foam (AFFF) in mixtures with fluoroalkylthioamido sulfonates [56], used as a PFOS replacement in metal plating applications [57] and is a transformation intermediate in the degradation of more complex fluorotelomer-based compounds [4].", "High concentrations of 6:2 FTS in WWTP effluents have been associated with AFFF use in catchments in the USA [33].", "It is possible that elevated levels of 6:2 FTS observed may be associated with AFFF use or PFOS substitution in metal plating, however, it more likely indicates the presence of a range of not yet measured precursor compounds with 6:2 FTS as an intermediate degradation product.", "The compound 6:2 Cl-PFESA was only detected in 4% of aqueous samples and 16% of solid samples between LOD and LOQ (Table 2).", "The compound 8:2 Cl-PFESA was not detected above LOD in any aqueous sample, and in 8% of solid samples between LOD and LOQ.", "These compounds have been demonstrated as the major components of the commercial product F–53B after purification, with a reported 6:2 Cl-PFESA content of 77.6% and 8:2 Cl-PFESA comprising an unreported percentage of the remaining fraction [19].", "F–53B is used as a PFOS alternative for mist suppression in metal plating applications used in China that has recently been detected in Chinese WWTPs and the environment [19, 24, 25].", "In Australia between 2006 and 2007, 99% of the directly imported PFOS was for use as a mist suppressant in metal plating which is listed as an approved, essential use [58].", "The Australian metal plating industry has no need to switch to alternatives like F–53B as PFOS is still approved for use.", "The low F–53B concentrations detected in this study may be a result of contamination of products sourced from markets that utilise F–53B.", "PFAS concentrations generally increased in both aqueous and solid matrices through the wastewater treatment process (Fig. 2).", "The mean concentration of ∑21PFAS increased as wastewater treatment progressed from influent, to primary effluent, secondary effluent, final effluent and recycled water (76, 89, 140, 140 and 120 ng L−1, respectively).", "PFCA concentrations in aqueous samples also increased from influent to final effluent, with levels persisting in recycled water, whilst PFSA concentrations within treatment plants varied.", "In influent, PFOS had the highest mean concentration (17 ng L−1) (Table S4).", "PFOA had the highest mean concentration in primary effluent (23 ng L−1), displaying an increase from mean influent concentration (7.9 ng L−1).", "PFHxA had the highest mean concentration from pooled aqueous samples in secondary effluent, final effluent, and recycled water; increasing in concentration from influent to primary, secondary and final effluent and recycled water (11, 16, 28, 28 and 32 ng L−1, respectively).", "Due to the delay of transmission of PFAS (caused by hydraulic retention time) within a wastewater treatment plant, the comparison of influent and effluent over the same 24-hour period may not be directly applicable.", "There was, however, a large variation in all PFAS concentrations between and within treatment plants from influent to final effluent.", "In 16 of the 19 WWTPs, ∑21PFAS concentrations in final effluent were greater than influent at the same WWTP, which is consistent with trends in WWTPs worldwide [11].", "At WWTPs-5, 6 and 9, ∑21PFAS concentration was greater in influent than final effluent and largely due to PFSA and FTS concentrations.", "Linear mixed-effects analysis of pooled influent and effluent data confirmed that some PFAS concentrations increased between influent and final effluent (Fig. 2).", "Between influent and final effluent, the compounds PFPeA, PFHxA, PFHpA, PFOA, PFNA, and PFDA (all of which are PFCAs) increased significantly.", "A number of transformation pathways with stable PFCA endproducts are known [18], this may explain some of the increase in PFCAs from influent to final effluent.", "Microbial degradation of the compounds 6:2 PAP and 6:2 diPAP using WWTP aerobic microbes has been shown to produce 6:2 FTOH, which was then degraded further to PFHxA [59].", "Furthermore, degradation of the compound 6:2 FTOH in activated sludge has been demonstrated to produce the corresponding 5:3 acid, which is then degraded further to PFHxA [34].", "Little transformation of 6:2 FTOH to PFPeA was observed as the intermediary product 5:2s FTOH was likely volatilized before biotransformation could occur.", "This may explain the high concentrations of PFHxA compared to PFPeA (whose precursor is partitioned to the gas phase) observed in this study.", "PFOA has been observed as a microbial transformation product of 8:2 diPAP in soil [60] and in gilthead bream [61].", "Furthermore, both PFOA and PFHxA have displayed net positive increases from influent to effluent, associated with diPAP and unknown PFAS precursor degradation in WWTP influent and sludge in three Swedish WWTPs [32].", "It is likely that similar precursor transformation processes are occurring within our studied WWTPs, contributing to increased PFCA concentrations as treatment progresses.", "The concentration of PFOS, PFDA, and PFDoA was higher in sludge, compared to other PFAS (Table S4).", "The median concentration of PFOS, PFDA, and PFDoA increased between primary and secondary sludge from 3,8 – 12, <LOQ – 17 and <LOQ – 14 ng g−1 dw, respectively (Fig. 2, Table S4).", "This increase between primary and secondary sludge was also reflected in the calculated distribution coefficients (Table S5); where coefficients increased between primary and secondary locations by 0.17–1.22 log units for PFOS and 0.37 to 1.34 log units for PFDA.", "Pearson correlation coefficients were positive for all PFAS measured in influent (Figure S1).", "In influent, positive, strong (r > 0.70) and significant (p < 0.05) correlations were displayed between compounds within the same compound class PFCAs (PFHxA-PFOA, PFHxA-PFNA, PFHxA-PFDA, and PFOA-PFDA) and between PFPeA-6:2 FTS and PFPeA-PFHxS.", "In final effluent, there were significant, positive, strong correlations between PFPeA-PFHxA, PFPeA-PFOA, PFHxA-PFOA, PFHxA-PFNA, PFHpA-PFHxA, PFHpA-PFNA, PFHpA-PFHxS, PFHpA-PFOS, and PFHxS-PFOS.", "There was only one significant negative correlation between 6:2 FTS-PFOA (r = 0.3).", "There were no significant correlations for the following: 1) PFBA and all other compounds; 2) 6:2 FTS and 5 of the eleven compounds; 3) PFBS-PFOA and PFBS-PFDA; 4) PFHxS-PFPeA and PFHxS-PFOA; 5) PFOS-PFPeA, PFOS-PFOA, and PFOS-PFDA.", "PFCAs and PFSAs were not strongly correlated in final effluent.", "This implies the distribution of PFCAs and PFSAs are WWTP specific and vary in final effluent independently of each other.", "There were four principal components in the influent data, and five principal components in the final effluent data with eigenvalues above 1 (Fig. 3).", "For the influent data, the first four components explained 93.2% of the variation (47.8, 20.2, 15.9 and 7.32%, respectively).", "In component 1, the PFSAs (PFBS, PFHxS, PFOS), odd chained PFCAs (PFPeA, PFHpA, and PFNA), and PFHxA displayed strong associations and accounted for a large proportion of the variation within the data.", "Short-odd chain PFCAs (<C8) and PFHxA have been associated as impurities, degradants and metabolites of the short-chain fluorochemistries used to replace PFOA [4, 18, 62] Furthermore, in Australia, PFOS is still employed in approved uses and there are no current restrictions on PFHxS or PFBS [13].", "The strong associations of these compounds and their contribution to the observed variation in influent data may reflect Australian PFAS usage trends and PFAS loading within specific WWTP catchments.", "Principal component 2 showed strong associations between WWTP daily inflow, percentage trade waste, and 6:2 FTS.", "This strong association was largely a result of the larger WWTPs accepting a higher proportion of trade waste, however, it shows the importance of 6:2 FTS as a possible trade waste indicator in these Australian WWTPs.", "In component 3, PFOA and PFDA were highly associated and in component 4, PFBA was the main contributor to the variation observed.", "This may indicate that these three compounds behave independently of each of in respect to PFAS loading in influent.", "For the final effluent data, the five principal components accounted for 89.5% of the variation (39.9, 21.5, 12.5, 7.82 and 7.74%, respectively).", "In component 1 of the final effluent, there were strong associations between PFSAs (PFBS, PFHxS, PFOS), odd chained PFCAs (PFPeA, PFHpA, and PFNA), and PFHxA.", "In principal component 2, there was again a strong association between daily inflow volume, percentage trade waste, and 6:2 FTS (an intermediate degradant from C6 based precursors), with the addition of PFOA.", "There are many demonstrated transformation pathways with PFOA as the terminal end-product [63], and the significant increase of PFOA from influent to final effluent may reflect this.", "The strong association of PFOA with 6:2 FTS (which showed no significant change between influent and final effluent), inflow and percentage trade waste may be a result of degradation of PFOA precursors (likely as impurities from the C6 manufacture process) associated with trade waste that are not yet measured in influent at these WWTPs.", "In component 3, there were strong associations between PFOS, PFHxS, PFOA, and PFDA, all of which have been used extensively in the past [4].", "In principle component 4, PFBA was the main contributor to variation, and behaved independently of the other PFCAs, reflecting the trend seen in influent.", "The compounds PFPeA and PFDA were strongly associated, accounting for a small amount of the variation in component 5.", "Calculation of the estimated annual discharge at a WWTP from a single sampling campaign contains a high uncertainty due to daily and seasonal variation [64].", "In Australian WWTPs, temporal variation of PFAS in influent [43] and effluent [37] has been shown to be low; with observed temporal variation in influent being more likely from pulse release as opposed to seasonal factors [43].", "It is, however, useful to estimate annual discharge to compare to similar Australian studies.", "Daily discharge rates from the 19 WWTPs in this study varied greatly, were similar to previous Australian studies, similar to studies worldwide and were influenced primarily by daily inflow (and as an extension WWTP size; Table S6).", "PFOS and PFOA concentrations were similar to those measured in 2014 from a study of nine PFAS in effluent from in 14 Australian WWTPs [37].", "In their study, they estimated a national ∑9PFAS discharge from Australian WWTPs as 175 kg per year in Australian WWTP effluent [37].", "Assuming the same annual discharge volume of 3013 GL and using mean annual discharge rates from the 19 WWTPs in this study, we calculated an estimated discharge of ∑21PFAS of 339 kg.", "When compared, their study and our study produce similar yearly mass discharged for PFOA, PFNA, PFDA, PFHxS and PFOS (Fig. 4).", "In our study the estimated yearly mass discharged was higher for PFHpA (8.8 vs 22 kg annually) and PFHxA (43 vs 87 kg annually).", "This difference may be a result of changing PFAS use patterns or bias introduced through the WWTPs selected and sampling design each study.", "The annual mass discharge of three compounds not measured in [37]: PFBA, PFPeA and 6:2 FTS (25, 27 and 19 kg annually), were similar to that of PFOS calculated for both studies (26 vs 26 kg annually).", "If this is the case for three compounds, and as there are now over 4700 listed PFAS in the environment [10], it is likely both studies have underestimated the total PFAS emissions from Australian WWTPs.", "Twenty-one PFAS from four classes (PFCAs, PFSA, FTS, F–53B) were measured in aqueous and solid samples from the 19 Australian WWTPs.", "PFAS was detected in every sample analysed.", "Many PFAS were highly correlated, suggesting similar sources of PFSAs and PFCAs and independent behavior of these compound classes within WWTPs.", "Statistical analyses showed an increase of PFPeA, PFHxA, PFHpA, PFOA, PFNA, and PFDA between influent and final effluent.", "When compared to Australian WWTP PFAS emission data measured in 2014, the estimated annual discharge for the newly reported compounds PFBA, PFPeA and 6:2 FTS (25, 27 and 19 kg annually) were similar to PFOS (26 kg annually).", "This demonstrated that it is likely both studies have significantly underestimated the total PFAS emissions from Australian WWTPs and future work is required to determine the risk profile of PFAS present and total PFAS loading at Australian WWTPs.", "The compounds 6:2 FTS and 8:2 FTS quantified, and F–53B components 6:2 Cl-PFESA and 8:2 Cl-PFESA were detected in Australian WWTPs.", "6:2 FTS was strongly associated with the proportion of trade waste in influent, was partitioned to the aqueous phase, had a similar estimated Australia-wide annual mass discharged in effluent to PFOS, and did not significantly decrease between influent and final effluent.", "Although the ecological risk of 6:2 FTS is considered low, there are many unknowns regarding the environmental fate and effects and its presence likely indicates the degradation of currently employed short-chain fluorochemistries.", "In Australia, the presence of 6:2 FTS may be an emerging concern in Australian WWTPs and aqueous environments receiving WWTP effluent.", "Timothy Coggan: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.", "Damien Moodie: Performed the experiments; Contributed reagents, materials, analysis tools or data.", "Adam Kolobaric, Drew Szabo: Performed the experiments.", "Jeff Shimeta, Nicholas Crosbie, Elliot Lee, Milena Fernandes: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.", "Bradley Clarke: Conceived and designed the experiments.", "This work was supported by an Australian Government Research Training Program (RTP) Scholarship.", "Funding support was provided by Water Research Australia Limited, Water Corporation (WA), SA Water and Melbourne Water.", "The authors declare the following conflict of interests: Nicholas Crosbie is an employee of Melbourne Water who provided samples and funding.", "Elliot Lee is an employee of Water Corporation who provided samples and funding.", "Milena Fernandes is an employee of SA Water who provided samples and funding.", "Timothy Coggan, Damien Moodie and Drew Szabo have scholarship support from Melbourne Water through Water Research Australia.", "Bradley Clarke receives some research funding from Melbourne Water.", "Jeff Shimeta and Bradley Clarke are PhD supervisors for Timothy Coggan, Drew Szabo and Damien Moodie.", "Adam Kolobaric declares no conflicts of interest.", "No additional information is available for this paper.", "Supplementary content related to this article has been published online at https://doi.org/10.1016/j.heliyon.2019.e02316." ]
[]
A theory of economic unions
After decades of successful growth, economic unions have recently become the focus of heightened political controversy. To understand why, we develop a theoretical model of the effects on trade, income distribution and welfare of economic unions that differ in size and scope. Our model shows that political support for international unions can grow with their breadth and depth provided member countries are sufficiently similar. However, differences in economic size and factor endowments can trigger disagreement over the value of unions. Our model is consistent with some salient features of the process of European integration and statistical evidence from survey data.
[ "ECON" ]
[ "Economic unions", "European integration", "Non-tariff barriers" ]
[ "2002", "2003" ]
[ "The development of global markets increasingly relies on international institutions providing common regulation to reduce or remove non-tariff barriers that hamper trade across national borders.", "The growing importance of these non-tariff barriers reflects, on the one hand, the decline of more obvious costs of international trade: progress in transportation and communication technology has steadily reduced shipping costs, while multilateral and regional trade agreements have brought tariffs down to an all-time low of 3% on average.1", "On the other hand, regulatory harmonization has become more important with the increasing complexity of world trade.", "Recent decades have witnessed the growth of trade in tasks, with different stages of production located in different countries along global value chains.", "In the future, growth in international trade must increasingly lie in the service sector.", "Its importance is steadily growing, and it already accounts for almost two thirds of world output; yet it only accounts for less than a quarter of world trade, in part because many services—especially professional and financial services—are bound by distinct national regulations.2", "The need for common policies to enable market integration and reap the gains from trade has led to the creation of international economic unions.", "Europe has been at the forefront of this institutional development.", "Establishing a common market was the core objective of the European Economic Community at its founding in 1957.", "Over the following six decades, what is now the European Union has grown from 6 to 28 member states, while steadily deepening economic integration and regulatory harmonization in its Single Market.", "Yet economic unions are far from an exclusively European phenomenon; on the contrary, they are found on every continent.3", "Moreover, recent trade agreements such as the European Union–Japan Economic Partnership Agreement have increasingly emphasized regulatory cooperation, common standards and impartial enforcement procedures for the protection of investors and intellectual-property owners.", "After decades of successful growth, however, economic unions have become the focus of heightened political controversy over the past few years.", "After the election of President Trump in 2016, the United States abandoned both the Trans-Pacific Partnership and the Transatlantic Trade and Investment Partnership.", "Also in 2016, the United Kingdom voted in a referendum to leave the European Union.", "Euro skepticism appears on the rise more broadly, and plans for the enlargement of the euro area are effectively on hold.", "These setbacks do not mean that international institution are now facing a universal backlash, just as they did not previously enjoy universal support.", "Yet, they have become increasingly polarizing.", "Both President Trump and Brexit won narrow and bitterly divisive victories at the polls.", "Eurobarometer surveys show that the share of European citizens who perceive net benefits of European Union membership has remained quite steady over the decades; however, the share who perceive net costs has been gradually catching up, as the share of undecided respondents fell (Fig. 1).", "Preference polarization over international economic integration is naturally linked to the changing nature of international trade.", "Throughout the second half of the twentieth century, the bulk of world trade was taking place between similar countries.", "Likewise, economic unions initially included countries with comparable levels of income and factor endowments.", "A key feature of this type of economic integration is that it does not imply the reallocation of resources predicted by conventional models of trade driven by differences in factor endowments.", "Accordingly, one of the original motivations for developing models of trade in differentiated varieties was the need to account for episodes such as the creation of the European Economic Community, in which trade liberalization had faced little political opposition because it had led to rises in real income for owners of all factors in all member states (Helpman and Krugman, 1985).", "Yet, the enlargements of the European Union to the East, trade liberalization in developing countries and the rise of China in global markets have brought distributional considerations to the forefront.", "As a result, many now fear that the negative consequences of import competition can overshadow the benefits of market size.", "To shed light on these phenomena, in this paper we develop a theoretical framework to study the effects on trade, income distribution and welfare of economic unions that differ in size and scope.", "We then apply our model to interpret the process of European integration.", "Section 2 presents the basic setup of our theory.", "It considers a multi-country, multi-industry and multi-factor framework that combines the Krugman model of trade and monopolistic competition with the Ricardo-Viner model of specific factors.", "In particular, countries produce differentiated varieties in a continuum of industries employing sector-specific human capital.", "They differ both in size and in the distribution of their factor endowments across sectors.", "These differences entail potential gains from both intra-industry and inter-industry trade.", "In Section 3, we add to our framework a theory of border costs.", "We assume that in some industries trade is possible only in the presence of union policies that overcome non-tariff barriers.", "For instance, common regulations or standards are often needed for firms to sell their products in foreign markets, especially in certain sectors such as financial services.", "We explicitly recognize that all economic unions are not the same.", "In particular, unions vary in their depth, i.e., the measure of industries covered by union policies; and their breadth, i.e., the set of countries included in the union.", "We study how these aspects of the union determine its impact on the world distribution of income and welfare.", "In Section 4, we determine the sources of political support for specific economic unions and derive predictions on how this political support varies across countries, and how it reacts to changes in the depth and breadth of the union.", "We find that some of the effects of union policies are homogeneous within each country because they reflect changes in prices that accrue entirely to consumers.", "This is the case for the benefits of increased variety that result from intra-industry trade.", "Inter-industry trade also yields consumer benefits, but in addition it has distributive consequences across workers in the same country.", "Workers in exporting industries reap an extra gain from accessing foreign markets, while workers in importing industries lose from the entry of foreign competitors into their home market.", "The model paints a rich and realistic picture, which yields the following main results.", "If union members are sufficiently similar, there is no disagreement either between or within countries.", "Support for the union unanimously increases with the size and scope of the union.", "The intuition for this result is simply that the value of trade-promoting policies increases with the number of potential trading partners and the industries they cover.", "However, disagreement over the value of unions arises if countries differ in size, income and endowments.", "Differences in economic size introduce disagreement over the value of the union between countries, as the benefit from the access to the larger union market is higher for small and poor countries.", "Comparative advantage due to differences in relative endowments introduces instead disagreement over the union within countries.", "Workers in comparative-advantage industries support the union because they stand to benefit as exporters.", "Workers in comparative-disadvantage industries benefit from lower prices, but experience a fall in their income due to import competition from other countries in the union.", "Hence, the effect of comparative advantage on the support for market integration is nuanced.", "On the one hand, inter-industry trade increases the value the union.", "On the other hand, it also generates winners and losers.", "Various scenarios may arise.", "For instance, adding dissimilar countries can weaken the support for the union, even when more market integration is beneficial for all countries as a whole.", "The reason is that, while winners gain more from such an enlargement, the number of sectors and workers threatened by import competition increases.", "Section 5 discusses how our theory helps interpret the history of European integration.", "In the twentieth century, the European Union steadily grew in size up to 15 members, while constantly deepening market integration and enjoying broad political support.", "This pattern is consistent with our prediction that economic unions can grow without triggering opposition when their members are not too dissimilar.", "In the twenty-first century, however, enlargement has brought into the European Union countries that are smaller, poorer and have a different mix of factor endowments.", "In keeping with our theory, this has led to growing political tensions and discontent among losers in larger, richer countries.", "We provide new statistical evidence that measures of economic size and exposure to import competition from other European Union countries correlate with survey data on attitudes towards the union precisely as predicted by our model.", "In particular, rich and large countries tend to have a more negative image of the European Union.", "Moreover, support for the European Union is weaker in countries with a larger share of workers employed in comparative-disadvantage sectors; finally, this share has increased in many member countries after the 2004-06 enlargements.", "There is a large literature on international and regional trade agreements: Freund and Ornelas (2010), Maggi (2014; 2016), Grossman (2016), and Bagwell and Staiger (2016) provide excellent surveys.", "This literature considers international agreements as coordination and commitment devices to prevent the escalation of negative externalities generated by trade policy and to protect governments from the influence of domestic pressure groups.", "It has studied the design of rules for achieving these goals, and the merits of multilateral relative to regional negotiations.", "In contrast, we study the effect of unions at eliminating non-tariff barriers to trade and removing the undesirable “border effect” that these barriers produce.", "Our focus is on the heterogeneous costs and benefits of these policies.", "We abstract from coordination and commitment problems, which have been studied extensively.", "A recent paper that also examines non-tariff barriers, but focuses instead on coordination problems in the adoption of product standards, is that of Grossman et al. (2019).", "Our paper is also related to the literature on the size of trade-promoting international unions.", "Several papers build on the insight from theories of federalism (Oates, 1972) that unions, like centralized jurisdictions, reap the benefits of coordination and market integration, but at the cost of imposing uniform policies on members with different preferences (Alesina et al., 2005; 2000; Alesina and Wacziarg, 1999; Bolton and Roland, 1996; 1997; Casella, 2001; Casella and Feinstein, 2002; Gancia et al., 2018).", "These papers have studied how the size of jurisdictions changes with exogenous changes in the costs of trade.", "In contrast, we study the effect of unions that vary in size and scope on countries that differ in size, productivity and factor endowments.", "In this respect, our model is also related to a small set of papers studying asymmetric unions (Berglof et al., 2008; 2012; Harstad, 2006).", "This literature has however focused on a different question, namely, whether the possibility of forming “inner clubs” is desirable and/or can sustain more cooperation in the presence of externalities.", "Finally, there is a new but fast-growing literature on the recent backlash against globalization.", "There is evidence that voters exposed to import competition become more protectionist (Feigenbaum and Hall, 2015), and that the opposition of import-exposed workers to international economic integration was one of the economic drivers of support for Brexit (Becker et al., 2017; Colantone and Stanig, 2018a).", "More broadly, a series of recent papers have investigated empirically the connection of import competition with economic nationalism and political extremism (Autor et al., 2017; Che et al., 2016; Colantone and Stanig, 2018b).", "Dippel et al. (2015) and Jensen et al. (2017) have shown that, just as import competition is positively linked to anti-establishment politics, export opportunities have the opposite political effect.", "From a theoretical perspective, political opposition to globalization has been associated mostly to a rise in inequality (Grossman and Helpman, 2018; Pastor and Veronesi, 2019).", "In this paper, we also consider inequality, but we show how it interacts with other factors giving rise to a rich set of results.", "Interestingly, Buera et al. (2011) warned that, in a model with uncertainty and learning, a large economic shock could trigger a reversal against market-oriented policies.", "We instead abstract from issues related to information frictions.", "While all the papers in this literature study unilateral policy choices, we focus on political support for existing international unions.", "This section develops a multi-country, multi-industry and multi-factor framework that combines the Krugman model of trade and monopolistic competition with the Ricardo-Viner model of specific factors.", "Each industry contains a continuum of monopolistic competitors producing differentiated products and earning zero profits.", "Labor is the only factor of production; but there are many different types of labor, one for each industry.", "As usual, country borders affect trade.", "In labor markets, border costs are prohibitive and producers hire domestic labor only.", "Thus, there is a local labor market for each country and labor type.", "In product markets, border costs vary across industries and country pairs.", "Thus, some product markets are local, some are global, and some are somewhere in between.", "What determines the matrix of bilateral border costs?", "How does an economic union affect this matrix?", "We now add to our framework a theory of border costs and the impact of an economic union on them.", "This theory recognizes that all economic unions are not the same.", "In particular, unions vary in their depth, i.e., the measure of industries covered; and their breadth, i.e., the set of countries included.", "We study how these features of the union affect the distribution of industry incomes and prices.", "In global industries, incomes reflect productivities.", "In local industries, factors are expensive in countries where they are scarce relative to the country’s aggregate income.", "In union industries, factors are expensive in industries whose union-wide endowment is scarce relative to the union’s aggregate income.", "Aggregate incomes reflect productivities in all tradable industries.", "For countries that do not belong to the union, and for the union as a whole, nominal income simply equals the average factor endowment in effective units across all global industries.", "For union members, nominal income can be intuitively decomposed as a weighted average of the country’s income in global industries and the country’s share of the union’s income in union industries.", "We are now ready to derive predictions about the political support for the union.", "To do this, we ask the inhabitants of our theoretical world whether a specific union raises their welfare relative to having no union at all.", "We then record their positive answers as support for the union, and the negative ones as opposition to the union.", "Thus, our exercise consists of counting winners and losers.", "Naturally, this second case of unequal sharing is the empirically relevant one.", "But we start by analyzing the case of equal sharing because it is easier analytically, and because it is a first step towards analyzing the case of unequal sharing.", "This decomposition highlights a few results that apply to our setting, some of which are familiar from trade theory.", "The analysis so far has assumed that union membership is accompanied by a redistributive policy that ensures that the gains from union membership are equally shared.", "This assumption allowed us to focus on the representative worker of country n.", "In the absence of redistributive policies, the gains from union membership are not equally shared.", "In particular, union membership also disrupts the domestic income distribution in every member country, so much so that it may leave some residents worse off.", "We turn now to these distributive tensions.", "Naturally, the trade balance in local industries is zero both without and with union membership.", "As a result, local industries do not experience a change in the share of national income.", "These results suggest that workers in all local industries and all exporting union industries support union membership.", "Whether workers in global industries and workers in importing union industries support union membership is ambiguous.", "But we can still say a few things about them.", "Workers in importing union industries are divided.", "Those with a weak comparative disadvantage still support the union since it lowers the cost of living.", "Those with a strong comparative disadvantage do not support the union.", "All workers in global industries are affected in the same way.", "Thus, all global industries support union membership if, as a group, they have a comparative advantage relative to union industries.", "Otherwise, they may or may not support union membership.", "They will support it because of its consumer benefits if the average comparative disadvantage of global industries relative to union industries is weak.", "They will oppose the union because of producer losses if their average comparative disadvantage is strong.", "Finally, it is worth saying a bit more about the redistribution caused by the union.", "In a sense, all our simplifying assumptions have been designed to “manage” the shape of this redistribution so as to obtain a clear theoretical benchmark.", "For instance, there is no redistribution between tradable (global and union) and non-tradable (local) industries.", "The reason, of course, is the unit elasticity of substitution between products of different industries.", "If this elasticity were larger than one, there would also be a redistribution from non-tradable industries to tradable ones.", "If this elasticity were less than one, this redistribution would go in the opposite direction.", "There is no redistribution either from non-members of the union to members of the union or vice versa.", "Even if we kept the unit elasticity assumption, we would observe such redistribution if border costs were not extreme.", "Since the union lowers the cost of trading among its members, it shifts demand towards union members and away from non-members.", "We do not observe this shift of demand away from non-members because there is no such demand with extreme border costs.", "If we made these costs less extreme, the creation of a union would lead to redistribution from non-members to members within the affected industries.", "All these complications might be important in quantitative applications.", "However, they seem likely to be less central than the already rich pattern of redistribution we focus upon here.", "In any case, even these additional redistributive patterns become clearer once we look at them from the perspective of our stylized benchmark.", "European integration started when six countries—Belgium, France, Italy, Luxembourg, the Netherlands, and West Germany—signed the Treaty of Paris in 1952 and the Treaty of Rome in 1957, creating the European Economic Community.", "Member countries removed customs duties and agreed on a common agricultural policy.", "The first enlargement happened in 1973, when Denmark, Ireland and the United Kingdom joined the union.", "During the 1970s, the union implemented regional policies to help the development of poorer areas and adopted laws to protect the environment.", "Over time, the European Parliament increased its influence and, from 1979, all citizens started to elect its members directly.", "In 1981, Greece joined the union.", "Spain and Portugal followed in 1986.", "In the same year, the Single European Act extended Community powers especially in the area foreign policy.", "In 1993, the Maastricht Treaty established the European Union and the Single Market with its “four pillars:” the free movement of goods, services, capital and persons.", "In 1995, the European Union gained three more new members: Austria, Finland and Sweden.", "In 1999, the euro as a currency was launched.", "Ten new countries—Cyprus, the Czech Republic, Estonia, Hungary, Latvia, Lithuania, Malta, Poland, Slovakia and Slovenia—joined the European Union in 2004, followed by Bulgaria and Romania in 2007.", "In response to the eurozone debt crisis, the so-called “banking union” was established, transferring responsibilities for banking policy from the national level to the European Union.", "In 2013, Croatia became the 28th member.", "Besides these major events, the European Union was built on a complex maze of treaties and agreements, steadily widening and deepening the economic integration between its members.", "Ever since its initial foundation, the European Union has also been accompanied by controversy.", "Clear examples of disagreement are various cases of failed expansions.", "For instance, the UK’s membership was vetoed by France in 1961; Spain’s application was rejected by the European Council in 1962; Norway’s citizens voted against joining the union in 1967 and 1992.", "Yet, until recently, the union has always grown in size and scope.", "In 2016, for the first time, a member state—the UK—voted to leave the union.", "Anti-European Union sentiment has also been on the rise in other countries.", "What is driving this growing dissatisfaction with European integration?", "Our specification of factor endowments implies that, for all countries and union types, the average income in union industries equals the average income in global industries.", "Thus, the share of domestic income of workers in global industries is not affected by the depth and breadth of the union.", "The latter creates redistribution across union industries only.", "The intuition provided by this example is that a union becomes unstable and fragile when its member countries have sufficiently different patterns of comparative advantage, and thus sufficiently large imbalances in intra-union trade.", "While such a union creates greater efficiency gains than a union among homogeneous countries, those gains accrue to consumers and to winners in each country’s export industries.", "Instead, losers emerge whose welfare is reduced by the union.", "A marginal reduction in union depth is likely to be one strategy to reduce their number.", "However, another and more efficient strategy may be a “big push” towards much closer integration.", "As Eq. (43) highlights, this second possibility exists when a fully integrated common market is valuable enough, i.e., when gains from intra-industry trade are high (σ is low) and trade without union policies is limited (τ is low).", "Both Eq. (43) and Eq. (41) show that opposition to a heterogeneous union is greater in larger member countries.", "The reason, intuitively, is that residents of larger economies stand to gain less as consumers from the common union market.", "Therefore, they are more sensitive to the losses they may suffer as producers in import-competing industries.", "Finally, note that Eq. (41) measures the share of industries in country n that oppose the union.", "We have assumed for ease of exposition that workers are uniformly distributed across industries, but it is straightforward to relax this assumption.", "Then, popular opposition to the union will be greater in countries with a larger share of the workforce in low-productivity, comparative-disadvantage sectors.", "These two forces—lower consumer gains in larger economies, and more numerous losers in countries with greater employment in import-competing industries—are the two predictions that we examine next.", "Before doing this, it is worth also noting that ultimately the union becomes unpopular because countries are not implementing redistributive policies that ensure an equal or fair sharing of the gains from union membership.", "With such policies in place, union membership would be universally supported.", "Fig. 2 plots the evolution over time of this measure of discontent.", "The solid line corresponds to the European Union simple average, the dashed line is the simple average for the sample of 15 member countries in 2000, and the dotted line is the European Union average weighted by population.", "A number of facts stand out.", "First, the figure shows a growing dissatisfaction that started around 2007 and continued until 2012.", "Clearly, one culprit is the eurozone debt crisis.", "However, the deterioration of the image of the European Union seems to predate the crisis.", "Second, the dashed line (EU 15 average) is above the solid line (EU average), implying that new members tend to have a more positive view of the European Union.", "Moreover the difference seems to have grown, at least until 2016.", "This means that dissatisfaction has grown especially in countries that were part of the European Union before the accession of new members and suggests that “enlargement fatigue” may have played a role.", "Third, the dotted line (population weighted average) is closer to the dashed line (EU 15 average), confirming that new members tend to be smaller in size.", "Finally, all lines display strong co-movements, suggesting that some of the driving forces may be common to many countries.", "As a next step, we do a simple variance decomposition exercise.", "We ask what fraction of the variance in attitudes towards the European Union is accounted for by time-specific common factors and country-specific factors.", "We do this by comparing the R-squared obtained by regressing the share of respondents with a negative image of the European Union on year and country fixed effects.", "This exercise shows that the largest source of variation is cross sectional: country fixed effects alone explain 49% of the data, while year dummies accounts for 17%.", "We now discuss the main factors that, according to our model, may explain the observed time and cross-sectional variation.", "Starting from the latter, our model suggests that an important determinant of preferences for the union is economic size: larger and richer countries benefit relatively less from adopting union policies.", "To have a first sense of whether this prediction is consistent with the data, Fig. 3 shows a scatter plot of respondents with a negative image of the European Union (vertical axis) against the logarithm of aggregate GDP (horizontal axis) in 2017.11", "The picture shows a clear positive association between size and a negative image of the European Union, highlighted in the graph by the linear regression line.", "The figure is also useful to visualize the cross-sectional variation in the data.", "It is immediate to see that countries differ substantially in their attitude towards the union.", "The most pro-Europe members states (less than 10% of respondents with a negative view) include small countries such as Malta, Lithuania and Ireland, but also Germany, despite its size.", "Countries with a strong anti-Europe attitude (more than 20% of respondents with a negative view) include several large economies, such as the UK and Italy, but also Greece.", "Regarding the time variation, our theory suggests that European Union enlargement may be an important factor.", "While a bigger union is better at promoting trade, it may lead to more disagreement if new members make the union more diverse.", "This is indeed what happened.", "As Fig. 4 shows, enlargements have made the European Union more heterogeneous in economic terms.", "The figure shows the average aggregate GDP of union members relative to the average GDP of the six founding members.", "The figure confirms that, almost invariably, each enlargement included countries with an economic size on average smaller than the initial members.", "Moreover, the sharpest drop in average country size happens after the 2004 enlargement.", "The European Union has also become more diverse in terms of factor endowments: since 2004, new member states were not just smaller, but also on average poorer and with lower levels of education.", "Our theory then suggests that enlargement fatigue could be driven, at least in part, by a dissatisfaction of the larger economies with the extent of economic integration with much smaller and poorer economies.", "Increased trade with countries with different factor endowments may have had redistributive effects that heightened disagreement over the union within countries.", "This argument is consistent with the simple observations that the UK has always been one of the largest economies in the European Union and that UK workers employed in sectors more exposed to import competition from Eastern Europe voted predominantly to leave the union.", "Besides enlargement, our theory also suggests that attitudes towards the European Union may also be affected by any other changes that make workers in a country more exposed to import competition from the union.", "We now study the relationship between economic size, import competition and discontent towards the European Union more systematically.", "To this end, we have collected a number of additional variables sourced from Eurostat.", "To measure aggregate economic size, we consider separately the logarithm of population and of GDP per capita.", "Breaking down the two components of size is useful as they exhibit significantly different variation over time and across countries.", "Next, we are interested in measuring workers’ exposure to import competition from other European Union members.", "Our model suggests that workers employed in sectors with a comparative disadvantage relative to other countries in the European Union should have a more negative view of the union.", "To test this prediction, we have collected data on employment and the value of production at a detailed sectoral level (NACE classification, level 2) from the Structural Business Statistics (SBS) database, describing the structure and performance of businesses across the European Union.", "Then, for each country-year, we computed the fraction of employment in industries with a share of domestic production lower than the income share of the country in the European Union.12", "According to our model, this variable captures the share of workers in industries with a comparative disadvantage who might lose income due to import competition from the European Union.13", "It measures aversion to the union net of size effects, which are instead captured separately by GDP, and other price effects.", "Exposure to import competition, which is available from 2000 to 2016, varies both because the structure of employment and production varies in all countries, but also because the composition of the European Union changes over time.", "For this variable, Fig. 5 plots the evolution of the European Union simple average (solid line) and the simple average for the sample of 15 member countries in 2000 (dashed line).", "The rise in the dashed line and its divergence from the solid line after 2004 indicate that enlargement has made workers in the initial EU 15 countries more exposed to import competition from new members, whose workforce is more concentrated in sectors with net exports.", "Interestingly, the fact that the dashed line (EU 15 average) keeps rising suggests that import competition from new member states has grown even after their accession.", "Moreover, comparing Fig. 5 to Fig. 2 it is easy to see that exposure to import competition in the EU 15 countries and a negative attitude towards the European Union display a similar evolution, both peaking in 2012.", "Fig. 6 shows instead a scatter plot of respondents with a negative image of the European Union (vertical axis) against the share of workers exposed to import competition from the European Union (horizontal axis) in 2016.", "The picture shows a clear positive association between the fraction of workers in comparative disadvantage industries and a negative image of the European Union, highlighted by the linear regression line.", "Besides these variables, which have clear counterparts in our model, we also consider a number of additional controls.", "Since our theory suggests that exporters are keener on economic integration than importers, we also consider net exports to the European Union, normalized by the volume of trade with the European Union.", "The existing literature suggests that negative attitudes towards the European Union are typically associated with economic hardship, low levels of education and inequality.", "There is also a popular view that immigration may have triggered an anti-Europe sentiment.", "Hence, to account for these complementary explanations, we control for the unemployment rate of the active population (in percentage points), as a measure of economic crises; for the share of working-age population (25–64) with tertiary education; for the Gini coefficient of the net income distribution, as a measure of inequality; and for the stock of immigrants as a share of population.", "Most of these variables are available for all 28 countries over the period 2000–2018.14", "We start by studying correlates of changes in negative attitudes towards the European Union, with the caveat that, for some variables of interest, the most meaningful variation is cross sectional.", "Table 1 reports panel regressions with country fixed effects and standard errors clustered at the year level.", "Column (1) shows that the coefficients for both components of economic size are positive, but only population is statistically significant (at the 5% level).", "This is not too surprising, given that changes in GDP per capita over time are likely to capture cyclical factors such as the severity of the crisis, which may in turn prompt more negative views.", "Column (2) shows, as expected, that changes in import competition from the European Union are strongly correlated with a negative image of the union.", "The coefficient is significant at the 1% level.", "Column (3) confirms the previous findings when all these variables are included simultaneously.", "In column (4) we include all the other control variables, except immigration, which has several missing values.", "The coefficients for population and import competition remain highly significant.", "Regarding the new controls, perhaps surprisingly, the coefficient for net exports to the European Union and tertiary education turn out to be positive and significant.", "However, as we shall see, these findings are not robust, which may reflect the fact that changes in these variables over time are hard to interpret.15", "An increase in the unemployment rate is associated with a more negative view of the European Union, consistent with the view that the crisis has fueled anti-Europe sentiments.", "Changes in the Gini coefficient have instead no statistically significant correlation.", "In column (5) we also include the share of immigrants.", "Despite the loss of observations, the coefficients for population and import competition remain significant.", "Finally, in column (6) we include year fixed effects, thereby removing any common factor.", "Despite the demanding specification, the positive coefficient for exposure to import competition remains highly significant.", "Interestingly, we now find that differential increases in inequality, as captured by the Gini coefficient, are associated with a more negative attitude towards the European Union.", "The same applies to the variables capturing macroeconomic performance: a fall in GDP per capita or an increase in the unemployment rate relative to other countries is correlated with a lower support for the union.", "Next, we focus on the cross sectional variation in the data.", "Table 2 replicates columns (1)–(5), but with year fixed effects instead of country dummies, and clustering standard errors at the country level.", "The coefficients for both components of economic size are still positive, but now only GDP per capita is statistically significant (at least at the 5% level).", "Hence, rich countries tend to have a more negative view of the European Union.", "The coefficient for import competition is also positive and significant (except in one case).", "Interestingly, in the cross section, net exports to the European Union are strongly correlated with a less negative image of the union.", "These results confirm that countries with higher employment in import-competing sectors and higher imports relative to exports to the European Union tend to dislike the union more.", "There is also some evidence that tertiary education is correlated with a more favorable view of the European Union.", "The relatively weak correlation between education and preferences for the European Union seems in contrast with existing evidence within countries.", "For instance, for the UK, Becker et al. (2017), Pastor and Veronesi (2019) and others find that voting for Brexit is associated with low educational attainment.", "A special survey by the European Commission (2006) also shows that education markedly affected the attitude of Europeans towards European Union enlargement: 51% of those who left full-time education at the age of 20 or later favored European Union enlargement, compared to 35% of those who left at age 15.", "Yet, a discrepancy between results within and across countries is consistent with our model, which highlights different sources of disagreement over union policies across and within countries.", "From this perspective, it is useful to look at how attitudes towards the European Union vary with the level of education by country.", "Data from the Eurobarometer stratified by education groups confirm that the share of respondents with a “very positive or fairly positive” image of the European Union in 2017 is on average much higher (48%) among individuals who left education at the age of 20 or later than among those who left at age 16–19 (36%) or below (29%).", "But do these differences between more and less educated people vary systematically with a country’s income level?", "In other words, are highly educated workers more pro-Europe in richer countries?", "The answer to this question is provided by Fig. 7, which shows the share of positive views among respondents who left education at age 20+ relative to those who left school at age 16–19, against the log of GDP per capita in 2017.", "This scatter plot confirms that in all countries but Hungary positive views are more frequent among better educated people.", "Strikingly, however, this difference increases with income.", "In particular, the coefficient of a linear regression is 0.13, with a standard error of 0.05.16", "What can we learn from these results?", "The evidence suggests that attitudes towards the European Union mirror the distribution of the gains from intra-EU trade.", "It broadly supports the predictions of our theory.", "Smaller and poorer countries, and countries with a larger share of employment in export-oriented sectors, reap greater benefits from accessing the larger union market.", "Within countries, educated workers are more likely to benefit from trade because they tend to be specialized in industries where products are more differentiated and gains from variety higher (e.g., Epifani and Gancia, 2008).", "At the same time, as in models of inter-industry trade, gains are larger for workers in export industries, such as the high-skilled in rich countries, then for those in import-competing industries.17", "In line with this view, there is a growing empirical literature suggesting that exposure to import competition has increased the support for parties and politicians with protectionist, populist, and nationalist agendas (Colantone and Stanig, 2018a; 2018b).", "While import competition is often perceived as a major threat of globalization, export opportunities can instead help explain the support for international unions.", "The German case is of particular interest here.", "Dauth et al. (2014) find that the rise in trade with Eastern Europe caused job losses in German regions specialized in import-competing industries, but that these losses were more than compensated by employment gains in export-oriented industries.", "Dippel et al. (2015) also find that German regions that gained better export opportunities reduced their vote share for extreme right-wing parties, at the same time as it increased in regions that faced sharper import competition.", "These results then offer some insight on the persistent support for European integration in Germany.", "While Germany is the largest economy in Europe, it is also an export powerhouse, with a trade surplus in a majority of industries.", "Our framework then suggests that German workers are uncommonly keen on the Single Market because they are more likely to be employed in sectors with positive net exports to the European Union.", "The expansion of the WTO from 23 to 164 countries, the enlargement of the European Union, especially after 2004, the rise of China and the emergence of global supply chains have all fostered trade between an increasingly diverse set of countries.", "While all these phenomena raise the potential gains from trade, our model highlights that they may also undermine the political support for the process of economic integration.", "We have shown how the support for international unions can become weaker as member countries become more dissimilar in economic size and factor endowments.", "If an international union comprises countries that are sufficiently similar, it can grow without facing any opposition.", "An intra-union pattern of comparative advantage due to differences in endowments, instead, increases the value of the union but also creates winners and losers within each country.", "This redistribution may undermine the support for the union even when the country as a whole would benefit from union membership.", "Moreover, differences in economic size imply that, in the presence of any cost of integration, larger economies would prefer a shallow union, while smaller ones prefer deeper integration.", "What policies could then be adopted to restore the support for more economic integration, and hence to reap additional gains from trade?", "If disagreement over the value of the union is between winners and losers within countries, domestic redistribution may suffice to solve it.", "This was clear in the theory developed here.", "A further complication can arise once we relax some of the assumptions of the theory and it becomes possible that some countries lose as a whole from further integration (relaxing the unit elasticity assumption across industries would do this).", "If then the disagreement is between countries that differ in size and income, solving it requires some form of international redistribution.", "This may be more difficult to achieve, given that such policies tend to be politically costly and hard to implement.", "Alternatively, support for the union may be restored by making the union more attractive, for instance by deepening integration in areas that are less contentious.", "In this paper, we have taken the size and scope of the union as given.", "However, our model shows how changes on one margin affect preferences for the other.", "For instance, as we have already argued, the accession of smaller and poorer countries, which want deeper integration, may trigger a deepening of the union which may go against the desire of larger and richer countries.", "Studying more formally the interaction between the size and scope of economic unions may help shed new light on recent phenomena and seems a fruitful avenue for future research.", "Finally, we cannot fail to notice that much of the political debate about European integration, and especially about Brexit, has focused on migration.", "Migration features prominently on the front pages of newspapers, in the narratives of politicians, and so on.", "At the same time, we cannot fail to notice either that migration flows within Europe are small relative to trade flows.", "According to Eurostat demography figures, out of 512 millions residents of the European Union in 2017, only 22 million (4.3%) were non-EU citizens and only 17 million (3.3%) were EU citizens living in a country other than their country of citizenship.", "These are very small numbers.18", "Instead, intra-EU trade in goods (19.6%) and services (7.4%) add up to 27% of the total GDP of the European Union.", "These are not small numbers.", "Clearly we still live in a world in which trade in goods and services is quantitatively more important than migration.", "These two observations jointly create a puzzle.", "Why does migration attract so much attention in the political debate and in the media, despite being such a small phenomenon in the European Union?", "Perhaps politicians and journalists simply blow up the issue of migration out of proportion to its true welfare impact.", "Supporting this view, in our regressions we do not find that migration flows predict support for the European Union across countries.", "However, this negative result might just reflect a limitation of our empirical specifications.", "Therefore, we cannot totally discard the alternative that small migration flows may have large welfare effects.", "One possibility is that these large welfare effects are of a non-economic nature.", "Assessing this hypothesis would require a very different modeling framework than the one we offered here.", "Another possibility, closer to our analysis, is that migration flows have more controversial economic consequences than trade flows of a similar magnitude.", "Although we have written a classic model of international trade without factor mobility, our setup suggests one reason why this could be the case.", "Trade in factors is as effective as trade in goods at reaping the gains from inter-industry trade.", "But those are precisely the gains that we have shown to be associated with acute distributive tensions.", "Trade in goods also delivers gains from intra-industry trade that are universally shared.", "Trade in factors cannot do that.", "When workers move from one country to another, they can reduce the differences in factor endowments between the two countries.", "However, the varieties these workers produce cannot be consumed by both countries simultaneously." ]
[ "Economic unions promote trade among their members by removing non-tariff barriers.", "Economic unions lower the cost of living but may cause distributive conflict.", "Differences in members’ size and factor endowments make economic unions controversial.", "Workers in large countries and import industries gain the least from economic unions.", "Survey data on support for the EU are consistent with the predictions of our model." ]
Mechanical behaviour and damping properties of Ni modified Cu–Zn–Al shape memory alloys
The microstructure, mechanical behaviour and damping properties of Cu–18Zn–7Al–xNi alloys (where x = 0.1, 0.2, 0.3 and 0.4) were investigated. The Cu–Zn–Al alloys were produced by casting and then subjected to a homogenization – cold rolling – annealing treatment scheme. Optical-, scanning electron-microscopy and X-ray diffraction analysis were utilized for structural characterization of the alloys, while tensile test, fracture toughness, and hardness measurement were used to assess the mechanical properties. The results show that all the alloy compositions consisted of the predominating CuZn phase. Sharp edged elongated grain structures were observed in the unmodified and the 0.4% Ni modified CuZnAl alloys, while the 0.1, 0.2 and 0.3 %Ni modified CuZnAl alloy compositions, had more of granular/curved/round grain edges and smaller grain widths. The hardness of the unmodified CuZnAl alloy (294.5 ± 2.08 VHN) was lower than that of the Ni modified CuZnAl ones with an increase in hardness ranging between 23.5 and 38.4%. The tensile strength, the percentage elongation (10.7–14.3%) and the fracture toughness of the 0.1, 0.2 and 0.3% Ni modified CuZnAl alloys were observed to be higher than those of the unmodified and the 0.4 %Ni modified CuZnAl alloys. The 0.2% Ni modified CuZnAl alloy had the highest damping capacity among all compositions under investigation, while the 0.4% Ni modified one showed the least capacity to serve as a damping material.
[ "MATE" ]
[ "CuZnAl alloys", "Damping properties", "Mechanical behaviour", "Micro-alloying", "Shape memory capacity", "Structural characterization" ]
[ "2501", "2502", "2503", "2504" ]
[ "There is a growing attention to the problems created by mechanical, structural and noise vibrations in the environment on the account of industrial processes, seismic events, excavation, construction, mining, and exploration activities [1–3].", "These vibration sources can generate diverse effects ranging from mild discomfort and general machinery inefficiencies to collapse of structures, loss of investments, lives and properties [3,4].", "In order to mitigate the potential hazards which could arise from such vibrations, there is growing interest in the development of damping materials for vibration control in engineering structures and systems [4–6].", "Damping materials possess the inherent capacity of attenuating vibrations by dissipating the energy absorbed during the vibration to a safe mode such as heat, usually by hysteretic actions [7].", "There is a wide range of engineering materials with damping ability among which shape memory alloys (SMAs) have been found extremely useful [8,9].", "Shape memory alloys are known principally for their shape memory effect and pseudoelastic properties, but have also been observed to possess high damping capacity – which makes them attractive for the design of vibration control devices [10,11].", "The high damping capacity observed in SMAs has been attributed to the high internal friction occurring during martensitic transformation, which manifests in the loss of energy by the movement between the martensite variant interfaces and the parent martensite habit planes [12,13].", "These remarkable damping properties have been observed principally in NiTi and Cu based SMAs.", "CuZnAl based SMAs are however the focus of this research because of its relatively cheaper processing cost and its relatively superior strain recovery compared to other Cu based SMAs [14].", "The damping properties of CuZnAl based SMAs have been the subject of several investigations [15,16].", "Nai-chao [17] showed that the damping performance of CuZnAl SMAs is dependent on whether it is in martensitic or austenitic state.", "The area enclosed by the hysteretic loop – which is a measure of the damping capacity – was observed to be larger in the martensitic state than in the austenitic state.", "The former was thus better suited for vibration control application.", "Wu et al. [18], studied the damping characteristics of the inherent and intrinsic internal friction of CuZnAl SMAs containing varied Zn content.", "It was reported that the damping capacity was sensitive to the Zn composition of the alloys.", "Compositions containing 7.5–8.5 wt.", "% Zn were found to have higher damping capacities compared to compositions containing 7 wt.", "% Zn (Cu7Zn11Al), because the γ′3 martensite phase they are characterized which possesses a 2H type structure with abundant movable twin boundaries.", "Cimpoesu et al. [19] studied the effect of stress on damping capacity of CuZnAl SMAs.", "It was observed that the internal friction peak in these SMAs, is influenced by the temperature and deformation.", "Also lean amounts of elements like iron, lead, and nickel present in the CuZnAl alloy, helped improve the damping capacity of the alloy.", "The role of these elements (iron, lead and nickel) when used independently as additions to the CuZnAl alloy was however not covered by the study.", "It is recalled that the use of elements such as Fe, B, Ni, Ti, among others as micro-alloying additions in Cu based SMAs, have been reported to modify the grain structure; and consequently, improve the toughness and the overall mechanical performance of the SMAs [14].", "Ni in particular, has been reported to help raise the transformation temperature of Cu based alloys, but its impact on mechanical and damping behaviour when used as micro-alloying addition in CuZnAl alloys has not received comprehensive reportage in literature.", "This happens to be the focus of the present study, which aims at assessing the potentials of Ni modified CuZnAl SMAs for structural and damping applications where there is a growing quest for functional and cheap alternatives to NiTi based SMAs.", "Conventional liquid metallurgy route was utilized for the production of the Ni modified CuZnAl alloys following procedures in accordance with Alaneme [20].", "Five compositions of the alloys were produced containing: Cu18Zn7Al as base alloy composition, 0.1, 0.2, 0.3 and 0.4 wt.", "% Ni additions to four different heats of the Cu18Zn7Al alloy – for the production of the four Ni modified alloy compositions.", "A crucible furnace was used for the melting operation, and the melts cast into cast iron metallic moulds.", "The chemical compositions of the five CuZnAl based alloys produced were determined by SEM-EDS analysis, and are presented with their respective sample designations in Table 1.", "A homogenization treatment performed at 800 °C for 4 h followed with air cooling, was undertaken to improve the chemical and microstructural homogeneity in the alloys produced.", "They were subsequently cold deformed to 10% reduction of the original thickness, using a miniature cold rolling machine; and thereafter, annealed at 450 °C for 1 h followed by air cooling to eliminate cold deformation induced internal stresses developed in the samples.", "The samples for mechanical and damping tests, microstructural and phase analysis, were then machined to standard specifications before a final annealing at 400 °C for 2 h followed by water quenching at room temperature, was performed to eliminate machining stresses on the samples.", "The structures of the produced alloys were characterized using optical microscopy (OM), scanning electron microscopy (SEM), and X-ray diffractometry (XRD) in accordance with Alaneme et al. [21].", "The microstructures of the CuZnAl alloys produced were studied using a Zeiss optical microscope.", "The samples were prepared to metallographic finish using a series of grinding and polishing processes.", "The mirror finish surface produced on each sample was etched by swabbing for 10–20 s using a solution containing 5 g ferric chloride, 10 ml HCl, and 95 ml ethanol, after which microstructural analysis was performed on the samples.", "The optical microscopy analysis was complemented with detailed microstructural and compositional studies using a TESCAN VEGA3 thermionic emission scanning electron microscope system with accessories for energy dispersive spectroscopy (EDS).", "The SEM analysis entailed the use of back scattered electron (BSE) and secondary electron (SE) modes imaging for assessing the phase and the grain distribution, while SEM-EDS analysis was used for the determination of the chemical compositions of the CuZnAl alloys.", "XRD analysis was finally utilised for the phase characterization of the CuZnAl alloys produced.", "The samples for the analysis were prepared following standard procedures.", "The crystalline phases present and their peak intensities were determined using a PANanalytical Empyrean diffractometer with PIXCEL detector and Fe filtered Co-Kα radiation source was used for the analysis.", "The analysis was performed from diffraction 2θ angle spectral range of 0° to 120° while the phases were identified using the X'Pert Highscore plus software.", "The crystal structures of the phases identified were determined by analyzing the pattern of the diffracting crystal planes (hkl) for the entire range of the 2θ diffraction angles [22].", "A Vickers hardness scale was used for the evaluation of the hardness of the alloys, using a hardness testing machine.", "The samples were prepared with fine finished plane parallel surface, while the testing procedure was in accordance with the ASTM E92-17 standard [23].", "The hardness test was performed using a 30 kgf load for a dwell time of 10 s.", "The hardness indentation was repeated for a minimum of five times and readings within the margin of 2% were taken for the determination of the average hardness values.", "The tensile testing was performed on the as-produced CuZnAl alloys using a universal testing machine.", "The samples for the test were machined to tensile test specification of 5 mm diameter and 30 mm gauge length.", "The test samples were mounted on the testing platform and pulled in tension to fracture at a strain rate of 10−3/s.", "The samples preparation, testing procedure and data analysis were performed following the recommendations of ASTM E8/E8M-15a [24] standard.", "Three repeated tests were performed for each CuZnAl alloy composition produced to guarantee the reliability and to assure the reproducibility of the test results.", "The ultimate tensile strength and strain to fracture were evaluated from the stress–strain curves developed from the test conducted.", "Three repeated tests were performed for each Cu–Zn–Al alloy composition to ensure that generated results are consistent and hence reliable.", "The optical micrographs of the unmodified and Ni modified CuZnAl alloys are presented in Fig. 1.", "Fig. 1a shows the optical micrograph of the unmodified CuZnAl alloy, which is observed to contain sharp edged directionally solidified grains.", "The elongated grain feature is very common in CuZnAl shape memory alloys within the composition range considered in this research, and specifically matches the structural features reported in [30–32].", "The CuZn Al alloy composition modified with 0.1% Ni (Fig. 1b), shows a significantly modified grain morphology from the sharp edged feature observed for the unmodified CuZnAl alloy to mostly granular/polygonal shaped grain structure.", "The round shaped grains appear smaller in size compared to those in the unmodified CuZnAl alloy.", "For the 0.2% Ni modified CuZnAl alloy (Fig. 1c), the grain structure consists of finer and sparsely distributed elongated grains.", "The 0.3% Ni modified CuZnAl alloy (Fig. 1d), also shows a directionally solidified grain structure, but with some curve/round edged grain features.", "The 0.4% Ni modified CuZnAl alloy (Fig. 1e), equally contains dominantly elongated grains although the longitudinal lengths of the grains are seen slightly shorter than those observed for the unmodified CuZnAl alloy (Fig. 1a).", "Table 2 shows the average grain width (transversal axis thickness) of the unmodified and Ni modified CuZnAl alloys.", "It is observed that the average grain width decreases from 7.9 μm for the unmodified CuZnAl alloy to as low as 3.8 μm for the 0.2% Ni modified CuZnAl alloy, which corresponds to approximately 52% decrease in the grain width.", "The increase in the Ni content above 0.2%, is observed to result in a marginal grain refinement and in the grain width coarsening for the 0.3% and 0.4% Ni modified CuZnAl alloys, respectively.", "Grain width increase as high as 22.7% was observed with the 0.4% Ni addition in comparison to the base CuZnAl alloy.", "The analyses from Fig. 1 and Table 2 show that the amount of Ni used as micro-alloying addition in CuZnAl alloy significantly affects its solidification patterns.", "Grain modification was more pronounced and distinct for the 0.1% Ni modified CuZnAl alloy where a dramatic transformation from sharp edged elongated grain structure to a predominantly granular structure was observed.", "The 0.2% Ni addition resulted in the finest size and least predominant presence of the needle-like structures and the 0.3% Ni addition only induces slight changes in the grain edge morphology, while the 0.4% Ni modified CuZnAl alloy composition shows changes in both grain growth and in grain edge morphology.", "Representative secondary electron mode images of the unmodified and selected modified Cu–Zn–Al alloys are presented in Fig. 2.", "They all demonstrate profoundly identical structure features as observed in the corresponding optical micrographs of the investigated alloys (Fig. 1).", "As it is seen in Fig. 2a, the unmodified alloy microstructure consists of predominantly elongated grains with sharp edges.", "In Fig. 2b, the micrograph of the 0.2% Ni modified CuZnAl alloy shows predominantly a finer structure with a few needle-like precipitated features, while in Fig. 2c the 0.4% Ni modified CuZnAl alloy is seen to consist of slightly elongated larger size grains compared to the unmodified one.", "These results confirm the observations from the optical microscopy investigation that the presence of Ni in the CuZnAl alloys significantly alters their solidification patterns with essentially varied grain morphology.", "This sort of influence of Ni as micro-alloy addition, has also been reported for several other micro-alloying elements [14,33,34].", "Fig. 3 with representative EDS spectra confirms the presence of Cu, Zn, Al and Cu, Zn, Al, Ni for the unmodified and the 0.4% Ni modified CuZnAl alloy, respectively.", "The X-ray diffraction analysis of the unmodified and Ni modified CuZnAl alloys is presented in Fig. 4.", "As it is observed from the patterns, all samples consist essentially of CuZn phase despite the differences in grain morphology (see Figs. 1 and 2).", "There are slight shifts in the peak positions for each of the alloy compositions (although less than 2°) which is a common feature in alloys with slightly altered alloying composition (due to the difference in lattice parameters).", "The analysis of the pattern of the diffracting crystal planes for the entire 2θ diffraction angles and the lattice parameters confirm the CuZn phase to be the β – phase, which from studies has been shown to be martensitic and displays shape memory capacity [35].", "There are very small peaks of CuAl and NiAl phases observed from the XRD scan for the Ni modified compositions.", "These peaks are, however, not well resolved in the plots because of the small intensities.", "The predominance of the Cu–Zn peaks in the difractograms as analyzed is in agreement with the EDS results presented in Fig. 3.", "The hardness values of the unmodified and Ni modified CuZnAl alloys are presented in Fig. 5.", "It is observed that the hardness values of the CuZnAl alloys with Ni addition, are basically greater than that of the unmodified CuZnAl alloy.", "The maximum increase is obtained in the 0.2% Ni modified CuZnAl alloy, which corresponds to a 38.4% increase in hardness.", "The hardness, however, is found decreased with the further increase of the Ni addition, albeit not congruently with Ni concentration.", "The improved hardness observed in the Ni modified CuZnAl alloys, is attributed to the presence of Ni as micro-alloying addition, which resulted in the reduction of the elongated grain width (i.e. grain thickness) and the modification of grain edge morphology.", "The 0.2% Ni modified CuZnAl alloy has the highest hardness value, as it is seen in Fig. 1 and Table 2, this sample also contains the finest size grain structure with the smallest grain width, respectively.", "This improved resistance to indentation with the decreased grain size is in agreement with the Hall–Petch relation [36].", "The stress–strain plots of the unmodified and Ni modified CuZnAl alloys are presented in Fig. 6, while the ultimate tensile strength and percentage elongation plots are presented in Figs. 7 and 8, respectively.", "As it can be seen in Fig. 7, the ultimate tensile strength (UTS) of the unmodified and the 0.4% Ni modified CuZnAl alloys show values of 450.85 MPa and 452.62 MPa, respectively; which are lower than the UTS values of the other Ni modified CuZnAl alloys.", "Compared to the unmodified CuZnAl alloy, tensile strength increases of 9.8, 12.1, 13.7, and 0.4% were obtained with the use of 0.1, 0.2, 0.3, and 0.4 wt.", "% Ni, respectively.", "The low tensile strength of the unmodified and 0.4 wt.", "% Ni modified CuZnAl alloys can be linked to the relatively larger width of the elongated grains in these samples.", "Also the sharp edges of the grain structure for both samples is another important factor.", "Sharp tip grain edges usually serve as stress concentration sites which would suggest that the nominal stress acting on the material is considerably amplified to values exceeding its maximum stress bearing capacity at the grain tips [37].", "Accordingly, the improved tensile strength of the 0.1, 0.2, and 0.3 wt.", "% Ni modified CuZnAl alloys, is linked to the finer elongated grain width coupled with the change in the grain morphology to the granular structure (for the 0.1% Ni modified alloy), and the round/elliptical grain edges (for the 0.3% Ni modified alloy).", "The finer grain structure, round/elliptical grain edges and granular structures observed in these Ni modified CuZnAl alloys, reduce the tendency of the grain edges to serve as stress concentration sites.", "Consequently, the nominal applied stress on the alloys must be high to attain the maximum stress bearing capacity values for the process of micro-crack formation and fracture to be heralded in the alloys.", "The significance of the results is that the addition of between 0.1 and 0.3 wt.", "% Ni to CuZnAl alloy can enhance the stress transmission/bearing capacity of the alloy.", "The percentage elongation of the unmodified and Ni modified CuZnAl alloys are presented in Fig. 8.", "It is observed that the elongation values of the unmodified and 0.4 wt.", "% Ni modified CuZnAl alloys are almost at the same level (8.5–8.6%).", "But there is an improvement in the elongation values for the other Ni modified CuZnAl alloys ranging between 10.7–14.3%.", "The CuZnAl alloy modified with 0.3 wt.", "% Ni, shows the highest elongation value of 14.3%.", "This implies that the addition of 0.1–0.3 wt.", "% Ni can result in improved ductility in CuZnAl alloys and hence enhanced plastic workability of these.", "The reasons for the lower percentage elongation of the unmodified and 0.4% Ni modified CuZnAl alloys, is tied to the sharp tip grain edges, which can restrain plastic deformation due to the triaxial stress state created at such sites.", "The plasticity restraint makes the alloys more resistant to yielding and hence exhibit less ductility [36].", "In the case of the 0.1, 0.2 and 0.3 wt.", "% Ni modified CuZnAl alloys, the development of a granular structure, fewer sharp edged grains, and curved/elliptical grain edges, respectively; are responsible for the high plastic strain sustaining capacity of these alloys.", "The fracture toughness of the unmodified and the Ni modified CuZnAl alloys are presented in Fig. 9.", "It is clearly seen that all the Ni modified CuZnAl alloys except the 0.4% Ni modified composition, show fracture toughness values that are higher than that of the unmodified one.", "The fracture toughness increase of 13.4, 28, and 12% are obtained for the 0.1, 0.2 and 0.3% Ni modified CuZnAl alloys, respectively, while a 4% decrease in fracture toughness is observed for the 0.4% Ni modified one.", "These observations imply that the 0.1–0.3% Ni microalloying addition in the CuZnAl alloy improves its resistance to crack propagation, while the 0.4% Ni microalloying addition, is detrimental to the toughness of the CuZnAl alloys.", "The same reasons attributed to the improved ductility are valid for the improvement in the fracture toughness – that is, the change in the grain edge shape from sharp edged to round/elliptical shape for the 0.1 and 0.3 Ni modified CuZnAl alloys and to the fewer elongated grain structure in the 0.2% Ni modified one.", "A preponderance of sharp edge grains is known to facilitate the triaxial stress state at the grain tip which suppresses yielding and accentuates brittle fracture susceptibility [21].", "Fig 10 shows the damping capacity, storage modulus, and loss modulus of the unmodified and Ni modified CuZnAl alloys.", "As it is seen in Fig. 10a, at 1 Hz test frequency only the 0.2% Ni modified CuZnAl alloy shows higher damping capacity values than the unmodified one for the test temperature range of 25 °C–250 °C.", "The 0.4% Ni modified CuZnAl alloy exhibits the least damping capacity of all the alloys under investigation.", "The Peak internal friction of 0.43 was obtained at 75 °C for the 0.2% Ni modified CuZnAl alloy, while the unmodified, the 0.1 and 0.3% Ni modified CuZnAl alloys show less obvious damping peaks.", "A peak internal friction of 0.026 was observed for the 0.4% Ni modified CuZnAl alloy at 225 °C; but this alloy maintains a constant low value of 0.001 from room temperature to about 200 °C.", "The same trend is observed for 2 Hz frequency (Fig. 10b) where also the 0.2% Ni modified CuZnAl alloy shows the highest damping capacity for the test temperature range of 25 °C–250 °C.", "However, a peak internal friction of 0.034, which is lower than 0.043 at 1 Hz is obtained at 50 °C for this 0.2% Ni modified CuZnAl composition.", "The unmodified alloy exhibits the next highest damping capacity among other alloy compositions, while the 0.4% Ni sample again exhibits the least damping capacity.", "Damping capacity is associated with the movement and reorientation of martensite variants and interfaces [14].", "The results described above, thus, imply that such movement and orientation of the martensite variants are better facilitated in the 0.2% Ni modified CuZnAl alloy than in others.", "The low damping capacity of the 0.4% Ni modified CuZnAl alloy will accordingly be linked to the restriction of the movement of the parent phase/martensite interfaces and the martensite variants [18].", "This may be on the account of the population of the Ni solute atoms which can wield a pinning effect on the boundaries and interfaces.", "This is why smaller internal friction peaks are observed for this composition.", "It should be noted that at 2 Hz, the 0.1 and 0.3% Ni modified alloys show peak internal frictions of about 0.015 and 0.013, respectively, at 225 °C.", "The E’ “storage modulus” (see Fig. 10c and d) as a measure of the capacity of a material to absorb and to store energy induced by vibrations [29] is observed to be stable for the temperature range of 25 °C–250 °C for all alloy compositions, with the exception of the 0.4% Ni modified one, which is observed to exhibit a drastic drop in storage modulus (energy absorption capacity) at about 200 °C.", "The 0.1% Ni modified CuZnAl alloy shows the highest storage modulus of 130,300 MPa, while the 0.2% modified one has the least storage modulus of averagely 4500 MPa.", "It is noted that the storage modulus was not affected by the frequency (either 1 or 2 Hz).", "The E” “loss modulus” (see Fig. 10e and f) meaning the energy dissipation capacity in form of heat of the material [29] shows an intermittent variability in the temperature range of 25 °C–200 °C, but beyond this temperature, there is continuous increase in the loss modulus with the increase in temperature.", "The 0.1 and 0.2% Ni modified compositions exhibit higher loss modulus values compared with the others above 200 °C.", "It is noted that at about 200 °C, there is a dramatic and significant drop in the loss modulus of the 0.4% Ni modified CuZnAl alloy.", "This suggests that this composition may not be suitable for damping applications.", "The microstructure, mechanical behaviour and damping properties of unmodified and 0.1–0.4 Ni modified Cu–18Zn–7Al alloys were investigated.", "Sharp edged elongated grain structures synonymous with the directional solidifications were observed in the unmodified and the 0.4% Ni modified CuZnAl alloys.", "The grain structure was, however, significantly altered in the 0.1, 0.2 and 0.3 %Ni modified CuZnAl alloys, where granular structure, small grain width with fewer sharp edge grains, and curved/round grain edges, respectively, were observed.", "The mechanical properties of the unmodified and the 0.4% Ni modified CuZnAl alloys were generally lower than those of the 0.1 and 0.3% Ni modified ones.", "The 0.4% modified CuZnAl alloy showing the lowest damping capacity, does not seem, thus, suitable to serve as a damping material, while the 0.2% Ni modified one exhibits the highest damping capacity among all the CuZnAl alloy compositions." ]
[]
Measurement of wind field data in Southeast China
The data presented in this article are the wind measurements acquired from a tower in Southeast China during typhoon Nesat (1709#) and typhoon Haitang (1710#). Three 3D ultrasonic anemometers Wind Master Pro were utilized to obtain 3D wind data. The anemometer works well with wind speed range of 0–65 m/s and wind angle range of 0–360°. Three direction wind speeds and wind angles were recorded per every 0.1 s. The present research analyzed wind characteristics based on recorded data. In this article, the detailed test set-up and data pre-processing methodology for the wind characteristics analysis are provided.
[ "MULT" ]
[]
[ "1000" ]
[ "The data presented in this article is acquired from the wind field measurement station (as shown in Fig. 1) at Pingtan in Southeast coastal of China when Typhoon Nesat (1709#) and Typhoon Haitang (1710#) attacked Pingtan during 30th and 31st of July in 2017.", "The recorded wind speed data during typhoon process is obtained.", "To ensure the data quality, the wind speed data were filtered by data control method.", "The filtered wind speed data during typhoon is available in the supplementary material.", "The filtered data was analyzed to obtain wind characteristics presented in paper “Analysis on the Wind Characteristics under Typhoon Climate at the Southeast coast of China.”", "The experiment station was set up in Pingtan, Fujian Province, China.", "The location of sensors installation can be seen in the Fig. 1.", "There 3D ultrasonic anemometers Wind Master Pro produced by Gill Company in UK, were installed for the record of wind speed data.", "The arrangement of sensors can be seen in the Table 1.", "The corresponding main parameters are: wind speed range: 0–65 m/s; resolution ratio: 0.01 m/s; wind direction range: 0–359°; resolution: 0.1° and frequency 10 Hz.", "The anemometers were installed on the tower by the designed steel holder as shown in Fig. 2.", "The data was collected and monitored by data acquisition system CR3000 as can be seen in the Fig. 3.", "Based on the above observation station, the wind speed data were obtained during typhoon Nesat and typhoon Haitang.", "However, the recorded wind speed data may include some bad or invalid data.", "The record data was filtered to ensure the validation by data controlled method (Fig. 4).", "The reliability of data was firstly diagnosed by comparing experienced wind speed observed by nearby meteorological station.", "A multiple truncation variance method [1–3] was used to determine the rationality of the original data.", "The smooth estimation of the original data was performed for each time series (30 s).", "By detecting the sudden change of the data, it was determined whether the value exceeded the range of the smooth estimation to ensure the validity of the data point.", "Data processing can be specified as follows:", "Moreover, to avoid the noise during collection process, the data was low-pass filtered at 3 Hz.", "Filtered data are available in the supplementary material which can be used for the wind characteristics analysis." ]
[]
Nitrogen oxidation consortia dynamics influence the performance of full-scale rotating biological contactors
Ammonia oxidising microorganisms (AOM) play an important role in ammonia removal in wastewater treatment works (WWTW) including rotating biological contactors (RBCs). Environmental factors within RBCs are known to impact the performance of key AOM, such that only some operational RBCs have shown ability for elevated ammonia removal. In this work, long-term treatment performance of seven full-scale RBC systems along with the structure and abundance of the ammonia oxidising bacteria (AOB) and archaea (AOA) communities within microbial biofilms were examined. Long term data showed the dominance of AOB in most RBCs, although two RBCs had demonstrable shift toward an AOA dominated AOM community. Next Generation Sequencing of the 16S rRNA gene revealed diverse evolutionary ancestry of AOB from RBC biofilms while nitrite-oxidising bacteria (NOBs) were similar to reference databases. AOA were more abundant in the biofilms subject to lower organic loading and higher oxygen concentration found at the distal end of RBC systems. Modelling revealed a distinct nitrogen cycling community present within high performing RBCs, linked to efficient control of RBC process variables (retention time, organic loading and oxygen concentration). We present a novel template for enhancing the resilience of RBC systems through microbial community analysis which can guide future strategies for more effective ammonia removal. To best of the author's knowledge, this is the first comparative study reporting the use of next generation sequencing data on microbial biofilms from RBCs to inform effluent quality of small WWTW.
[ "ENVI" ]
[ "Ammonia oxidising archaea", "Ammonia oxidising bacteria", "Biofilm", "Nitrification", "Rotating biological contactor", "Wastewater treatment" ]
[ "2300" ]
[ "The removal of ammonia remains essential for purification of wastewater prior to discharge which is difficult to achieve at small wastewater treatment works (WWTWs) (e.g. <2000–10,000 population equivalents) due to conflicting requirements for low energy and maintenance, land, capital expenditure and need for decentralised rural networks compared to conventional activated sludge processes (ASP) (Dutta et al., 2007; Hassard et al., 2015).", "Rotating biological contactors (RBC) are one technology which is commonly used within this small works context and are maintained based on decades of observed relationships between performance and operating parameters.", "However, differences underlying RBCs which perform well and RBCs which do not perform well are poorly understood, particularly for ammonia removal (Cortez et al., 2008; Hassard et al., 2018).", "A deeper analysis of the dynamics of RBC microbial communities and performance with respect to ammonia removal has the potential to highlight unprecedented insight into the performance of these biofilm reactors.", "Ammonia-oxidizing bacteria (AOB) and ammonia oxidising archaea (AOA), which together are known as ammonia oxidising microorganisms (AOM) along with nitrite-oxidizing bacteria (NOB) are involved in nitrification processes (Bollmann et al., 2014; Daims et al., 2015).", "These chemolithoautotrophs obtain energy for growth by oxidizing either ammonia to nitrite (AOM) or nitrite to nitrate (NOM) using oxygen as a terminal electron acceptor.", "The AOM and NOB do not normally oxidize both substrates simultaneously.", "Complete nitrification is also possible by some members of the genus Nitrospira, however these complete ammonia oxidizers (Comammox) do not tend to dominate the nitrogen cycle within secondary treatments within WWTWs (Daims et al., 2015; Fitzgerald et al., 2015; Van Kessel et al., 2015).", "Recently comammox bacteria have shown to be abundant in tertiary RBC biofilms (i.e. low ammonia concentration), however their role in governing performance remains unknown particularly in highly loaded RBCs (Spasov et al., 2019).", "In other nitrifying organisms the separation of the two nitrification steps in different groups of organisms leads to process coupling and co-aggregation of AOM with NOB into reciprocal feeding nitrifying consortia harnessed for secondary wastewater treatment (Wijeyekoon et al., 2004).", "Both the AOB and AOA possess the amoA gene which codes for the alpha-subunit of ammonia monooxygenase protein (AMO), which catalyses ammonia oxidation (Könneke et al. 2005) and the dominant pathway in conventional WWTW.", "This gene therefore was used to ascertain whether abundance of such organisms does or does not correlate with nitrification activity and therefore performance (Hassard et al., 2015).", "Since both AOB and AOA are responsible for ammonium removal in WWTWs, it was important to understand which environmental factors / process conditions influence the AOB and AOA abundance and performance of the RBCs.", "Here, we focus on environmental factors which play important roles in maintaining stable nitrification as (i) AOM are sensitive to changes in their environment such as fluctuations in pH, salinity, ammonia concentration, light, oxygen and the presence of toxic or inhibitory compounds present within wastewater.", "(ii) AOA can outcompete AOB especially in low ammonia loading environments (e.g. distal through process) due to higher specific yield despite slower growth rate (You et al., 2009).", "Finally, (iii) AOB dominate over AOA when ammonia loading rates are high and the substrate-influx limits the growth within a spatial clustering of biomass which is common within microbial granules, aggregates or biofilms due to their higher specific growth rates (Wijeyekoon et al., 2004).", "In this study we consider acclimation to substrate and oxygen limited concentrations would (1) lead to reduced biofilm ammonia removal rates and (2) result in a permanent change to the reactor community reflected in the distribution of AOA and AOB within biofilm reactors.", "These questions are studied through analysis of AOB and AOA ecology in seven full-scale RBC systems using high throughput sequencing of 16S rRNA gene and quantitative polymerase chain reaction (qPCR) of the AOB and AOA amoA genes and phylogenetic analysis.", "The insights into the microbial community dynamics in RBC systems will help to guide optimisation strategies for more effective ammonia removal.", "To our knowledge, this is the first study to compare the microbial ammonia oxidising communities across several different RBCs operating at small WWTW.", "Biofilm was sampled from seven small WWTWs in the Midlands area of the United Kingdom (Table 1).", "At each WWTW, a 36 cm2 circular area of biofilm was sampled from the surface of the individual RBC disks using a sterile disposable plastic spatula and microcentrifuge tube.", "The media could not be removed; therefore this represented a surface sample of biofilm.", "Subsequently the biofilm samples were transported on ice to the laboratory.", "Biofilm was sampled approximately twice monthly over a period of one year in two phases between November 2014 and February 2015, and August 2015 and November 2015 (Table S1).", "Samples were collected every two weeks whilst the RBC was temporarily stationary.", "During this period the RBC was operated under increasing load of municipal settled wastewater from 12 m3 day−1 to 38 m3 day−1.", "Access hatches enabled samples to be taken from the front and rear of the two biozones from the pilot scale RBC located WWTW 1 (Fig. 1A).", "Biofilm samples were taken from WWTW 2 – 7 over a period of 1 month between November 2015 and December 2015, access hatches permitted samples to be taken from the front and / or the rear of the RBC biozone (Fig. 1B).", "Sampling of front and rear of RBC was undertaken to understand if any differences between the biofilm was evident during the dynamic reactor conditions.", "Sampling of biofilm from WWTW 1 – 7 was undertaken in an identical way and details are provided within Table S1.", "The weight of the wet sample was recorded, the samples were homogenised prior to being snap frozen in liquid nitrogen and stored at −80 °C.", "Samples storage occurred within 4 h of sampling from the RBC.", "Rotor speed was fixed at 2.4 rpm at each RBC.", "This led to differences in the media tip speed due to different RBC media diameter between different WWTW.", "Fixed rotor rpm (and variable tip speed) is typical for most water utilities due to complexity of operating variable rpm RBCs and mechanical issues which can arise.", "The media submergence was set at 38% for all the RBCs studied.", "No wastewater or solids recycle was undertaken at any of the WWTW studied.", "Therefore, denitrification is not expected to any significant degree.", "Ammonia oxidation assays were performed according to standard methods (details provided within Supplementary Material).", "Total nucleic acids were extracted from 0.3/0.0025 g (wet weight/dry weight [VSS]) biofilm samples using the Powersoil DNA isolation kit (Mo Bio Laboratories, USA) according to the manufacturer’s instructions and the DNA were re-suspended in 50 μL of molecular grade water (Ultrapure, Sigma, UK).", "These isolates were stored at −80 °C until further analysis.", "Procedural blanks were included throughout.", "qPCR for amoA genes of AOB and AOA were performed on a CFX96 Real-Time Detection System (Bio-Rad, USA).", "The amoA gene of AOB was amplified using the primers amoA-1F and amoA-2R (Rotthauwe et al. 1997).", "The amoA gene fragment of AOA was amplified using the primers CrenamoA23F and CrenamoA616R (Tourna et al. 2008).", "Full details of primers and sequences are provided in Table S2.", "For both AOB and AOA amoA genes, each 15 μL reaction mixture contained 1 μL of DNA template, 7.5 μL of 2 × SensiFAST SYBR No-Rox Kit (Bioline, UK), and 200 nM concentration of each primer.", "A dissociation curve analysis was performed at the end of each reaction to verify amplification of a single PCR product.", "The qPCR conditions for both AOA and AOB reactions were set as follows: initial denaturation for 3 min at 95 °C, followed by 40 cycles consisting of 95 °C for 5 s, then 60 °C for 30 s. Negative controls were performed with UltraPure Distilled water as the template.", "The samples were quantified against the corresponding standard curve using CFX Manager version 2.0 software (Bio-Rad).", "All purified DNA from each sample were subjected to duplicate qPCR assays.", "DNA standards of known quantity were created using a dilution series 10−1–10−7 of previously extracted amoA clones obtained from the PCR amplification of sediment DNA extracts (obtained from University of Essex).", "The abundance of gene copies for standards was calculated by assuming a molecular mass of 660 Da for double-stranded DNA using the following formula: gene abundance = 6.023 × 1023 (copies mol−1) × standard concentration (g ml−1)/molecular mass (g mol−1).", "The DNA standards were diluted over a range from 102 to 107 target genes ml−1 for both AOB amoA and AOA amoA.", "Standards, samples, and no-template controls (NTC) were amplified in duplicate with each primer set.", "The standard curve correlation coefficient (R2) and amplification efficiencies (%) for the AOB were 0.981 and 95% respectively and for AOA were 0.997, 96% respectively.", "If CQ values between duplicate sample reactions exceeded 1 then data was discarded, and nucleic acids extraction and qPCR analysis was repeated on similar biofilm samples taken during the same sampling event.", "The qPCR data was normalised as gene copies per gram of wet biofilm (GC.g−1 biofilm).", "For bacterial 16S rRNA gene libraries, primers Bakt_341F and Bakt_805R were applied (Herlemann et al., 2011).", "For archaeal 16S rRNA gene libraries, primers 344F (Raskin et al., 1994) and 915R (Stahl and Amann, 1991) were applied.", "All primers were flanked with the Illumina Nextera overhang sequences on the 5́ end Forward overhang: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG; Reverse overhang: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG.", "Samples were PCR amplified with a Veriti 96-Well Thermal Recycler (Applied Biosystems, UK) using the following conditions: initial denaturation for 3 min at 95 °C, followed by 28 cycles consisting of 95 °C for 30 s, then 55 °C for 30 s, and 72 °C for 30 s and finally 72 °C for 5 mins.", "Each reaction mixture contained 1 μL of DNA template, 12.5 μL of 2 × RedTaq Ready Mix (Sigma, UK), and a 5 μL of each primer (1 μM).", "Following the PCR amplification, the amplicons were cleaned using AMPure XP beads in conjunction with a SPRI super magnetic plate (Beckman Coulter, UK).", "Nextera XT identification indexes (Illumina, UK) and flow cell adapters were then applied to the 16S rRNA amplicons by PCR, using the same conditions, but limited to 8-cycles.", "Each reaction contained 5 μL of 16S rRNA amplicon product, 25 μL of 2 × RedTaq Ready Mix (Sigma, UK), and a 5 μL of each index primer.", "The PCR products were cleaned using the AMPure XP beads in conjunction with a SPRI super magnetic plate (Beckman Coulter, UK).", "A 1:10 dilution of PCR samples was the used to quantify the products using the PicoGreen (ThermoFisher, UK) assay and fluorescence was measured using FLUOstar Omega microplate reader (BMG, UK) before pooling the amplicon in equimolar concentrations.", "The final pooled 16S rRNA amplicon library was sequenced by The Earlham Institute, Norwich, UK) using a MiSeq Reagent Kit v3 (600-cycle) on an Illumina MiSeq instrument.", "Sequence reads were quality trimmed using Sickle (Joshi and Fass, 2011), error corrected within SPAdes (Nurk et al. 2013) using the Bayes Hammer algorithm (Nikolenko et al. 2013).", "Pair-end assembly was performed with PEAR (Zhang et al., 2014) within PANDASeq (Masella et al. 2012) with a minimum 50 base pair (bp) overlap for bacterial sequences.", "Archaeal amplicons were analysed on the forward read only as the amplicons were too long to overlap.", "The quality filtered (all sequences below 200 bp were removed) and error corrected sequences were then depreplicated, sorted by their abundance and operational taxonomic unit (OTU) centroids were picked using VSEARCH (Rognes et al., 2016) at the 97% level.", "Singleton and doubleton OTUs were removed, along with all chimeric sequences using reference-based chimera checking with UCHIME (Edgar et al., 2011) against the 16S rRNA RDP (Ribosomal Database Project).", "The number of sequences obtained for each group is presented within Table S3.", "Rarefaction analysis was performed to ensure sufficient observations were made to estimate sequence quantity that was measured by sampling and sequencing.", "Bacterial rarefaction curves were compared at a sampling effort of 7000 sequences to maximise the number of samples which could be used within the study (Fig. S1).", "Asymptotic accumulation curves were found indicating that the amplicon libraries represented dominant individuals within the RBC biofilm communities.", "Taxonomy assignment was performed with the RDP Classifier (Wang et al., 2007).", "16S rRNA sequences of bacterial AOM obtained from this study were deposited to the GenBank Database under the accession numbers: MN326757- MN326769.", "Archaeon sequencing did not have even coverage across samples evidenced by rarefaction analysis (data not shown) therefore this data was not analysed further.", "16S rRNA gene sequences of AOM (AOB, NOB from this study and complete nitrification organisms from database inclusive) were aligned using MUSCLE (Edgar, 2004).", "The evolutionary history was inferred using the Neighbour-Joining method (Saitou and Nei, 1987).", "The optimal tree with the sum of branch length = 1.2798 was constructed.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (values over 70 shown) (Felsenstein, 1985).", "The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The evolutionary distances were computed using the Jukes Cantor method (Jukes and Cantor, 1969) and are in the units of the number of base substitutions per site.", "The analysis involved 62 nucleotide sequences.", "There was a total of 435 positions in the final dataset.", "Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013).", "Performance and operating condition data were measured on a weekly basis, the flow rate to each of the RBCs studied was dependent on WWTW loading rates and was not controlled as part of this study.", "The substrate loading rate (feeding rate) was normalized to surface area of discs (total including wetted and non-wetted surface) and volume of wastewater per time, also referred to as disc loading rate.", "The following wastewater constituents were considered soluble and total chemical oxygen demand (sCOD and tCOD) concentrations (a measurement of how much soluble or total organic material is in wastewater), 5 days biochemical oxygen demand concentrations (a measure of degradation potential and oxygen consumption over 5 days – BOD5), total suspended solids (TSS) and ammonia (NH4-N) concentrations of water.", "The TSS was measured gravimetrically and the NH4-N was measured using cell tests.", "The water pH and temperature were also recorded.", "Together, this dataset yielding a wide range of substrate / RBC removal performance values and relative percentage of the nitrogen oxidation pathway compared with the total removal of carbonaceous compounds.", "The hydraulic retention time (HRT) was calculated based on the wetted reactor volume/average dry weather flow rate (unit day).", "All chemical wastewater analysis was undertaken according to standard methods in APHA-AWWA-WEF (2012).", "Wastewater characteristics are presented within Table S4.", "Multivariate distant based linear model (DistLM) were constructed to assess the effect of different reactor physicochemical features on the AOM abundance and overall microbial community composition.", "First, AOM data were log10 + 1 transformed and environmental variables were normalised.", "Subsequently, a resemblance matrix based on Bray-Curtis similarity index was compiled using a step-wise forward procedure with 9999 permutations and Euclidian distance for physicochemical feature (PERMANOVA + for PRIMER).", "BEST analysis was used to distinguish most suitable characteristics for predictive analysis, by maximising a rank correlation between their respective resemblance matrices.", "Assumptions required for DistLM were met.", "Correlations between predictor variables was assessed and variables with autocorrelations R > 0.8 were excluded from the analysis, by taking the most suitable variable forward for the model construction.", "A canonical analysis of principal components was undertaken by constraining the community data and using it to predict positions along an ‘X axis’ using the multivariate data cloud.", "A Welch’s t-test was undertaken using SPSS v25 (IBM, USA) to test for significance of different in AOM abundance from qPCR data between different RBC biofilm.", "Ammonia concentrations in the WWTW 1 and 2 were similar at 28.8 mg L−1 (WWTW 1) and 33.2 mg L−1 (WWTW 2) on average.", "WWTW 1 had higher organic load, represented by a COD of 546 mg L−1 compared to 463 mg L−1 for WWTW 2.", "Generally the wastewater treated by WWTW 2 was more biodegradable evidenced by a COD:BOD5 of 3.1 compared to 4.8 at WWTW 2.", "WWTW 2 had RBCs which had 2.2 fold greater ammonia loading to the front of the RBC biozone than WWTW 1 (data not shown).", "The wastewater quality of WWTW 4–7 were not sampled frequently as part of this research programme and thus inferences were not made about the performance of these biofilm reactors.", "WWTW 1–3 were sampled for influent and effluent quality for a period of 7.5 years.", "WWTW 1 and 2 had water sampled weekly for influent, within reactor (biozone sample) and effluent for a period of 6 months.", "The biozone sample represented a composite of three samples taken interspaced within the biozone.", "Temperature strongly influenced the ammonia removal performance of RBCs.", "A decline in performance from a peak of 85.1% ammonia removal (WWTW 1) occurred at a temperature of 17.5 °C compared to a low of 68% ammonia removal (WWTW 2) which occurred at a temperature of 8 °C (Fig. 2A).", "Effluent ammonia concentrations were relatively stable over the 7.5 years sampling with characteristic elevated ammonia levels in effluent which typically occurred in winter months (notably WWTW 1).", "Average ammonia levels were 4.4, 0.36 and 1.61 mg L−1 for WWTW 1, 2 and 3 respectively (Fig. 2B).", "Inter-stage sampling of WWTW 1 revealed intermediate removals across the RBC with higher concentrations than the final effluent (Fig. 2C).", "Superior performance occurred within WWTW 2 with indistinguishable concentrations within the biozone and the effluent quality which was attributed to the higher loading rates and nitrification activity of these biofilm (data not shown).", "In this work, we use qPCR to assess the abundance of key functional groups of AOB and AOA and Next Generation Sequencing of the 16S rRNA gene to assess the diversity of bacterial AOM present within RBC biofilms across 7 operational WWTW.", "Biofilm samples were collected over a 1-month period from seven full-scale RBCs.", "Performance data was collected weekly (approx.)", "for a period of 1 year.", "A total of 24 biofilm samples were taken from RBCs in triplicate and analysed by qPCR and next generation sequencing of 16 s rRNA amplicons 72 samples were analysed from WWTW 1 and a further 15 samples from WWTW 2–7 (see Materials and Methods section).", "The mean AOA amoA gene copy number was between 3.8 × 104 (GC.", "g−1 biofilm) in WWTW 7 and 1.9 × 108 GC.g−1 in WWTW 6.", "For AOB, the mean amoA gene abundance was between 2.6 × 104 to 2.6 × 108 (GC.", "g−1 biofilm) in the same WWTW.", "WWTW 6 exhibited the highest abundance of both AOA and AOB in contrast to WWTW 7 which had the lowest abundance of both AOA and AOB (Fig. 3A).", "The ammonia effluent concentration was similar between WWTW 6 and 7, despite nine-fold difference in dry weather flow between the two WWTW (Table 2).", "WWTW 7 received a very low flow (<5 m3 day−1) and furthermore this flow was intermittent.", "Therefore, the AOM in WWTW 7 was subjected to frequent drying / wetting periods and ammonia starvation, which could contribute to the observed low total amoA abundance (AOB + AOA).", "WWTW 4 and 6 ranked as first and second in terms of lowest ammonia effluent concentration (circumstantial evidence of high nitrification activity), these two sites exhibited significantly higher AOA gene abundance with an average of 1.3 × 107 and 1.9 × 108 GC.", "g−1 biofilm respectively, compared to an overall average of 2.6 × 105 for WWTW 1, 2, 3, 5 and 7 (Fig. 3A).", "Welch’s unequal variances t-tests revealed that AOB significantly varied across WWTW (tWelch = 4.6, p < 0.001) whereas AOA was similar on average between WTWW (tWelch = 2.8, p > 0.05; Fig. 3A).", "Post hoc test revealed that most of this difference was attributed to differences between WTWW 6 and the other WWTW which were similar (Games-Howell test, p < 0.001).", "Dominance in terms of abundance of each group of AOM in each RBC was tested.", "In most WWTW there was no significant difference between the AOB and AOA abundance.", "A Welch’s t-test showed that WWTW 1 and 2 were dominated by AOA (WTWW 1, tWelch = 8.1, p = 0.05) and (WTWW 2, tWelch = 2.3, p < 0.001).", "In contrast, WWTW 4 was dominated by AOB (WTWW 4, tWelch = 3.5, p < 0.001)", "The WWTW with the highest ammonia effluent concentration (17.4 ± 10.8 mg L−1) had the lowest AOA:AOB ratios (0.034) in terms of amoA gene abundance (Table 2).", "WWTW 4 exhibited the lowest ammonia effluent concentration (0.29 ± 0.4 mg L−1) and was associated with the highest AOA:AOB ratio (38.7).", "WWTW 7 and 6 had the next highest AOA:AOB ratios (1.451 and 0.75, respectively) and these sites also had the third and second lowest average ammonia effluent concentration (2.4 ± 4.5 and 2.6 ± 2.5 mg L−1, respectively) (Table 2).", "This suggested that a high AOA:AOB ratio corresponds to better ammonia effluent quality and therefore AOMs could represent bio-sentinels of reactor performance.", "The suitability of this is discussed in more detail in subsequent sections.", "The high ammonia and oxygen affinities (Stahl and de la Torre, 2012; Pan et al., 2016), may lead to the AOA dominance that has been reported in domestic WWTWs in China (Bai et al., 2012), municipal WWTWs in Thailand (Kayee et al., 2011; Limpiyakorn et al., 2011), industrial WWTWs in Europe (Mußmann et al., 2011), and a RBC treating municipal wastewater in Canada (Sauder et al., 2012).", "In contrast, other studies found that the abundance of AOB is greater than AOA in municipal WWTWs in the USA (Wells et al., 2009), a lab-scale WWTWs in China (Jin et al., 2012), industrial WWTW in China (Bai et al., 2012).", "This study reports that abundance varies according to the conditions within individual WWTWs, however AOB abundance usually outweighs AOA abundance in optimal conditions where excessive load is not present and oxygen availability is sufficient.", "Similar biomass activities were found between two intensively sampled RBCs (Fig. S2).", "However different AOM abundance was found between RBCs resulting in approx. 2.5 fold greater specific ammonia oxidation activity (Fig. S3).", "Differences in specific ammonia oxidising activity could account for the observed ammonia treatment performance at each WWTW (Fig. 2).", "Yao and Peng (2017) has reviewed the per cell AOB activity which ranged from 1.1 to 123 fmol-N.", "cell−1h−1 which is a similar order the the per GC AOB activity reported here (Fig. S3).", "All RBC biozones in this study consisted of at least 4 zones, with zone 2 and 3 separated by a baffle.", "This spatial separation of wastewater and biofilm is thought to enhance performance by selecting for specialist organisms adapted to wastewater in each biozone (Hassard et al., 2018; Petrie et al., 2014).", "Access hatches at WWTW 1 enabled samples to be taken at each zone (see Materials and Methods section), therefore this WWTW was selected for detailed sampling of biofilm and reactor physicochemical character.", "Mean AOB amoA gene abundance steadily increases from 2.77 × 105 to 1.48 × 108 GC.", "g−1 biofilm from the front of zone 1 to the rear of zone 2 (Fig. 3B).", "On the other hand, AOA amoA gene abundance per gram of biofilm was similar throughout the process, with mean gene abundance ranging from 4.89 × 105 to 2.39 × 106 (Fig. 3B).", "The difference between AOA and AOB was significant in WWTW 1 (tWelch, t = 23.4, p < 0.001; Fig. 3B) with most of the difference attributed to the rear of zone 2 both within and between AOA and AOB (Games-Howell, p < 0.001).", "This suggests that either the AOA population was more resilient to fluctuating load and oxygen availability, or that the AOB are readily outcompeted under lower HRTs.", "This finding is interesting as it indicated that AOA maintain activity in conditions that normally reduce AOB abundance.", "Also, as AOA have greater oxygen affinity than AOB, they can fill oxygen-limited niches within the deep layers of the biofilms and maintain a stable population where there is less competition.", "In contrast, AOB become more competitive in low loading conditions towards the distal end of the RBC biozone (zone 2 front and rear, Fig. 3B) where BOD5 concentration is at its lowest and dissolved oxygen is at its greatest.", "Biofilm was sampled on a monthly basis from the front of zone 2 of WWTW 1 in two sampling phases (see materials and methods).", "The first phase WWTW 1 had 12 m3 d−1 flowrate into the RBC.", "During this phase the RBC was nitrifying well (between 96 and 80% NH4-N removed in December 2014 and January 2015) as the RBC was acclimatising and steady state was reached in December 2014.", "In the second phase, the flow was 38 m3 day−1 and the RBC appeared to heavily be loaded for nitrification as removal rates of 65 and 15% was achieved from March to October 2015 respectively (Fig. 4A).", "The bulk water dissolved oxygen concentration in phase 1 exceeded 4 mg L−1 which diminished to <2.5 mg L−1 during the increased organic loading which occurred in phase 2.", "High ammonia removal in phase 1 correlated with the peak in AOB amoA with 9.2 × 107 GC.g−1.", "This 1–2 orders of magnitude greater than the AOA which was between 6.1 × 104 and 9.8 × 105 amoA GC.g−1 biofilm irrespective of wastewater loading (Fig. 4B).", "This agrees with previous studies which have shown that in most WWTW, AOA do not contribute in a significant way to overall ammonia removal performance (Fitzgerald et al. 2015).", "The amoA gene is involved in the process of microbial ammonia oxidation (Tourna et al. 2008) and therefore can provide a useful indicator of ammonia oxidation dynamics in RBCs.", "In controlled lab-scale bioreactors variable AOB abundance has been shown in reactors with equivalent performance (Ahn et al. 2008) which could be explained by large variability in specific activity of AOB which was reviewed in Yao and Peng (2017).", "Reported specific AOB activity ranged from 1.1 to 123 fmol-N.", "cell−1h−1 but had an average of 23 fmol-N.", "cell−1h−1.", "However it is considered that the greater the number of AOM the higher the nitrification potential of a wastewater system (Almstrand et al. 2011).", "Whilst other studies have suggested limited correlation between nitrification rates and either AOA or AOB activity (Santoro et al., 2010).", "Despite the apparent lack of correlation, resilience of AOA to adverse conditions has been noted, compared to AOB, in a study where 50 mg L−1 of silver nano-particles had a significant and detrimental effect on AOB amoA, but limited impact on AOA amoA gene abundance (Beddow et al., 2017).", "Differences could, in part, be due to differences in the ammonia monooxygenase (AMO) protein (e.g. active site for ammonia oxidation) between AOA and AOB species (Walker et al., 2010).", "Contribution to total ammonia removal is a balance between diversity, abundance and activity of ammonia oxidisers which ultimately contribute to the nitrogen oxidation processes in wastewaters (Egli et al., 2003).", "Here we revealed which AOB species are found in RBCs and how the abundance of AOA and AOB relates to full-scale RBCs performance.", "Ultimately microbial community dynamics in RBC systems can be used as a tool to engineer more resilient ammonia oxidation in small WWTW.", "Overall the data indicate that bacteria dominated at WWTW 1.", "However, in situations where load was high and dissolved oxygen was low, for example, at the beginning of the process or in high flow of 38 m3 day−1, archaea are competitive and may even play the dominant role.", "AOB appear to be more sensitive to changes in environmental conditions, with the amoA gene abundance increasing in low load, high dissolved oxygen conditions.", "DistLM revealed that a combination of HRT, organics loading, removal rate of NH4-N and temperature were important factors governing the ammonia oxidizing community variability in WWTW 1 (Table 3).", "The HRT was the most important reactor variable (DBLM, Pseudo-F = 6.2, proportion = 0.32, p = 0.002) governing the AOM ecology suggesting this important process condition should be optimized for robust ammonia removal in RBCs (Table 3).", "Together the NH4-N and BOD5 loading rates accounted for ~ 25% each of the abundance of ammonia oxidizing community (p < 0.05).", "Whereas, the NH4-N % removal accounted for 17% in the variability of the ammonia oxidizing community (DBLM, Pseudo-F = 2.71, proportion = 0.17p < 0.05), demonstrating the role of AOM and reactor performance (Figs. 5 and 6).", "Similarly, the role of BOD5 removal was responsible for 21% of the variability in AOM (DBLM, Pseudo-F = 3.37, proportion = 0.21, p < 0.05) reinforcing previous studies which show the competition linked spatial localization between heterotrophic organisms and AOM.", "Modelling revealed a distinct nitrogen cycling community present within high performing RBCs operating at small WWTW, linked to efficient control of RBC process variables (mainly retention time, organic loading effects) and environmental variables e.g. temperature.", "The ability of AOA to outcompete AOB at low ammonia concentrations is partly due to their superior ammonia affinity (Km), reported as being 0.02 mg N L−1 which is 100-fold lower than reported for AOB (Pan et al., 2016; Prosser and Nicol 2012).", "As well as higher ammonia affinity, AOA also possess higher oxygen affinity than AOB (Stahl and de la Torre, 2012; Mußmann et al., 2011).", "Consequently, AOA have been shown to exhibit higher activity than AOB in municipal WWTWs under DO of < 0.1 mg L−1 (You et al., 2009; Fitzgerald et al., 2015).", "A similar trend of increased importance to nitrogen cycling and therefore wastewater reactor performance has been demonstrated for single step complete nitrification organisms (Daims et al., 2015).", "In contrast, another study showed that AOA only represent 8% of the AOM and have lower activity compared to AOB across 52 conventional WWWTs (Mußmann et al., 2011).", "The differing affinities for ammonia and oxygen, along with competition from heterotrophs can explain the dynamic state of ammonia oxidising communities and the ecological niche they occupy within biofilm (Pan et al., 2016).", "Phylogenetic analysis of sequences obtained by next generation sequencing showed that the AOB had diverse evolutionary ancestry from RBC biofilms and were similar to themselves and to reference OTUs.", "AOB-otu 586 was 97.2% similar to an isolate from a groundwater aquifer.", "Unsurprisingly, a very high similarity of 99.57% occurred between an isolate from a down-flow biofilm reactor and AOB-otu 887.", "In contrast, NOBs from RBC biofilm had a similar heritage with two distinct branches evident.", "NOBs was very similar (99.87%) to an uncultured Nitrospira sp. clone isolated from a submerged aerated biological filter (Fig. 5).", "These organisms were present on a separate branch to other NOBs and contained Nitrospira-like OTUs and separately, Nitrobacter-like organisms.", "High similarity was observed between RBC NOBs and database OTUs.", "For example, NOB-NOB-OTU 27 had a 99.34% similarity to a Candidatus Nitrospira defluvii and a clone isolated from a conventional WWTW.", "Previous studies also reported Nitrososphaera genus to be dominant in WWTWs (Kayee et al., 2011; Limpiyakorn et al., 2011; Mußmann et al., 2011; Park et al., 2006; Gao et al., 2014).", "Nitrososphaera members are slow growing and their ammonia oxidation activity (1.4 fmol cell−1 day−1) is less than other AOAs including which ranged between 2.5 and 12.8 fmol cell−1 day−1) (Li et al., 2016).", "Therefore, Nitrososphaera members are significant beneficiaries of the protective biofilm structure where they can exploit niches of low ammonia concentration (Hassard et al., 2018).", "Microbiome studies may help elucidate complex interactions between key functional groups (e.g. AOM and heterotrophs) and their impact on the nitrogen cycle which governs the performance of biofilm systems.", "Some operational RBCs have shown ability for elevated ammonia removal.", "In this work, long-term treatment performance of seven full-scale RBC systems along with the structure and abundance of the ammonia oxidising bacteria (AOB) and archaea (AOA) communities within microbial biofilms were examined.", "The 90 month study revealed the following new insights into small biofilm based wastewater treatment processes:", "Long term data showed the dominance of AOB in most RBCs, although two RBCs had demonstrable shift toward an AOA dominated AOM community.", "Similar biomass activities were found between two intensively sampled RBCs.", "However different AOM abundance was found resulting in approx. 2.5 fold greater specific ammonia oxidation activity.", "This was thought to be a principal reason behind differences in observed ammonia treatment performance at each WWTW.", "Next Generation Sequencing of the 16S rRNA gene revealed a few new OTUs of AOM from RBC biofilms which were for the most part similar to reference databases.", "A", "AOA were more abundant in the biofilms subject to lower organic loading and higher oxygen concentration found at the distal end of RBC systems.", "Modelling revealed a distinct nitrogen cycling community present within high performing RBCs, linked to efficient control of RBC process variables (retention time, organic loading and oxygen concentration).", "We show that distinct AOM community exist between different RBC reactors operating at different WWTW.", "Nitrogen cycling organisms were present in all RBCs but differed in a significant manner between the RBCs.", "The study of fundamental process conditions (notably HRT, organic loading and NH4-N removal rate) suggested that these acted as determinants governing variability in AOM.", "The major issue remains to be solved in future studies, how to increase the specific ammonia oxidation activity at small WWTW which are often treating dilute wastewater, are subject to shock loading, low temperatures, are for the most part remotely operated.", "Continued focus is recommended on stabilising ammonia loading rate to increase the resilience of ammonia oxidising microorganisms." ]
[ "Ammonia removal performance and community influenced by RBC process.", "7 full scale RBCs microbial community showed community influence performance.", "Reactor dissolved oxygen and substrate loading governed performance.", "Nitrogen removal community change manifested in dominance of archaeon over bacteria.", "Overloading RBCs result in suppression of ammonia oxidising bacteria and archaeon.", "Ratio of archaeon to bacterial ammonia oxidisers useful predictor of performance." ]
Two approaches to forecast Ebola synthetic epidemics
We use two modelling approaches to forecast synthetic Ebola epidemics in the context of the RAPIDD Ebola Forecasting Challenge. The first approach is a standard stochastic compartmental model that aims to forecast incidence, hospitalization and deaths among both the general population and health care workers. The second is a model based on the renewal equation with latent variables that forecasts incidence in the whole population only. We describe fitting and forecasting procedures for each model and discuss their advantages and drawbacks. We did not find that one model was consistently better in forecasting than the other.
[ "IMMU", "MEDI" ]
[ "Compartmental model", "Epidemics", "Forecasting", "Renewal equation" ]
[ "2404", "2405", "2406", "2713", "2725", "2739" ]
[ "The scale of 2014 West African Ebola epidemic took the world by surprise.", "Although several Ebola outbreaks erupted in Africa over the past 40 years, none reached more than several hundred cases, far from the estimated 28,000 cases and 11,000 deaths observed during this last epidemic.", "As national and international health organizations rushed to control an unprecedented epidemic, the need to forecast case incidence mounted daily in order to support difficult decisions about limited resource allocation.", "Mathematical and statistical tools developed over the last two decades have improved epidemic forecasting (Lekone and Finkenstädt, 2006; Ferguson, 2001; Shaman and Karspeck, 2012).", "Yet it is still notoriously difficult to accurately predict an epidemic trajectory, because key features of the epidemic may be poorly known (e.g., pathogen natural history, routes of transmission, observation errors, etc.).", "Indeed, the 2014 West African Ebola epidemic was challenging to forecast because (i) it was the first time an Ebola epidemic hit an African urban centre, (ii) the natural history of the pathogen was poorly known and (iii) the quality of epidemiological data reported was poor.", "Forecasting incidence with a mathematical model involves two steps.", "First, the model parameters are fitted to observed data, then incidence is simulated forward using the inferred values for these parameters, while propagating uncertainty.", "The difficulty of each step depends on the modelling framework chosen.", "Here, we explore two different modelling approaches in the context of the Ebola Challenge, a forecasting exercise on synthetic Ebola epidemics (Viboud et al., 2018).", "The first approach uses a classical compartmental model with population and transmission structure, fitted with approximate Bayesian computation (Toni et al., 2009); the corresponding forecasting step uses a simple filtering technique.", "The second model uses the renewal equation with latent variables.", "The fit uses Markov chain Monte Carlo, which is less straightforward than the filtering methods of the compartmental model, but provides a more robust statistical framework for inference.", "In this section, we describe the theoretical framework, the fitting and forecasting procedure for the two mathematical models used in the Challenge.", "The first model is a standard compartmental model; the second is based on the renewal equation.", "The compartments were chosen to reflect the natural history of Ebola virus disease and to use the data that was going to be made available by the challenge organizers.", "The data included contact-tracing data and incidence and death counts among the general population and health care workers (HCW).", "More generally, this model is a discrete-state, compartmental and stochastic model.", "Unlike canonical compartmental models, however, it keeps track of individuals in order to make use of contact-tracing data.", "The model was implemented in C++ and interfaced with R (R Core Team, 2011) for data analysis and calibration.", "The choice of parameters to fit to data or to fix to a pre-specified value is based on the literature and on the modeler's prior knowledge given the scenario and the timing of the forecast relative to the whole epidemic (WHO Ebola Response Team, 2014).", "Two calibration methods were implemented.", "First, we followed the methodology described in Wood (2010) where a synthetic multivariate normal likelihood is calculated from summary statistics of selected outputs of the model.", "Priors on the parameter values are set, first using a relatively non-informative distribution, then narrowing the distributions based on the posteriors from the first phase.", "We used the R package synlik to implement this calibration method.", "The second method implemented was an approximate Bayesian computation (ABC) where priors were defined similarly to the synthetic likelihood method (Beaumont, 2010).", "The summary statistics for both synthetic likelihood and ABC were based on incidence and death counts.", "More precisely, for both the general population and HCW, we used the sum of squared errors between the quadratic interpolations of the observed and modelled log-incidence.", "We also used the squared error between the observed and modelled cumulative death counts.", "The choice to use cumulative counts was driven by the small number of weekly deaths observed, which were challenging to fit on a time-interval basis, but more manageable on a cumulative one (the time interval death counts often consisted in sparse “spikes”; hence the error between data and simulated deaths was easier to handle when considering the cumulative counts).", "The contact-tracing information, when available, was used to fit the distribution of the generation interval (via nE and nI).", "We used both calibration methods for the two first prediction points but ended up using only ABC because this implementation was more robust (synthetic likelihood would occasionally fail because of very small variances of the summary statistics; this was likely a solvable problem but we did not have the time to find and implement a solution).", "Table 2 summarizes all model parameters and their fitting method, if any.", "Briefly, we chose to estimate contact parameters between infectious and susceptible individuals (βFS, βIS), the mean duration of latency (σ), infectiousness (γ) and the effective population size.", "All other parameters were either fixed to values taken from the literature or set proportional to the estimated parameters.", "The contact-tracing data, when available, were used to fit the backward generation interval distribution (Champredon and Dushoff, 2015).", "The number of initial infectious individuals was arbitrarily set to five, so not fitted.", "The effective total size had a Beta distribution as prior, where the mean was arbitrarily set at 40,000 (which represents about 1% of the total population of Liberia, the country used for this challenge), and boundary values set at 0 and 100,000.", "Once the model parameters were fitted to data, we simulated the epidemic forward to forecast incidence and deaths in both the general population and among HCW.", "We generated 1000 simulations with parameter values sampled from the posterior distributions.", "For a given observation time, the total incidence is T = O + U, with O the observed cases and U the unobserved ones.", "In order to infer a confidence interval for T, we assumed that U had a negative binomial distribution with U as its overdispersion parameter (the number of “failures” in the failure-counting definition of the distribution) and a constant reporting rate arbitrarily chosen to be 30% for its probability parameter.", "In other words, the reported data were modelled with an observation error following a negative binomial distribution.", "The associated 95% confidence interval was used to filter, from the 1000 simulations generated, the trajectories where the incidence value at the last observed time was contained in this confidence interval.", "The forecast statistics were then calculated based on these remaining simulations.", "The generalized renewal equation (GRE) model is a Bayesian, latent-incidence model.", "The true incidence in each time period is treated as a latent (unobserved) variable.", "These variables are used to calculate the likelihood of the observed data and of the latent-incidence time series itself: each latent incidence is assumed to arise from a force of infection due to the infected individuals stemming from incidence in preceding time periods.", "The force of infection is reduced to account for endogenous behaviour change and for the estimated effects of interventions.", "The number of infections in a time period results from the force of infection being applied to an effective number of susceptibles, which is also a latent variable.", "Fig. 2 shows a diagram of the model.", "We fit the GRE model using JAGS (Plummer, 2003), a program for Bayesian inference that takes a model structure and explicit priors for all parameters and uses Markov chain Monte Carlo sampling to generate samples from the posterior distribution of the parameters.", "We assumed that a proportion ρ of the population was a priori susceptible to reported infections.", "This proportion consists of two components: the effective proportion of the population which is susceptible ρE, and the reporting fraction ρr (thus ρ = ρEρr).", "To aid convergence, we fit the relatively stable value of ρ and parameterized ρE as ρpE (thus ρr = ρ1−pE), with Beta-distributed priors on both ρ and pE.", "The starting number of susceptibles was therefore S(0) = ρEN, where N was the approximate population of Liberia.", "At each subsequent time step, we decreased the effective number of susceptibles by the latent incidence i: S(t) = S(t − 1) − i(t).", "For the Challenge, we used a time step of one week, and 5 time lags (thus, a maximum generation lag of L = 5 weeks).", "Given more time, we would have preferred to use a time step of 0.5 weeks, aggregating model-predicted incidences to match the observed incidence data.", "We modeled the generation-interval distribution phenomenologically using the functional form of the negative binomial (but truncated at L), with a position parameter (expressed as a function of the maximum generation time) and a shape parameter (analogous to the negative-binomial shape parameter).", "Process error is incorporated through the Gamma distribution in (3), and observation error is incorporated through the negative binomial distribution (6).", "Both of these use shape parameters fitted with broad prior distributions (Table 3).", "Both models were developed during the Challenge.", "SHERIF being the first ready to fit and forecast, our contributed forecasts were made with this model for the first two prediction time points.", "We did not have a formal method to select between models for later prediction time points.", "Model selection was done based on visually assessing the two models’ performance in previous rounds and on our opinions about the plausibility of their predictions.", "Because many parameters of the SHERIF model could not be directly inferred using the data provided by the Ebola Challenge, we decided to fix the values for those that could be reasonably supported by the literature (Table 2).", "We also set some parameters proportional to others in order to reduce the high dimensionality associated with fitting the SHERIF model.", "Furthermore, we had to give informative priors (obtained with sequential fits, as explained in Section 2).", "Fig. 3 shows an example of the forecasting process.", "Note that our method requires a large number of simulations because the filtered ones represent only a small fraction (in our case, only about 10% of simulations were kept).", "One difficulty we faced was when the incidence data showed “double bumps”, probably indicative of an effect of spatial structure where a sub-epidemic in one region has already reached its maximum incidence while a second sub-epidemic is still growing.", "Because we did not have the resources to develop a spatial version of SHERIF in time for this challenge, capturing a double bump was challenging.", "We artificially accounted for this suspected spatial structure by changing the time-varying transmission rates in order to mimic these observed incidence patterns.", "The model calibration and forecast were insensitive to most of the HCW parameters, most likely because HCW cases and deaths represented a small proportion of the overall epidemic so their contribution to the force of infection could not be clearly identified in this modelling framework.", "The calibration process took about 2 h on a small cluster of 12 CPUs at 2.1 GHz and the forecasting process (simulating forward based on fitted parameters values) took less than 10 min.", "Neither calibration nor forecasting demanded large amounts of memory.", "An advantage of the GRE model is that, once the priors are set, it can fit observed data without intervention or supervision; we simply ran the MCMC algorithm generated by JAGS long enough to achieve convergence (as assessed based on visual assessment of trace plots and standard diagnostics such as the Gelman–Rubin statistic (Gelman and Rubin, 1992)).", "A drawback of our particular implementation (due to time constraints) is that the GRE was fit only to incidence data, and only forecasted incidence.", "Deaths were reported for the Challenge as a fixed proportion of incidence (we used the same ratio as the one found in SHERIF).", "Examples of typical forecasting outputs are shown in Fig. 4.", "As with the SHERIF model, we did not have time to add simple spatial structure.", "We expect it would help fitting incidence curves, although it would probably increase forecast uncertainties.", "The GRE model fit all of the scenarios reasonably well without any specific tuning or calibration.", "Memory demands were not large, and convergence was typically obtained for a single scenario with a 30-min run on a laptop.", "For the contest, we ran each scenario for one to two hours; this improved convergence in some cases.", "For this Challenge, we used two models with distinct advantages and drawbacks.", "The main advantage of the SHERIF compartmental model is its use of a structured population that can identify incidence and the impact of targeted interventions in specific communities (here, general population and HCW).", "But this comes at the price of having 24 parameters, for many of which no calibration data were available, forcing us to make arbitrary choices during the fitting process.", "The GRE model provided a theoretical framework particularly well adapted for forecasting incidence.", "It has relatively few parameters, and once finished was able to robustly make forecasts without supervision.", "However, we experienced implementation and numerical difficulties with the fitting process.", "In particular, we found it was necessary to implement a “hybrid approach” to aid MCMC convergence.", "Our efforts to use more powerful MCMC algorithms (e.g., Hamiltonian MCMC (Neal, 2011)) failed during the Challenge time frame, but we have pursued them further in other venues (Li et al., in review).", "The simplicity of the GRE framework means that we are optimistic about our ability to add further structure, such as explicit tracking of deaths and funerals and representing health-care workers and hospital settings in the model.", "However, some of these added complexities will no doubt add to the computation difficulties of fitting the model, which will require further innovations analogous to the hybrid model described above.", "The approach taken with SHERIF – that is, a compartmental model with parameters estimated from reporting data – is fairly standard in the literature: the natural history of the pathogen is translated in differential equations involving relevant disease states, then a statistical inference method is chosen, usually one that can deal with a relatively high-dimensional parameter estimation problem (see for example Lekone and Finkenstädt, 2006; Shaman and Karspeck, 2012).", "The renewal equation used in the GRE framework has historically been used for the estimation of the reproductive number of an epidemic (WHO, 2014) rather than forecasting because, in the simplest formulation of the renewal equation, the epidemic grows exponentially to infinity.", "In this regard, the GRE is relatively innovative.", "But in its current form, the GRE can only forecast incidence, which may be a limitation if more detailed forecasts (e.g., deaths, number of hospital beds needed) are required.", "In the context of the Challenge, we found that both models suffered from the lack of spatial structure that could have provided a more principled framework to fit “double-bump” incidence patterns.", "In closing, we want caution about the differences between short- and long-term incidence forecasting.", "While a mathematical model can make detailed, and often reasonably correct, short-term epidemiological forecasts, it neglects much larger phenomena like knowledge transfer and behaviour changes, which can significantly affect longer term forecasts." ]
[]
Key Challenges and Countermeasures with Railway Accessibility along the Silk Road
[ "CENG", "COMP", "ENER", "ENGI", "ENVI", "MATE" ]
[]
[ "1500", "1700", "2102", "2200", "2305", "2501" ]
[ "The Silk Road originated in Chang’an (Xi’an today) and linked the land routes of countries from Transoxiana, the Persian Gulf, and the Mediterranean Sea.", "It is the trunk road over which China historically carried out economic, trade, and cultural exchanges with Central Asia, West Asia, South Europe, and North Africa; the diligent and courageous people living in this region worked together with a friendship and cooperation that is still widely praised today.", "Trade, investment, cooperation, and exchange have once again become active along the Silk Road.", "In order to rejuvenate the Silk Road, the Chinese Government has announced its vision and action to build the “Silk Road Economic Belt and the 21st Century Maritime Silk Road” (also known as the “Belt and Road” initiative) [1].", "The announcement specifically points out that one of the focuses of the Silk Road economic belt construction is to unblock the road from China to Central Asia, West Asia, the Persian Gulf, and the Mediterranean Sea.", "This major strategic focus is to jointly build a China/Central Asia/West Asia international economic cooperation corridor by relying on major international corridors, using central cities along the route as underlying support, and using key economic and trade parks as the cooperation platform.", "The “Belt and Road” initiative will focus on the key corridors, key nodes, and key projects of the transportation infrastructure.", "It will complete missing segments, unblock bottleneck segments, provide sound road safety protection facilities and traffic management facilities, and improve the accessibility of the road.", "It will also establish a unified full-course transport coordination mechanism; promote organic linkages between international custom clearance, transshipments, and multi-model combined transportation; gradually develop a compatible and standardized transportation plan; and bring convenience to international transport.", "The strategic plan for the “Belt and Road” initiative has received broad recognition and an active response from the international community, and China will inevitably play an important role in promoting its construction.", "Railway is an important infrastructure and a popular mode of public transport.", "Boasting merits such as robust transportation capacity, relatively low energy consumption, minor pollution, and low costs, railway holds a dominant position in the comprehensive transportation system, making it an important tie connecting countries along the Silk Road and a critical contributor to economic growth and people’s livelihood.", "To support and facilitate the joint construction of the China/Central Asia/West Asia international economic cooperation corridor, it is necessary to build a trunk railway along the Silk Road.", "The current railway network along the Silk Road faces challenges such as being “open but blocked” in the eastern regions and missing segments in the western regions.", "Despite several rounds of research and negotiations, no progress has been made regarding the accessibility issue.", "Therefore, it is necessary and urgent for an in-depth study to be carried out on an accessibility solution for the Silk Road.", "The proposed railway along the Silk Road runs through many countries.", "Given the different technical standards and management systems involved, and considering factors such as economy, political systems, and railway development, we are currently faced with two major challenges.", "First, some areas are “open but blocked.”", "Due to the different gauge standards, the Chinese railway cannot seamlessly connect with the railways in Central Asia and Iran, and the existing transport capacity and service quality are inadequate to meet the requirements of modern freight and passenger transport.", "Second, there are missing segments.", "In certain West Asian countries, there are not only dead-end rails, but also blank spaces without rail.", "These countries have not resolved the interconnection of railway infrastructures, not to mention the basic formation of a railway network.", "Thus, they cannot benefit from the driving effect of railway and its competitive advantage on the transportation market.", "(1) Bogie change stations limit the transport capacity of the railway.", "Bogie change stations currently exist at railway junctions between China, Iran, and Central Asia.", "China has two bogie change stations located at the ports of entry of Alashankou and Khorgas.", "All five Central Asian countries employ the 1520 mm broad gauge, while China and Iran employ the 1435 mm standard gauge.", "Given such a gauge difference, the train must change its steering bogie or transship the cargos at the port-of-entry railway station—hence making the port-of-entry railway station into a bottleneck hampering the transport capacity of the railway.", "Taking the Alashankou–Dostyk port-of-entry railway station as an example, the freight volume transferred in 2014 was less than 1.7 × 107 t, the bogie change capacity at the station is near saturation, and transit cargos are frequently delayed.", "Even though many measures have been taken, the limitations in technology and equipment mean that the transport capacity on both sides cannot be adequately coordinated and integrated to form a corridor with greater transport capacity.", "(2) Bogie change operations significantly prolong the arrival time.", "Bogie change operations require changing the steering bogie (including the wheelset), transshipping cargos, using an expensive variable-gauge, or transferring the passengers.", "Such operations increase not only the transportation time but also the operating costs.", "In particular, transshipment operations at the port of entry may take as long as 10 days due to the different types of train and cargos to be handled, and given the detention of cargos at the port of entry.", "Taking the time-sensitive China-Europe block train as an example, on November 18, 2014, with a full journey stretching 13 052 km, the Yiwu–Xinjiang–Europe route (82 twenty-foot equivalent unit (TEU)) initiated operation, passed through eight countries, underwent three bogie change operations, and took 21 days to reach its destination.", "Clearly, bogie change operations significantly impair the advantage and competitiveness of railway transport.", "According to the International Union of Railways (UIC), broad gauges are above 1520 mm (inclusive), standard gauge is 1435 mm, and narrow gauges are below 1067 mm (inclusive).", "Different gauge systems have led to drawbacks such as low efficiency and high operating costs.", "Various solutions exist for international railway lines.", "(1) Normal-speed railways can maintain the existing gauge status and newly constructed railways such as high-speed railway can employ the standard gauge.", "Japan used to employ the 1067 mm gauge for its normal-speed railways.", "When constructing the Tokaido Main Line, which was put into operation in October 1964, Japan selected the standard gauge without hesitation, and continued to use the standard gauge for the construction of subsequent main lines.", "Railways with two different gauges are geared to different but explicit purposes, and the passenger transfer facilities are reasonable in arrangement and satisfactory in operation.", "Taiwan of China employs narrow gauge for its normal-speed railways but standard gauge for high-speed railways, such as the Taipei–Kaohsiung Line and the high-speed rails constructed in Taipei, Taoyuan, and Kaohsiung.", "Thailand, Malaysia, and Indonesia all employ narrow gauge for their normal-speed railways and standard gauge for the high-speed railways constructed in Bangkok and Kuala Lumpur.", "All three countries chose standard gauge for the future construction of high-speed railways.", "(2) Railway networks can unify the gauge in order to achieve unblocked interconnection.", "Spain, Portugal, Lithuania, Latvia, and Estonia employ broad gauge for their normal-speed railways, and are planning to change the broad gauge into standard gauge in order to achieve interconnection with European Union (EU) railways, so as to enable cross-line operations and reduce operating costs.", "Spain has developed a variable-gauge train that can automatically shift to another gauge at an operating speed of 15 km∙h−1; however, the technologies involved are complex, the manufacturing costs are high, and the operation will not be cost-effective.", "During the construction of its high-speed railways, Spain chose the standard gauge, and its total length of high-speed rail tracks now ranks second in the world.", "The historic gauge systems in Canada, the US, and Mexico were disorderly, making interconnection and combined transport impossible.", "Since February 1886, the US has uniformed the rail track to standard gauge gradually, rail tracks exceeding 20 000 km have been rebuilt.", "After the gauge was unified across North America, remarkable progress was made in straight-through combined transport, freight transport efficiency, and freighter satisfaction.", "Standard-, broad-, and narrow-gauge tracks all existed in the mainland of China in the past.", "During the construction of the Tangshan–Xugezhuang Railway and the Beijing–Zhangjiakou Railway, respectively, both Mr. Da Jin and Mr. Tianyou Zhan insisted on using standard gauge.", "Today, standard gauge has become a uniform gauge on the railway network.", "(3) Newly built high-speed railways can employ the same gauge as existing railways.", "In Russia and the member states of Commonwealth of Independent States (CIS), the rail gauge is 1520 mm.", "This gauge standard has been widely applied on the gigantic railway network across Europe and Asia, which ranks as the second largest railway network in the world.", "In order to take full advantage of existing resources (except for specific railway lines intended for specific purposes), newly built railway lines will employ the same gauge as existing railways.", "The Moscow–Kazan High-speed Railway, to be constructed in Russia, will also employ the 1520 mm gauge.", "In conclusion, regarding the solution to international railway lines with different gauges: The gauge system is generally a historical and cultural heritage of the railway development in respective countries, and different gauges will remain for quite a long time.", "Applying old standards to old lines and new standards to new lines is the best strategy, and can be worked out based on the social and economic development needs and the technological and economic level of respective countries.", "Accelerating the construction of the “Belt and Road” undertaking will contribute to economic prosperity and regional economic cooperation along the Silk Road, strengthening exchanges and mutual learning between different civilizations and promoting peaceful global development.", "It is a great cause, benefiting billions of people.", "The interconnection of the Silk Road is a top priority for the “Belt and Road” initiative.", "On the basis of respecting the sovereignty and security concerns of relevant countries, countries along the Silk Road are expected to strengthen their infrastructure construction and planning, and the linkage of their technical standards and systems; to jointly push ahead the construction of international trunk corridors; and to gradually shape the infrastructure network connecting Asian regions and connecting Asia with Europe and Africa.", "These visions have drawn high levels of concern and broad recognition from the international community, and relevant actions have been taken.", "The Gulf Cooperation Council plans to construct a railway network connecting six member states, with an anticipated total length exceeding 2000 km.", "Turkey, Iran, and Iraq will speed up the implementation of their respective national railway network development plans, and are actively working on railway interconnection with neighboring countries, marking a preliminary accomplishment in the goal to connect the missing segments.", "Therefore, the key to an unblocked railway along the Silk Road is to complete the missing segments in Central Asia.", "Given the geographic limitations posed by the Tian Shan and Kunlun Mountains, we have studied the natural, traffic, and economic characteristics of the China–Central Asia–Iran Railway, as well as the multilateral relations and the interest concerns of the relevant countries.", "We have proposed three valuable solutions for expanding or constructing railway lines through Khorgas, Torugart, or Irkeshtam.", "To mitigate the transportation pressure at Alashankou, the Jinghe–Khorgas Railway has been interconnected with Kazakhstan railways, and its role in promoting regional economic cooperation and cultural exchanges between China and Central Asian countries is becoming increasingly evident.", "Despite its current issues, the Khorgas port-of-entry railway station has great potential and is bound to develop into an international logistics hub, integrating passenger and freight transport, warehousing, and e-commerce.", "It will play a vital role in the construction of the Silk Road economic belt.", "However, in order to build a cross-line, unblocked, and high-throughput railway, we must address the bottleneck of the Khorgas bogie change station.", "The China–Kyrgyzstan–Uzbekistan Railway stretches over 500 km and passes through Kashgar, Torugart, and Andijan.", "The construction of this railway allows interconnection between China, Kyrgyzstan, and Uzbekistan, and adds one more option to the international railway network.", "It also helps to avoid the detour of cargo and passenger transport between China and Central/West Asia, and even changes the transportation landscape in Southern Kyrgyzstan.", "Given the different gauge standards, bogie change stations must be set up at the port of entry between China and Kyrgyzstan.", "In order to build a cross-line, unblocked, and high-throughput railway, the bottleneck caused by the bogie change station must be addressed.", "In December 2014, the China–Kyrgyzstan–Tajikistan–Afghanistan–Iran Railway was proposed at a railway conference held in Dushanbe.", "The preliminary route will pass through Kashgar, Irkeshtam, Karamyk, Dushanbe, Mazari Sharif, Maimana, Herat, and Taybad; then, at an appropriate node, it will split into a south line reaching the Persian Gulf directly and a west line reaching Turkey and the Middle East.", "The proposed railway stretches more than 2300 km and will run through mountainous areas characterized by complex geological conditions and posing notable engineering challenges.", "Having perceived its importance and potential, Iran has been an active promoter of this project.", "From the perspective of Kyrgyzstan, Tajikistan, and Afghanistan, this project can help to construct a regional railway transport platform that will facilitate international commodity exchanges and prosper the regional economy through constant transit shipment.", "For China, this project will create a new international railway and make direct transport more convenient.", "This project almost fills all the railway gaps within the region.", "If it is built using the expected standard gauge, a cross-line, unblocked, and high-throughput corridor will take shape.", "In view of the railway interconnection between China and its neighboring countries and the need for railway-oriented territorial development, the China-Kyrgyzstan-Uzbekistan Railway and the China–Kyrgyzstan–Tajikistan–Afghanistan–Iran Railway are both necessary and cannot replace each other.", "Given the complex geographic conditions, natural characteristics, market demands, economic characteristics, security considerations, interest concerns, and political situations involved, the possibility, feasibility, and urgency of project implementation remain in some doubt.", "In response to these major and key challenges, and abiding by the vision of the “Belt and Road” initiative as well as the principle of joint construction and cooperation, we have proposed a solution based on analysis and preliminary study.", "This solution is to construct a high-speed railway over the Central Asia section of the Silk Road, while simultaneously considering container lines.", "We suggest that a cooperative feasibility study be launched, through which we can carry out multi-level and multi-channel communication and consultation in order to consider the interests and concerns of all parties and seek common ground for cooperation.", "The Silk Road high-speed railway is based on the following premises:", "It will be differentiated from the existing Central Asian railway transport market, which is dominated by the 1520 mm gauge system.", "The two railways will complement each other’s advantages.", "Given the northwest-southeast trend of most existing railways in Central Asia, the proposed high-speed railway will improve the railway network by adding a northeast-southwest trend.", "It is expected to attract most of the population (60 million people) from five Central Asian countries, as well as international tourists.", "Incremental container cargos and transferred container cargos from sea transport will ensure the cargo supply for international container lines.", "It will contribute to the formation of a brand-new and efficient comprehensive transport system.", "It will contribute to the formation of a railway-driven economic belt, along which investment will be attracted to railway stations, cities will be built upon investment, people will accumulate wealth in the cities, and comprehensive developments will be rolled out.", "The proposed route will pass through Urumqi, Yining, Almaty, Bishkek, Tashkent, Samarkand (Dushanbe), Ashkhabad, and Teheran.", "This route not only covers major cities in Central Asia, but also stands apart from the existing railway trends and other proposed railways with a north-south trend in the region.", "In addition, the proposed route avoids complex mountainous areas and geological conditions.", "Carrying massive quantities of passengers and cargo, the existing 1520 mm gauge railways are important parts of the infrastructure of Central Asian countries.", "The Silk Road high-speed railway will be an important part of the modern comprehensive communication and transport system in Central Asia.", "Upon completion, it will effectively complement the existing normal-speed railway transport, road transport, and air transport.", "It will not only significantly boost the travel efficiency of passengers, but also greatly expand their travel options.", "The Silk Road high-speed passenger trains and international container lines will run on the same track.", "This model is different from that of the normal-speed railways on the railway transport market, and permits complementary advantages, thus promoting and serving socioeconomic development and a higher quality of life.", "Apart from the existing feasibility studies on the high-speed railway, the study on the Silk Road high-speed railway will have the following focuses:", "Convenient transfer between the standard-gauge Silk Road high-speed railway and the existing passenger transport system, and seamless linkage of container transport;", "A highest running speed of 250–300 km∙h−1;", "A highest running speed for international container lines of 120 km∙h−1, or an appropriate speed;", "One deck or two decks for the containers’ carriage;", "A selection of sleeper carriages or a running plan;", "A transport-oriented development (TOD) model and a development plan;", "Project implementation and a comparison of valuable business models.", "The Silk Road high-speed railway is an important solution to the construction of a high-throughput railway corridor along the Silk Road in Central Asia and railway interconnection between China, Central Asia, and West Asia.", "Taking this high-speed railway as the “axle” and treating city stops along the route as the “points,” the “point-axle” theory indicates that this initiative will help to develop an economic belt of co-prosperity within Central Asia, catering to economic development, the improvement of people’s livelihood, and future trends.", "As long as we hold to the principle of joint construction, focus on the “five links,” and make persistent efforts, this great vision will be achieved." ]
[]
Effect of chromium content on the corrosion resistance of ferritic stainless steels in sulfuric acid solution
Due to recent increases in the price of Ni, steel use is currently undergoing a global shift from austenitic stainless steels to ferritic stainless steels. In this study, the corrosion behavior of four types ferritic stainless steels with different Cr contents was investigated to study the effect of Cr content on the corrosion resistance in a sulfuric acid solution. The polarization curves of the ferritic stainless steel with the highest Cr content indicated the best corrosion resistance. No corrosion was observed for the stainless steel with 24 mass% Cr after a potential sweep based on ex-situ SEM images. Corrosion resistivity was improved for high Cr content (>24 mass%) stainless steel because it is considered to form a stable passivation layer.
[ "MULT" ]
[ "Electrochemistry", "Materials science", "Metallurgical engineering" ]
[ "1000" ]
[ "Stainless steels are classified by the three main types: austenitic, ferritic, and martensitic [1, 2, 3, 4, 5].", "Among these stainless steels, the corrosion resistances were superior for the Cr-Ni-type austenitic and Cr-type ferritic stainless steels.", "These stainless steels have been used as kitchen tools, architectural materials, mechanical and chemical-industrial equipment, fuel cell materials, etc. [6, 7, 8].", "However, due to the increase in Ni price because of resource exhaustion, use of stainless steels is currently undergoing a global shift from austenitic to Ni-less ferritic stainless steels [9, 10, 11, 12].", "The ferritic stainless steel SUS400 series, especially SUS430 in the 1960s and SUS445 in the 1990s, were developed for high corrosion resistance [13, 14].", "SUS430 contains 16–18% Cr and SUS445 contains 22% Cr and 1–2% Mo as the corrosion-resistant elements.", "The corrosion resistance of SUS445 in an acidic solution is as high as that of SUS316, which is a Ni-containing austenitic stainless steel [15].", "However, further improvement of corrosion resistance is needed for the application of these stainless steels in acidic and electrochemical environments, such as fuel cell materials.", "To realize corrosion resistance of the stainless steels, the natural oxidation of Cr and Fe on the surface to form passivation films composed of oxides/hydroxides is an important factor [16, 17, 18, 19].", "The Cr content significantly affects the corrosion resistance; there are a number of papers concerning the relationship between the Cr content amount and the corrosion resistance of stainless steel [20, 21, 22, 23, 24], although few among them report the use of ex-situ SEM observation.", "Recently, we found that the ferritic stainless steel can show higher corrosion resistance than SUS316 by increasing the Cr content, without any surface treatment, such as nitriding or coating [25, 26, 27].", "Ferritic stainless steel without surface treatment should be studied from the viewpoint of materials cost for application as bipolar plates which constitute one of the key components in fuel cells.", "Investigation of this high-Cr-containing ferritic stainless steel in greater detail revealed the possibility that materials with high corrosion resistance and appreciable electrical conductivity in even harsher environments than usual can be supplied without any surface treatment [28, 29, 30, 31, 32].", "In this study, corrosion evaluation experiments were conducted in an acidic solution using a proprietary high-Cr-content ferritic stainless steel in addition to the three SUS 400 series types with the different Cr contents of commercially-available products.", "The bulk properties were evaluated by X-ray diffraction (XRD) and glow discharge spectroscopy (GDS), and a surface scientific approach was conducted by ex-situ scanning electron microscopy (SEM).", "Corrosion resistance was highly improved at 24 mass% Cr without Ni, while exhibiting a useful level of electrical conductivity (e.g. in fuel cells).", "The four types stainless steels used in this study were SUS410 (t = 3.9 mm), SUS430 (t = 1.0 mm), SUS445 (t = 0.1 mm) and 24 mass% Cr-content stainless steel (24CrSS, t = 0.1 mm).", "The chemical compositions (in mass%) of these four types of stainless steels are shown in Table 1.", "The SUS445 stainless steel contains small amounts of aluminum (Al), molybdenum (Mo), titanium (Ti) and niobium (Nb) elements compared to the other stainless steels.", "All the four types of stainless steels contain no Ni and contain different Cr amounts.", "The electrochemical measurements were carried out at room temperature using the Electrochemical Analyzer Model 802B (ALS/[H] CH Instruments).", "To evaluate the corrosion resistance of the SUS410, SUS430, SUS445 and 24CrSS stainless steels, corrosion behavior was studied using an electrochemical three-electrode glass cell.", "The working, counter, and reference electrodes were the stainless steel specimen, a platinum coil, and an Ag/Ag2SO4 electrode, respectively.", "All measured electrode potentials were converted from Ag/Ag2SO4 to the SHE in this study.", "A 0.5 mol dm-3 H2SO4 solution was used as the electrolyte.", "The stainless steels were washed with acetone and distilled water for 5 min during sonication prior to the linear sweep voltammetry (LSV) measurement.", "A 30-min Ar gas bubbling was also performed.", "Next, the cathodic treatment for 1 min was conducted at a potential of -0.47 V vs. SHE, then kept for 5 min at the rest potential in the cell.", "During this holding time, Ar bubbles nucleated on the surface of the sample to remove generated H2 gas.", "The potential sweep was conducted from the rest potential to 1.1 V vs. SHE at a scan rate of 0.33 mV s-1 [15].", "After the electrochemical measurement, the samples were carefully removed from the cell and cleaned with ethanol.", "The corrosion resistance of all the samples was evaluated based on the Japanese Industrial Standards (JIS) G0579: 2007 measurement method [33].", "To evaluate the depth profiles of the elements on the surface of the four types of stainless steels, glow discharge optical emission spectroscopy (GDS) was carried out using a Horiba GD-Profiler 2 instrument.", "The measured elements included Fe, C, N, Cr, Al, etc.", "In this study, the Cr contents of the four types of stainless steels is the main data to be analyzed.", "The X-ray diffraction (XRD) experiments were performed by an XRD-6100 made by Shimadzu.", "The measurements were carried out in reflection geometry using Cu Kα radiation (λ = 1.5406 Å) generated at 40 kV and 30 mA; 2θ was scanned from 20° to 110° at a rate of 2°·min−1.", "Scanning electron microscopy (SEM) (JSM-6060A, JEOL Ltd.) was used to observe the surface morphology of the stainless steels before and after the LSV measurements were stopped at the peak of the polarization curves.", "Micrographs of were obtained at 15 kV.", "Finally, the electrical conductivity of the bipolar plate is very important, so the electrical conductivity of the four types of stainless steels was measured by a Mitsubishi Chemical Loresta HP (MCP-T410) electrometer using a four-point probe resistivity technique.", "As a standard measurement, four-point probe characterization is used to measure the electrical properties of solids and thin films [24, 34, 35, 36].", "The resistivity reported here for each stainless steel of this study is the average data of five measurements in different locations.", "Fig. 1 shows the GDS depth profiles of the SUS410, SUS430, SUS445 and 24CrSS stainless steels.", "The main compositions of these four types of stainless steels are Fe and Cr; a small amount of C was also detected.", "The GDS depth profiles of the SUS410, SUS430, SUS445 and 24CrSS stainless steels show different Cr contents in the bulk.", "The depth profiles of the Cr element of the SUS410, SUS430, SUS445 and 24CrSS samples are shown in Fig. 2.", "The GDS results show that the Cr contents are increased in the order of SUS410, SUS430, SUS445 and 24CrSS stainless steels.", "The 24CrSS stainless steel contains the highest content of corrosion-resisting Cr.", "Fig. 3 shows the XRD patterns of the SUS410, SUS430, SUS445, and 24CrSS stainless steels.", "The XRD patterns of all the stainless steels show the same diffraction peaks at 44.66°, 64.82°, 82.13° and 98.18°, relative to α-Fe.", "The peak intensity ratio for (110), (200), (211), and (220) are different at each stainless steel, because of the different rolling directions when they are produced [37].", "The XRD results also show that the four types of stainless steels have the ferritic structure, which is a body-centered cubic (BCC) structure.", "The polarization curves of the SUS410, SUS430, SUS445 and 24CrSS stainless steels in the Ar-saturated 0.5 mol dm-3 H2SO4 electrolyte are shown in Fig. 4.", "For the SUS410, SUS430 and SUS445 stainless steels, although the polarization characteristics for these three specimens are almost similar in shape, the polarization curve of the SUS445 stainless steel shows the same current density as SUS316 stainless steel in the potential region of 0.25–0.6 V vs. SHE [15].", "The formation of passive-current peak shifts due to a negative potential by increasing the Cr content, in increasing order; SUS410, SUS430, and SUS445.", "Lower current densities are observed at higher Cr-content levels.", "The SUS410 stainless steel shows the highest current densities due to it having the lowest Cr content.", "In the case of the 24CrSS stainless steel, the onset potential shifts toward the positive direction, and there is no active current peak compared to SUS410, SUS430 and SUS445 [24].", "This can be explained by the existence of a stable passivation layer even after the cathodic treatment of 24CrSS.", "The current densities of the 24CrSS sample also decreased indicating a better corrosion resistance of the 24CrSS stainless steel.", "Because the stainless steel contains a higher Cr content, the best corrosion resistance was observed [27].", "To investigate how the corrosion occurred on the surface of the four types of stainless steels, SEM measurements were carried out to observe the surface morphology of the four types of stainless steels.", "Fig. 5 shows the SEM images of the four types of stainless steels before and after the LSV measurements stopped at the peak (Fig. 4) and the end of the polarization curves.", "Compared to before the LSV measurement (Fig. 5(A-1)), the LSV measurement of SUS410 after the rest potential shifts to -0.02 V vs. SHE (Fig. 5(A-2)), we can clearly see that the grain of SUS410 was corroded, so the corrosion occurred on the surface of the SUS410 sample during the active region.", "For the SUS430 stainless steel, compared to before the LSV measurement (Fig. 5(B-1)), the LSV measurement after the rest potential to -0.13 V vs. SHE (Fig. 5(B-2)), the surface of the SUS430 stainless steel was corroded during the active region and the grain boundaries became visible due to the chemical attack of the acid solution.", "Comparing the SEM images of SUS445 before (Fig. 5(C-1)) and after the LSV measurement from the rest potential to -0.22 V vs. SHE (Fig. 5(C-2)), after the LSV measurement, the grain boundaries can still be observed, but not as clearly as that of SUS430 (Fig. 5(B-2)).", "The surface of the SUS445 stainless steel was also corroded during the active region.", "The corrosion damage to SUS445 was less than that of the SUS430 stainless steel.", "At the end of the LSV measurements (Fig. 5(A-3), (B-3), (C-3)), these three stainless steels were significantly corroded.", "As for the 24CrSS stainless steel, with better corrosion resistance, there was no change to the surface morphology before (Fig. 5(D-1)) and after the LSV measurements (Fig. 5(D-2), (D-3)).", "No apparent corrosion occurred on the 24CrSS stainless steel.", "The conjectured reason is that the 24CrSS stainless steel contains the highest content of the corrosion-resisting Cr element, and a passive layer already existed on the surface of the 24CrSS stainless steel before the experiments, thus the polarization curve of the 24CrSS stainless steel has no active region.", "It is important for the application of fuel cells to evaluate the materials' electrical conductivity.", "Therefore, electrical conductivities of SUS410, SUS430, SUS445 and 24CrSS were measured using a Mitsubishi Chemical “Loresta HP” (MCP-T410) electrometer by the four-point probe resistivity technique, and the results are listed in Table 2.", "The volume resistivity of the four types of stainless steels decreased one order of magnitude compared to that of the graphite carbon.", "The 0.1 mm-thick 24CrSS stainless steel (with a high corrosion resistance) showed the same electrical conductivity as that of the 0.1 mm thick SUS445 stainless steel.", "Therefore, we conjecture that it can be used as a bipolar plate, replacing the current graphite carbon.", "In this study, the corrosion behavior of the four types of ferritic stainless steels with different Cr contents have been experimentally investigated to study the effect of the Cr content on the corrosion resistance of ferritic stainless steels in a sulfuric acid environment.", "The results revealed the following:", "The ferritic stainless steel containing a higher amount of Cr (24 mass%) showed the best corrosion resistance based on its stable passivation layer.", "Based on the SEM images, no corrosion occurred on the 24CrSS stainless steel after the LSV measurement from the rest potential to 0.26 V vs. SHE due to its having the highest Cr content of the stainless steels tested in the sulfuric acid solution.", "Yang Yu: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.", "Sayoko Shironita: Analyzed and interpreted the data; Wrote the paper.", "Kenichi Souma: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.", "Minoru Umeda: Conceived and designed the experiments; Analyzed and interpreted the data.", "This work was supported by the Cross-ministerial Strategic Innovation Promotion Program (SIP), Cabinet Office, Government of Japan.", "This work was supported by JSPS KAKENHI Grant Number JP16K06770 and JP18K19127.", "The authors declare no conflict of interest.", "No additional information is available for this paper." ]
[]
Long-Fiber Carbon Nanotubes Replicate Asbestos-Induced Mesothelioma with Disruption of the Tumor Suppressor Gene Cdkn2a (Ink4a/Arf)
Mesothelioma is a fatal tumor of the pleura and is strongly associated with asbestos exposure. The molecular mechanisms underlying the long latency period of mesothelioma and driving carcinogenesis are unknown. Moreover, late diagnosis means that mesothelioma research is commonly focused on end-stage disease. Although disruption of the CDKN2A (INK4A/ARF) locus has been reported in end-stage disease, information is lacking on the status of this key tumor suppressor gene in pleural lesions preceding mesothelioma. Manufactured carbon nanotubes (CNTs) are similar to asbestos in terms of their fibrous shape and biopersistent properties and thus may pose an asbestos-like inhalation hazard. Here we show that instillation of either long CNTs or long asbestos fibers into the pleural cavity of mice induces mesothelioma that exhibits common key pro-oncogenic molecular events throughout the latency period of disease progression. Sustained activation of pro-oncogenic signaling pathways, increased proliferation, and oxidative DNA damage form a common molecular signature of long-CNT- and long-asbestos-fiber-induced pathology. We show that hypermethylation of p16/Ink4a and p19/Arf in CNT- and asbestos-induced inflammatory lesions precedes mesothelioma; this results in silencing of Cdkn2a (Ink4a/Arf) and loss of p16 and p19 protein, consistent with epigenetic alterations playing a gatekeeper role in cancer. In end-stage mesothelioma, silencing of p16/Ink4a is sustained and deletion of p19/Arf is detected, recapitulating human disease. This study addresses the long-standing question of which early molecular changes drive carcinogenesis during the long latency period of mesothelioma development and shows that CNT and asbestos pose a similar health hazard. Manufactured carbon nanotubes (CNTs) show structural similarities to asbestos, strongly associated with the fatal tumor mesothelioma. Chernova et al. show that CNTs and asbestos indeed pose similar health hazards and address the long-standing question of which early molecular changes drive carcinogenesis during mesothelioma's long latency period.
[ "AGRI", "BIOC" ]
[ "CDKN2A", "asbestos", "carbon nanotubes", "epigenetics", "hypermethylation", "mesothelioma", "toxicity", "tumor suppressor genes" ]
[ "1100", "1300" ]
[ "Malignant mesothelioma is an aggressive, incurable tumor strongly associated with asbestos exposure and with increasing incidence reported worldwide.", "Length-dependent retention of asbestos fibers in the pleural cavity is crucial for disease development, with chronic inflammation playing an important role in carcinogenesis [1, 2].", "The latency period of this disease extends to 40 years, and molecular events leading to malignant transformation are poorly understood.", "Mesothelioma in humans is considered to be a “disease of gene loss,” rather than being associated with driver mutations [3].", "Genetic analyses identified several key genetic alterations in end-stage disease, with most common deletions or mutations in CDKN2A, NF2, and BAP1 genes [4], and suggested that two main pathways, p53/DNA repair and PI3K-AKT, were associated with mesothelioma progression [5].", "Compromised antioxidant response due to mutation of DNA repair genes may also contribute to oncogenesis [6].", "Epigenetic alterations play a gatekeeper role in cancer as they are the earliest observable genetic change [7].", "DNA hypermethylation is commonly associated with tumor suppressor gene silencing.", "Transcriptional inactivation of these genes and the loss of their functions most frequently occur during the early stages of the carcinogenesis and at the pre-cancerous stages of tumor [7, 8].", "Although disruption of the CDKN2A locus, including p16/INK4A silencing, has been reported in end-stage disease [4, 9], the key molecular events that occur during the long latency period of mesothelioma are unknown.", "Due to their high aspect ratio and biopersistent properties, carbon nanotubes (CNTs) may pose an asbestos-like inhalation hazard [10–12].", "Several thousand tons of CNTs are produced each year, and these new compounds have many commercial and medical applications, including their incorporation into sports equipment, computers, and building materials.", "In vivo studies have indicated that CNTs can cause sustained inflammation and fibrosis of the pleura [1] and can also induce tumor development; however, this has only been shown in genetically susceptible or peritoneally exposed rodent models [12–15].", "Data comparing prolonged pleural exposure of wild-type animals to occupationally relevant doses of CNTs or asbestos is lacking, and the molecular mechanisms underlying fiber-induced carcinogenesis have not been explored [16].", "Here we investigate the effect of instillation of long CNTs and long asbestos into the pleural cavity of mice, the major site of tumor development in humans.", "We show that long CNTs and long asbestos induce mesothelioma with deletion of p19/Arf and silencing of p16/Ink4a, highlighting that epigenetic alterations play a gatekeeper role in mesothelioma.", "Importantly, this study identifies, for the first time, key molecular events underlying progression of long-CNT-induced inflammatory lesions to malignant mesothelioma and addresses the long-standing question of which molecular changes drive carcinogenesis during the latency period of asbestos-induced mesothelioma.", "In three parallel studies, we exposed mice to low, occupationally relevant doses of CNTs, consecutively reducing the dose and increasing exposure time: 5 μg/mouse for 1–12 weeks; 2.5 μg for 1 year; and 1 μg, 0.5 μg, and 0.2 μg for up to 20 months.", "In each study, a comparator group of animals was subjected to 25 μg amosite asbestos fiber exposure.", "Long (pathogenic) and short (not associated with disease) fiber amosite asbestos (LFA and SFA, respectively; [17, 18]) and long and short CNTs (LNTs and SNTs, respectively) were injected into the pleural cavity of mice, the major site of mesothelioma development (Figure S1 and STAR Methods), and the responses along the pleura were assessed at time points up to 20 months post-injection.", "This approach reflects the localization of fibers in the pleural space observed after translocation of fibers from the lungs [19].", "Direct instillation of long, but not short, asbestos and CNTs into the pleural cavity of mice resulted in the development and marked progression of inflammatory lesions along the pleura (Figure 1A).", "The cellular profile of the lesions, comprising mesothelial cells and stromal cells as assessed by immunostaining, reflected the transition from acute to chronic inflammation [20] and was similar for both LFA and LNTs at 1 week, 12 weeks, and 6 months post-injection (Figures 1B and 2A).", "mRNA array analysis (GEO: GSE51636) showed a common pattern of gene expression changes in both LFA- and LNT-induced lesions.", "When analyzed using hierarchical clustering, the samples from LFA- and LNT-exposed mice clustered together, whereas vehicle control (VC), SFA, and SNT groups formed a separate cluster (Figure 1C).", "The major network identified was associated with the inflammatory response (Table S1).", "To examine which signaling pathways were activated in CNT-exposed tissues, we determined the status of 64 kinases in pleurae from fiber-exposed and control animals.", "Antibody-based array analysis showed activation of pro-oncogenic signaling pathways, including Src family kinases, Akt, mTOR, ERK1/2, and STAT3, that was sustained in the pleurae of animals exposed to long, but not short, fibers (Figure S3).", "Acute and sustained kinase activation in both LFA- and LNT-exposed pleurae was strikingly similar, both in terms of the specific pathways identified and in their degree of activation (Figure 1D).", "Importantly, stimulation of these pathways was also observed in human mesothelioma tumor tissue from five patients compared to normal primary mesothelial cells (Figures 1E and 1F).", "Immunostaining of mesothelioma patient tissue demonstrated positive staining for the mesothelial marker HBME-1 and phosphorylated mTOR, 4EBP1, and Src in mesothelioma cells, as well as phospho-ERK1/2 in mesothelioma cells and fibroblasts, while phospho-STAT3 was predominant in lymphocytes and fibroblasts (Figure 1G).", "Similar to that observed in mesothelioma patient samples, specific signaling pathways were activated in different cell types within LFA- and LNT-induced lesions.", "Thus, activation of ERK was restricted to the mesothelial cells overlaying the inflammatory lesions; Src, mTOR, and Akt were stimulated in both mesothelial cells and stroma, whereas STAT3 activation was most apparent in the stroma (Figure S4).", "STAT3 expression is known to link inflammation and cancer [21]; therefore, to dissect the role of Stat3 and related genes, we isolated mRNA from specific areas of the lesions by laser microdissection (Figure 2B), thus enabling quantification of gene expression to be correlated to different cell types (mesothelial cells and stroma).", "Stat3 was upregulated (>3-fold) in both the mesothelial layer and stroma isolated from animals exposed to either LFA or LNTs, and its expression correlated with mRNA levels of IL-6, a well-characterized inducer of STAT3 in the IL-6/JAK2/STAT3 cascade [22, 23].", "Signaling via the PI3K/mTOR axis is also important in asbestos-induced carcinogenesis [24]; consistent with this, the Pik3cg gene was upregulated in both LFA- and LNT-treated mice (Figure 2B).", "At 6 months post-injection, both LFA- and LNT-induced lesions expanded to become contiguous with the surface of the chest wall and diaphragm (Figure 3A), containing zones of dense fibrous stroma, active fibroblasts, and scattered mononuclear cells.", "Again, a distinct pattern of signaling pathway activation was common to both LFA- and LNT-induced lesions (Figure 2C).", "To assess the extent of proliferation resulting from sustained aberrant signaling, we examined expression of the proliferation marker Ki-67 and the mitotic marker p-Histone H3.", "Increased phosphorylation of Histone H3 is also associated with chromatin accessibility and forms part of an epigenetic regulatory mechanism required for malignant transformation [25, 26].", "A sustained increase in proliferating cells was seen throughout both LFA- and LNT-induced lesions (Figure 3B).", "Oxidative DNA damage is often characteristic of chronic inflammation [27] and facilitates epigenetic modifications, thereby promoting carcinogenesis [28].", "Importantly, the level of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of oxidative stress and genotoxicity, progressively increased to a similar degree in DNA samples from both LFA- and LNT-induced lesions (Figure 3C).", "To explore the carcinogenic potential of long fibers in the pleura, we examined fiber-induced pathology at later time points (12–20 months) following exposure to LNTs or LFA.", "All fiber-exposed animals displayed advanced pleural lesions with mesothelial hyperplasia, fibrosis, and chronic inflammation (Figures 4A and 5A).", "In 10%–25% of animals across three independent studies, LNT-induced lesions progressed to pleural mesothelioma (1/4 wild-type animals exposed to 2.5 μg LNTs for 1 year; 1/4 animals exposed to 1 μg LNTs for 18 months, 1/5 animals exposed to 0.5 μg LNTs, and 1/12 animals exposed to 0.2 μg LNTs for 20 months) (Figures 4A, S5, and S7).", "Out of 32 animals exposed to asbestos (25 μg or 50 μg) for 18–20 months, three mice developed mesothelioma (Figure S6).", "This incidence of mesothelioma is consistent with the 15%–37% (overall or high levels of exposure, respectively) reported in humans exposed to asbestos [29].", "Both LNT- and LFA-induced tumors, involving the chest wall, diaphragm, and pericardium, stained positive for the mesothelial markers pan-cytokeratin and WT1 and exhibited histopathology consistent with malignant mesothelioma [30] (Figures 4B, S6A, and S7A).", "Aberrant signaling pathway activation, observed in earlier lesions (Figures 2C and S4), was sustained in the LNT-induced tumors (Figure S5C).", "To explore the molecular events underlying progression of LNT- and LFA-induced inflammatory lesions to mesothelioma, was examined the status of the tumor suppressor gene Cdkn2a and its products p16 and p19, known to be disrupted in asbestos-induced mesothelioma in humans [31, 32] (Figures 4B–4E, S6, and S7).", "Large areas of LNT-induced tumor (#610) or LFA-induced tumor (#398), which were predominantly negative for p16 or p19 protein expression, were micro-dissected and examined for p16Ink4a and p19Arf status (Figure 4C).", "In LNT-induced tumors, relative quantification of gene copy number [33] confirmed loss of the p19Arf locus in the p19-negative areas, as evidenced by ∼60% reduction in p19Arf genomic DNA (gDNA) compared to controls (Figure 4D).", "Loss of p16Ink4a, which is frequently co-deleted with p19Arf [34], was not detected at this stage (Figures 4D), suggesting that allelic deletion of p19Arf is an early event in LNT-induced carcinogenesis and indicating an alternative mechanism for p16 protein loss.", "Similarly, LNT tumor (#461) induced by a lower fiber dose (1 μg versus 2.5 μg) displayed loss of p16 and p19 protein expression, a reduction in Cdkn2a mRNA, and allelic loss of p19Arf (Figure S7).", "In LFA-induced mesothelioma, loss of p16 and p19 protein was evident by a patchy pattern of immunostaining (Figure 4E) and reduced Cdkn2a mRNA levels (Figure S6B), although no reduction in p19Arf gene copy number was detected at this stage (Figure S6C).", "The molecular changes that occur during the latency period of mesothelioma and drive the transition from pre-neoplastic to neoplastic stage of disease are largely unknown.", "To explore whether disruption of Cdkn2a occurred prior to tumor development, we examined the status of Cdkn2a in laser-dissected mesothelial cells from advanced inflammatory lesions.", "LNT-induced chronic inflammatory lesions from animals that did not develop tumors at the 1 year study end point (Figure 5A) displayed no reduction in p16Ink4a or p19Arf gene copy number (Figure 5D); however, mRNA levels were reduced (data not shown), and both p16 and p19 protein expression was absent in the majority of mesothelial cells in LNT-induced lesions (Figures 5B and 5C).", "Consistent with loss of the CDKN2A-encoded proteins p16/p14 in human mesothelioma, loss of p16 and p19 protein expression was also evident in advanced LFA-induced lesions from animals that did not develop mesothelioma at the 1 year study end point (Figures 5B and 5C).", "No loss of NF2-encoded Merlin expression was detected in either LFA- or LNT-induced tumors (Figure 5E).", "Mesothelial expression of Merlin in the LFA- and LNT-induced inflammatory lesions was not altered (data not shown).", "DNA hypermethylation is frequently associated with tumor suppressor gene transcriptional inactivation.", "Importantly, loss of function often occurs during the early stages of carcinogenesis and predominately at the pre-cancerous stage [7, 8].", "To explore possible epigenetic mechanisms of fiber-induced carcinogenesis, we examined gDNA from animals with advanced LNT-induced inflammatory lesions and inflammatory lesions and/or tumors from animals with LNT-induced mesothelioma (#610 and #461), as well as inflammatory lesions and/or tumors from animals with LFA-induced mesothelioma (#398), for p16Ink4a or p19Arf methylation status.", "Bisulphite sequencing confirmed hypermethylation of CpG islands in p16Ink4a and p19Arf (located in exon 1α and the 5′ region flanking exon 1β, respectively) in mesothelial cells in advanced LNT- and LFA-induced lesions, as well as in LFA- and LNT-induced tumors, compared with VC (Figures 6 and S7E).", "Here, we show for the first time that common molecular events underlie the development of LFA- and LNT-induced pleural lesions that progress to mesothelioma (Figure 7).", "Aberrant cell signaling was detected as early as 1 week post-instillation and sustained during the entire time of disease progression, including end-stage disease, in mice.", "Importantly, this pattern of pro-oncogenic signaling was strikingly similar in mesothelioma patients.", "The potential role of the stromal component of human mesothelioma in tumor progression has been highlighted previously [35, 36].", "In this regard, we now show that sustained activation of pro-oncogenic signaling pathways in fiber-induced lesions occurs largely in non-mesothelial (stromal) cells as fiber-induced lesions progress.", "Our data therefore suggest that proliferation of the mesothelial cell layer and the presence of “reactive” (changed morphology) mesothelial cells within the lesions occurs as a consequence of the crosstalk between stromal cells with activated pro-oncogenic pathways and target mesothelial cells.", "Inflammatory signals cause alterations in the cellular epigenetic program and induce hypermethylation [37].", "The presence of oxidative DNA damage could also contribute to pro-oncogenic events within the microenvironment, favoring aberrant DNA methylation in target cells [38], with hypermethylation of Cdkn2a (Ink4a/Arf) in LNT- and LFA-induced lesions prior to tumor development leading to allelic loss of p19Arf, as has been suggested in other cancers [17].", "Consistent with a role for hypermethylation, DNMT3a and DNMT3b mRNA levels were increased in mesothelial cells from LFA- and LNT-induced tumors (data not shown).", "Alteration of the CDKN2A locus in human malignant mesothelioma has been reported with inactivation of p16 in more than a half of mesothelioma patients [9] and, in more recent studies, with hypermethylation and silencing of p19 in 44% of patients [39].", "Significantly, we discovered that epigenetic silencing of Cdkn2a (Ink4a/Arf) and deletion of p19Arf observed in LNT-induced tumors recapitulates common features of human asbestos-induced mesothelioma (Figure 7).", "Overall, these findings provide important new insights into the early molecular changes that occur during the long latency period between fiber exposure and mesothelioma development and identifies epigenetic and/or genetic disruption of Cdkn2a as a key event in long-fiber-induced malignant transformation (Figure 7).", "Importantly, these findings facilitate the identification of potential biomarkers for earlier detection of asbestos-induced mesothelioma, as well as the development of new therapeutic avenues by which to tackle early-stage disease.", "Immunostaining of both LFA-and LNT-induced mesotheliomas for the tumor suppressor protein BAP1 revealed positive cytoplasmic staining in all tumor cells and positive nuclear staining in ∼10% tumor cells (data not shown).", "In view of the latest findings of separate activities of BAP1 in the nucleus and cytoplasm, as well as a possible requirement for both cytoplasmic and nuclear forms to exert a tumor suppressor function, the molecular involvement of BAP1 in carbon nanofiber-induced carinogenesis will be a focus of our future work.", "The long latency of malignant mesothelioma, with decades-long chronic inflammation accompanied by an aberrant microenvironment and the presence of ROS and oxidative DNA damage, would advocate a multifactorial mechanism of disease development, with a clear contribution via loss of tumor suppressor genes such as p16 and p19, as reported here.", "Overall, the common signature of LFA- and LNT-induced pathology demonstrates that there is a conserved molecular mechanism through which long fibers induce pleural disease, including mesothelioma, and crucially our data place long CNT fibers on the same adverse outcome pathway as asbestos.", "Notably, other nanofibers have been shown to produce length-dependent inflammatory effects in the pleura similar to LNTs [40], suggesting that any respirable long fiber that is biopersistent may pose a similar hazard.", "Given that the increasing manufacture of long CNT fibers raises the potential for human exposure, our findings reinforce the need for caution when using these agents if long-term harm is to be avoided.", "Further information and requests should be directed to and will be fulfilled by the Lead Contact, Marion MacFarlane ([email protected]).", "Eight-week-old female C57BL/6 strain mice (Charles River Laboratories, UK) were used in this study.", "Mice were kept in a maximal group size of five in standard caging with sawdust bedding within a pathogen-free Home Office approved facility.", "Mice were maintained on a normal 12 hr light and dark cycle.", "Prior to treatment, mice were kept for 7 days in the facility to acclimatize.", "The work was carried out by staff holding a valid UK Home Office personal license under a Home Office approved project license.", "Informed consent was obtained from all subjects with Ethical Committee Approval – LREC 08/H0406/226.", "Tissues from 13 patients were analyzed in this study, including 1 female and 12 males aged 45–78 years [41].", "The fiber panel consisted of one sample of short, straight CNT (SNT), one sample of long, straight CNT (LNT), short-fiber amosite asbestos (SFA) and long-fiber amosite asbestos (LFA).", "The SNT sample, produced by the catalytic vapor discharge method (CVD), was purchased commercially (Nanostructured & Amorphous Materials, TX, USA).", "The LNT sample was produced in an academic research laboratory (University of Manchester, Manchester, UK) using the CVD method.", "Mixed-length amosite asbestos enriched for long fibers (LFA), and shortened amosite asbestos [17] were used to link the response to asbestos pathogenicity.", "Both LFA and SFA were created from the same batch of South African amosite [18] obtained from the Manville Corporation, United States.", "SFA was prepared by grinding long fibers in a ceramic ball mill, and the resulting fiber preparation sedimented in water.", "Physical characteristics (diameter and length) were measured from scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of the dispersed fiber samples, as previously described by Poland et al. [11] (Figure S1).", "Trace soluble metal contaminants previously tested and reported by Poland et al. [11] were low and, thus, not considered to play a role in these studies.", "Injection directly into the pleural space without perforating the lungs was enabled by the addition of a sleeve over the tip of the 27-Gauge needle, which prevented the needle from passing through the pleural space into the lungs [1].", "Mice were randomly allocated to the treatments and received injection of one of the following: vehicle control (VC), SFA, LFA, SNT or LNT.", "For 1 and 12 weeks exposure, CNT samples were injected into the pleural cavity at a dose of 5 μg/mouse, n = 4 in each treatment group.", "For 6 months and 1year exposure, the treatments were as follows: VC, LFA and LNT, with n = 4 in each treatment group, at a dose of 2.5 μg/mouse (100 μL) of LNT.", "For prolonged (up to 20 months) exposure, mice were injected with 1 μg/mouse of LNT (n = 4), 0.5 μg/mouse of LNT (n = 5) or 0.2 μg/mouse of LNT (n = 12).", "Asbestos fiber samples were administered at a dose of 25 μg/mouse (100 μL) (n = 4 for short-term studies and n = 16, prolonged exposure) or at 50 μg/mouse (n = 16, prolonged exposure).", "0.5% BSA/saline (100 μL) was injected into mice as a vehicle control (VC).", "After 1 week, 12 weeks, 6 months, 1 year and 18-20 months, mice were humanely killed and tissues were collected for further examination.", "Animal IDs, indicated in the Figures, were as follows: #610 - LNT-exposed mouse that developed mesothelioma at 1 year study completion point; ## 621, 622, 623 - LNT-exposed mice that did not have a tumor at 1 year study completion point; ## 612, 613, 614, 615 - VC; #398 - LFA-exposed mouse; #461 - low-dose LNT-exposed mouse that developed mesothelioma after 17 months exposure; ## 616-620 - LFA-exposed mice with inflammatory lesions.", "All experiments were reproduced in at least three biological repeats.", "For in vivo experiments, sample sizes were chosen on the basis of prior experiments which have elicited significant results with a similar number of mice.", "With the exception of histological analysis, data collection and analyses were not performed blind.", "Data were considered statistically significantly different at p < 0.05.", "No data were excluded from any dataset.", "Genomic DNA from selected areas of tumor or from mesothelial cells in the inflammatory lesions was isolated using GeneJET Genomic DNA Purification Kit (Fisher Scientific, Loughborough, UK) and was bisulphite converted and recovered with EZ DNA Methylation-Direct Kit (Cambridge Bioscience, Cambridge UK), as recommended by manufacturer.", "The PCR to amplify CpG islands in the genes of interest was performed on a 3Prime Thermal Cycler (Bibby Scientific Limited, Stone, UK) using EpiMark Hot Start Taq DNA Polymerase (New England Biolabs Ltd, Herts, UK).", "The PCR conditions were as follows: 95°C for 3 min, then 40 cycles of 95°C for 30 s, 55°C for 30 s and 67°C for 2 min.", "A final incubation at 67°C for 10 min concluded the PCR.", "Positive control (Universal Methylated Mouse DNA Standard, Millipore Ltd, Watford, UK) and control (normal mesothelial tissue from VC) samples were included in each PCR reaction.", "PCR products were verified by gel electrophoresis, and a small aliquot of the PCR reaction was used with the TOPO-TA cloning system (Thermo Scientific, Paisley, UK) as suggested by the manufacturer.", "Clones were picked from Lysogeny Broth-Ampicillin cultures, and were then screened using restriction analysis.", "Positive clones were sequenced using M13 primer by the University of Leicester DNA Sequencing Facility.", "25-100 clones were analyzed for each described experimental condition.", "The lower-right posterior portion of the chest wall and half of the diaphragm were carefully removed from the mice.", "The tissue was washed in ice-cold saline, fixed in 10% formalin for 4 hr and transferred to 70% ethanol.", "Samples were embedded in paraffin, sectioned, and used for H&E, immunostaining and PALM microdissection.", "The pleurae were dissected from the surface of the chest wall and snap frozen for kinome profiling.", "The other half of the diaphragm was snap frozen for RNA extraction and PALM microdissection.", "Kinome profiling was performed using Phospho-Kinase Array Kit according to manufacturer’s protocol (R&D Systems, Oxford, UK).", "Pleural tissues from fiber-exposed and control mice were collected by dissection, homogenized and lysed, the lysates from the pleurae of 4 animals were pooled for each treatment group.", "Lysates, cleared by centrifugation, were loaded onto the provided membranes pre-coated with capture antibodies, and the presence of bound phospho-proteins was determined by western blotting with a mixture of detection antibodies.", "The signal intensities for kinase phosphorylation were determined in duplicate by densitometry and normalized on the provided positive control.", "The status of kinase phosphorylation in the pleurae of fiber-exposed animals is reported relative to the vehicle control.", "Total RNA from control and fiber-exposed animals (n = 4) was extracted by TRIzol (Fisher Scientific, Loughborough, UK) and then used for labeling and hybridization.", "Hybridization to 60K whole mouse genome microarray gene expression chips was conducted following manufacturer’s protocol (Agilent Technologies, Berkshire, UK).", "Briefly, total RNA from control and fiber-exposed mice, 12 weeks post-injection, was used for labeling and hybridization.", "RNA samples were Cy3-labeled using Agilent Low Input Quick Amp 1-color Labeling Kit (Agilent Technologies, Berkshire, UK).", "The level of dye incorporation was evaluated using a spectrophotometer (Nanodrop ND1000, LabTech).", "Labeled RNA was then fragmented in the appropriate buffer (Agilent Technologies, Berkshire, UK) for 30 min at 60°C before dilution (v/v) in hybridization buffer.", "Hybridization to 60K high-density oligonucleotide microarray slides was performed in a microarray hybridization oven (Agilent Technologies, Berkshire, UK) overnight at 65°C.", "Following hybridization, the slides were rinsed in gene expression wash buffers 1 and 2 and immediately scanned using a DNA Microarray Scanner (Model G2505C, Agilent Technologies, Berkshire, UK).", "Chest wall or diaphragm sections (5 μm) were deparaffinized with xylene and rehydrated.", "Antigen retrieval was carried out by incubating the slides in citrate buffer (pH 6.0) at 95°C for 15 min.", "Sections were blocked with 10% goat serum for 30 min at room temperature.", "Primary antibodies were diluted in 1% goat serum/0.1% BSA/PBS.", "Sections were incubated with primary antibodies (KEY RESOURCE TABLE) overnight at 4°C.", "Sections were washed with Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated with 3% H2O2 for 10 min at room temperature to block endogenous peroxidase activity.", "Sections were washed before incubation with biotin-labeled secondary antibodies (anti-Rabbit: Abcam, pre-diluted; anti-Mouse: DAKO LSAB2 System-HRP, anti-Rat: Invitrogen Histostain Kit pre-diluted) for 30 min at room temperature.", "Staining was visualized using a HRP/DAB detection system Abcam (Cambridge, UK) or Histostain-Plus detection System (Life Technologies).", "Control IHC experiments (data not shown) were performed without primary antibody.", "All sections were counterstained with Gill’s hematoxylin and mounted for digital slide scanning using a Hamamatsu slide scanner (NanoZoomer-XR Digital slide scanner C12000-01; Welwyn Garden City, Hertfordshire, UK).", "An ImageJ Color Balance Plugin, set with the same scaling factor for all images, was applied to all scanned images to normalize scanner background.", "The proportion of each cell type in the lesions was determined by counting the number of positively stained cells in 5 random fields of view and dividing by the total number of cells (between 700-1000 cells per cell-marker per treatment group were counted).", "Quantification of Ki-67 and phospho-Histone H3-positive cells was carried out by counting the number of positively stained cells in 5 random fields of view and dividing by the total number of cells (between 700-1000 cells per animal were counted, n = 3 per group).", "Data presented as mean ± SD.", "Statistical significance of data was estimated using two-tailed Student’s t test.", "Collection of tissues for RNA from selected areas of normal diaphragms (VC) and fiber-induced lesions (LFA, LNT) and collection of tissues for genomic DNA (gDNA) from tumor areas, predominantly positively or negatively stained for p19 or p16 protein, and from control tissues was performed by contact-free laser microdissection using the PALM Robot-MicroBeam system (P.A.L.M. Microlaser Technologies AG, Bernried, Germany), Motorised Zeiss Observer Z.1 inverted microscope (Zeiss, Germany) with high precision XY stage, pulsed UV laser and Robomover (PALM RoboSoftware, P.A.L.M. Microlaser Technologies AG, Germany).", "Laser catapulting was used for non-contact capture of excised samples in microcentrifuge tubes.", "Total RNA was isolated by using TRIzol (Fisher Scientific, Loughborough, UK).", "First strand cDNA synthesis was carried out using Superscript III (Fisher Scientific, Loughborough, UK).", "PCR primers were selected using the Primer Express v3.0.1 Software program (Fisher Scientific, Loughborough, UK).", "Primer sequences are shown in Table S4.", "Primers were designed to cross exon-exon boundaries and the concentration optimized (300 - 900 nM) to ensure that the efficiency of the target gene amplification and the efficiency of the endogenous reference amplification are approximately equal.", "Real-time PCR was performed using SYBR Green PCR Master Mix, primers, and 10 ng of reverse-transcribed cDNA in the ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA).", "The thermal-cycler protocol was: stage 1, 50°C for 2 min; stage 2, 95°C for 10 min; and stage 3, 40 cycles at 95°C for 15 s and 60°C for 1 min.", "Each sample was run in triplicate.", "The CT values for the target amplicon and endogenous control β2-microglobulin were determined for each sample.", "Quantification was performed using the comparative CT method ΔΔCT).", "Data presented as mean ± SD (n = 4).", "Statistical significance was assessed as p < 0.05 using two-tailed Student’s t test.", "Proteins were extracted from snap frozen freshly-resected mesothelioma tumor samples using lysis buffer (0.5% NP-40, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 10 μg/μL leupeptin, and 10 μg/μL aprotinin) and brief sonication and run on SDS-PAGE (20 μg protein).", "Proteins were transferred onto nitrocellulose membranes (Bio-Rad laboratories, Hemel Hempstead, UK) using electrophoresis.", "Membranes were pre-incubated with 5% skimmed milk in TBS-T.", "After incubation with primary and secondary antibodies, bands were detected by enhanced chemiluminescence (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and visualized by exposure to X-ray films (Hyperfilm ECL; Amersham Biosciences, Chalfont St Giles, UK).", "Primary antibodies were from the following sources:anti-STAT3, anti-phospho-Akt (Ser 473), anti-Akt, anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), anti-p44/42 MAPK (ERK1/2), anti-phospho Src (Y418), anti-Src from Cell Signaling Technology, (Buckinghamshire, UK); rabbit anti-phospho-Stat3 (Tyr705) from Abcam (Cambridge, UK), mouse anti-β Actin (loading control) was from Sigma–Aldrich (Gillingham, UK).", "Genomic DNA was isolated from the diaphragms of mice exposed to LFA and LNT for 12 weeks or 6 months using a DNA isolation kit GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Paisley, UK); 8-OHdG was measured using an EpiQuik 8-OHdG DNA Damage Quantification Direct Kit (Colorimetric) (Insight Biotechnology Ltd, Wembley, UK) according to manufacturer instructions and compared to VC (n = 4 per group).", "Briefly, 300 ng genomic DNA was bound to strip wells with high DNA affinity and 8-OHdG was detected using capture and detection antibodies.", "The detected signal was enhanced and then quantified colorimetrically in a microplate reader.", "Quantification was performed by using a standard curve and plotting the OD values versus the amount of positive control at each concentration point.", "Data presented as mean ± SD.", "Statistical significance of the data was estimated using a two-tailed Student’s t test.", "An assay based on the paralog ratio test and real-time PCR was used to determine the DNA copy number in formalin fixed paraffin embedded (FFPE) tissue as described previously [33].", "Genomic DNA (gDNA) was extracted using PureLink Genomic DNA Mini Kit (Fisher Scientific, Loughborough, UK) accordingly to the manufacturer’s instruction, from tumor areas positively and negatively stained for p16 and p19, and control tissues including unaffected tissue (heart) from fiber-exposed mice, diaphragm tissue from VC group, and liver tissue from a C57/Bl6 mouse.", "Commercially available murine gDNA from Swiss-Webster albino (CFW) mice (Promega, Southampton, UK) was also included as a control.", "Briefly, PCRs were designed in which two target sequences were amplified by a pair of primers.", "The first gene in each pair was from a region of a frequent loss or deletion (p16 or p19), whereas the second gene was the “housekeeping” gene, Supervillin, from a different chromosome.", "The Primer Express v3.0.1 Software program (Fisher Scientific, Loughborough, UK), was used to identify primer pairs and to design minor groove-binding hydrolysis probes.", "Primer sequences (Sigma) and TaqMan Probes (Life Technologies) are listed in Table S4.", "Real-time PCRs were performed on a 96-well plate on the ABI 7500 Fast system (Applied Biosystems Foster City, CA).", "Each of 10 μL reactions contained 3.4 μL of gDNA (5 ng), 0.3 μL of each of the two primers required for each locus (20 pmol/L), 0.2 μL of each of the probes required (50 to 200 nmol/L), and 5 μL of Genotyping MasterMix (Applied Biosystems Foster City, CA).", "Master mixes were prepared for each reaction, and the master mixes and DNA samples were transferred to 96-well plates using an electronic multi-channel pipette.", "The PCR conditions were as follows: 2 min at 50°C; 10 min at 95°C; 15 s at 95°C and 60 s at 60°C.", "Fifty cycles were performed.", "CT values were determined using the automatic threshold settings.", "Four samples of gDNA (unaffected heart tissue from the same animal, diaphragm tissue from a VC mouse, liver from C57/BL6 mouse, and commercial gDNA from Swiss-Webster albino (CFW) mice (Promega, Southampton, UK) were used as controls.", "Data were verified to be normally distributed.", "The raw data was uploaded into Agilent’s GeneSpring Software, normalized and fold changes calculated.", "For each group of animals the probes with an absolute 2-fold-change in mRNA expression between VC and fiber-exposed mice were included in subsequent analyses.", "These were subjected to ANOVA unequal variations test with Benjamini-Hochberg corrections.", "Significant 2-fold or more changes (p < 0.05) were subjected to hierarchical clustering with average linkage.", "The clustered heat-map was visualized using GeneSpring.", "The network pathways were identified using Ingenuity Pathways Analysis software (Ingenuity Systems, Redwood City, CA).", "Quantification of Ki-67 and phospho-Histone H3 positive cells was carried out by counting the number of positively stained cells in 5 random fields of view and dividing by the total number of cells (between 700-1000 cells per animal were counted, n = 3 per group).", "Data presented as mean ± SD.", "Statistical significance of data was estimated using two-tailed Student’s t test.", "Quantification of DNA damage was performed by using a standard curve and plotting the OD values versus the amount of positive control at each concentration point.", "Data presented as mean ± SD.", "Statistical significance of the data was estimated using a two-tailed Student’s t test.", "Quantification was performed using the comparative CT method (ΔΔCT).", "Data presented as mean ± SD (n = 4 for each group).", "Statistical significance was assessed as p < 0.05 using two-tailed Student’s t test.", "The ΔCT score was calculated for each of the reactions using the following equation: CT gene 1 − CT gene 2 = ΔCT, where gene 1 is that of common DNA loss and gene 2 is the reference.", "The mean ΔCT from the triplicate reactions was used for subsequent analysis.", "A series of four gDNA samples acted as control for each PCR run.", "The relative quantification of copy number was calculated using the 2-ΔΔCT value.", "Z scores for the controls and predominantly p19 negative tumor areas were generated using the mean and SD of a reference population comprising the four control ΔCT values.", "This was used to determine which samples were outside of the 99% reference range for control, with those outside of this range being considered aneuploid.", "The dataset for mRNA array, the “gene changes in response to asbestos and carbon nanotube exposure in the pleural cavity, measured using microarrays,” reported in this paper is NCBI GEO: GSE51636 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51636).", "T.C. and F.A.M. designed experiments and co-wrote the paper.", "T.C., S. Galavotti, and F.A.M conducted the majority of experiments and prepared the manuscript.", "X.-M.S., I.R.P., S. Grosso, and A.S. conducted experiments and helped with manuscript preparation.", "A.N. and J.B. recruited patients and contributed to discussions.", "C.A.P. and K.D. contributed to experimental design, data interpretation, and writing of the paper.", "P.G., J.Z.-C., D.D., K.M.D., and J.L.Q. performed data analysis.", "M.M., T.C., A.E.W., and M.B. conceived the study, designed experiments, and wrote the manuscript." ]
[ "Long-fiber carbon nanotubes induce malignant mesothelioma with loss of Cdkn2a", "Long-fiber carbon nanotube tumors replicate asbestos-induced mesothelioma in humans", "Cdkn2a hypermethylation during disease latency precedes fiber-induced mesothelioma", "Long-fiber carbon nanotubes pose an asbestos-like hazard" ]
Cytotoxic and antiinflammatory activities of the chemical constituents isolated from Baccharoides schimperi DC
A phytochemical investigation on aerial parts of Baccharoides schimperi DC. (Astraceae), led to the isolation of six secondary metabolites namely spinasterol (1), stigmasterol (2), 3′,4′,5,7-tetrahydroxy-3-methoxyflavone (3), vernolepin (4), vernadolol (5) and 11β,13-dihydrovernodalin (6). Their structures were determined by comprehensive analyses of 1D and 2D NMR spectra and comparison with available literature data. To the best of our knowledge, this is the first report on the isolation of compounds (1–6) from B. schimperi. Isolated compounds were subjected to biological assays to screen cytotoxic and antiinflammatory activities. Among the isolates, compound 4 was the most cytotoxic towards a panel of selected cell lines followed by 6 and 5. Compounds 4–6 also showed antiinflammatory activity in terms of inhibiting NF-kB and iNOS. The activity of 4 was comparable to the activity of parthenolide.
[ "AGRI" ]
[ "Antiinflammatiory activity", "Baccharoides schimperi", "Constituents", "Cytotoxicity" ]
[ "1110" ]
[ "The Vernonieae is one of the major tribes of the biggest family of flowering plant (Asteraceae) with ca. 25,000 species, found worldwide and the huge number of species more than 1000 comes under a single genus Vernonia (Keeley et al., 2007).", "Presently genera of Vernonieae reported from southern Africa includes Baccharoides, Bothriocline, Cyanthillium, Distephanus, Erlangea, Ethulia, Gymnanthemum, Hilliardiella, Oocephala, Orbivestus, Parapolydora, Polydora, Vernonella, Vernoniastrum, with two new genera listed as Namibithamnus and Pseudopegolettia.", "(Robinson et al., 2016).", "The genus Baccharoides has been reported to have about 23 species (Flan, 2013).", "The species now under the genus Baccharoides earlier belonged to Vernonia section Stenigelia (Isawumi et al., 1996).", "Literature survey revealed that one of the species of genus Baccharoides i.e. Baccharoides adoensis var.", "kotschyana Sch.", "Bip.", "Ex Walp.", "has been used in the treatment of gastric ulcers or externally as a wound healing remedy.", "The active principle was found to be the acidic polysaccharide fraction, with pectic arabinogalactan type II structures (Nergard et al., 2004, 2005).", "Baccharoides schimperi DC.", "(Syn.", "Vernonia schimperi DC) belongs to the genus Baccharoides, distributed in the Asir region of Saudi Arabia (Chaudhary, 2000; Flan, 2013).", "To our knowledge there are no reports about the phytochemical investigation of this plant except our previous work reporting acute toxicity and antiinflammatory activity of the ethanolic extract of B. schimperi and its fractions in experimental animals (Ahmed et al., 2015).", "However, antimicrobial activity (Abdel-Sattar et al., 2008), antiprotozoal and cytotoxic activity (Abdel-Sattar et al., 2010; Almehda et al., 2012) and antiinflammatory activity (Abdallah et al., 2014) have been reported for V. schimperi DC.", "The present investigation reports the isolation and structure elucidation of six compounds namely, spinasterol (1), stigmasterol (2), 3′,4′,5,7-tetrahydroxy-3-methoxyflavone (3), vernolepin (4), vernadalol (5) and 11β,13-dihydrovernodalin (6) The cytotoxic activity of compounds (1–6) and antiinflammatory activity of compounds (4–6) are also discussed.", "The 1H NMR, and 13C NMR spectra were recorded on a Bruker Avance spectrometer at 700 and 175 MHz respectively.", "For analytical purposes Thin Layer Chromatography (TLC) plates coated with UV254 fluorescence indicator (Merck) were used.", "Compounds were visualized under UV radiation in CAMAG UV cabinet dual wavelength, 254/366 nm and also by spraying with p-anisaldehyde reagent followed by heating with a heat gun.", "Silica gel 60 (230-400 μm; Merck, Darmstadt) and LiChroprep RP-18 (40–63 μm; Merck, Darmstadt) was utilized for column chromatography (CC).", "The plant material was collected in February, 2013 from the Jabal Shada Al-aala hills, Saudi Arabia.", "The collected plant material was identified by taxonomist and the voucher specimen of plant (# 16012) was deposited at the herbarium of the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.", "The aerial part was dried under shade and grinded to powder form.", "The powdered material (1 kg) was extracted at room temperature in acetone and ethanol 96% (10 L × 3), respectively.", "The extracts were evaporated to dryness at 40 °C under reduced pressure using Buchi Rotavapour which yielded green syrupy mass of 16.2 g and 26.9 g, respectively.", "The acetone extract was partitioned between n-hexane and acetonitrile (pre saturated with each other).", "The fractions obtained were evaporated to dryness at 40 °C under reduced pressure.", "The n-hexane fraction (2.8 g) and acetonitrile fraction (9.9 g) thus obtained were kept in screw capped bottles in refrigerator for further use.", "The n-Hexane fraction 2.6 g was applied for column chromatography.", "The column was packed with silica gel 80 g and first eluted with n-hexane and then mixture of ethyl acetate and n-hexane (ranging from 1:99 to 50:50).", "The fractions obtained were analyzed by using normal phase TLC plates coated with UV254 fluorescence indicator (Merck).", "The TLC solvent system n-hexane/ethyl acetate and acetone/toluene in different ratio were used.", "The fraction showing the same behavior on TLC plates were combined and finally it gave forty fractions labeled as 1 to 40.", "The fraction 26 on crystallization with acetone and fraction 28 on crystallization with methanol afforded compounds 1 (34.5 mg) and 2 (7.1 mg), respectively.", "The acetonitrile fraction 7.0 g was applied for column chromatography.", "The column was packed with silica gel and first eluted with chloroform and then a mixture of methanol and chloroform (ranging from 0.5:99.5 to 5:95).", "Fractions were examined by using normal phase TLC plates as described above and combined to afford twenty six fractions (A-Z).", "Fraction M (221.6 mg) on crystallization with methanol gave the yellow crystalline compound 3 (55.0 mg).", "Fraction I showed one major spot which on crystallization with EtOAc gave compound 4 (197.7 mg) as a colorless crystalline material.", "Fraction O (230.0 mg) again chromatographed over RP 18 column eluted with water and then a mixture of methanol:water ranging from 10:90 to 40:60 gave ten subfractions (I-X).", "Subfraction VI gave compound 5 (29.4 mg) as a colorless sticky material.", "Subfraction IX (131.8, mg) on repeated chromatography gave compound 6, (12.5 mg) as colorless sticky material.", "To determine the cytotoxicity of the isolates (1–6) a cell proliferation assay was performed in a panel of four human cancer cell lines (SK-MEL, KB, BT-549, SK-OV-3) and two mammalian kidney cell lines (LLC-PK1 and VERO).", "The cells were seeded in the wells of 96-well plates (25,000 cells/well) and incubated for 24 h for confluency.", "Test samples at various concentrations were added (25, 8.33, 2.78 μg/mL) and the cells were further incubated for 48 h.", "At the end of incubation, cell viability was determined by a colorimetric assay as described earlier (Al-Rehaily et al., 2014).", "A decrease in cell viability of sample treated cells was calculated in comparison to untreated controls.", "IC50 value (the concentration responsible for 50% decrease in cell viability) was calculated from the concentration response curve.", "Doxorubicin was included as the positive control for cytotoxicity.", "A reporter gene assay was performed in human chondrosarcoma (SW1353) cells to determine the inhibition of NF-κB activity as described earlier (Xu et al., 2015).", "In brief, cells transfected with NF-κB luciferase plasmid construct were plated at a density of 1.25 × 105 cells/well in 96-well plates and incubated for 24 h for confluency.", "Various concentrations of test compounds were added and after incubating for 30 min, phorbol 12-myristate 13-acetate (PMA, 70 ng/mL) was added and cells were further incubated for 6–8 h. Luciferase activity was measured using a Luciferase Assay Kit (Promega, Madison, WI, USA).", "Percent decrease in luciferase activity in sample treated cells was calculated relative to the vehicle treated control cells.", "Parthenolide was included as a positive control.", "The effect of test samples on a control transcription factor Sp-1 was also determined in the same assay to evaluate the general toxicity and specificity towards NF-κB as described earlier (Tabanca et al., 2007).", "The assay for inhibition of iNOS activity was performed in mouse macrophages (RAW264.7) as described earlier (Xu et al., 2015).", "Cells were plated at a density of 50,000 cells/well and incubated for 24 h for confluency.", "Test compounds were added first to the cells and after 30 min of incubation cells were induced with LPS (5 μg/mL) for 24 h.", "The level of nitrite in the cell culture supernatant was measured by using Griess reagent.", "Percent inhibition of nitrite production by the test compounds was calculated in comparison to the vehicle control.", "Cell viability was also measured as described above to determine the cytotoxicity of test samples towards macrophages during the assay.", "Parthenolide was included as a positive control.", "The antioxidant activity in terms of a decrease in cellular oxidative stress was measured in ABAP induced HepG2 cells as described earlier (Xu et al., 2015).", "HepG2 cells were seeded in the wells of a 96-well plate at a density of 60,000 cells/well and incubated for 24 h.", "The confluent cells were washed with PBS and treated with the test compounds diluted in serum-free medium containing 25 μM DCFH-DA for 1 h.", "After removing the medium, the cells were treated with 600 μM ABAP and the plate was immediately placed on a SpectraMax plate reader for kinetic measurement every 5 min for 1 h (37 °C, emission at 538 nm and excitation at 485 nm).", "The percent decrease in oxidative stress was calculated.", "Quercetin was used as positive control.", "The phytochemical investigations on n-hexane and acetonitrile fractions of B. schimperi led to the isolation of six compounds (1–6), shown in Fig. 1.", "Structures of isolated compounds were established by interpretation of their 1D and 2D NMR spectroscopic data as well as by comparison with available values in literature.", "The isolated compound are identified as spinasterol (1) (Ragasa and Lim, 2005), stigmasterol (2) (Pateh et al., 2008), 3′,4′,5,7-tetrahydroxy-3-methoxyflavone (3) (Ozgen et al., 2010), vernolepin (4) (Alqasoumi, 2009), vernadalol (5) (Erasto et al., 2007) and 11β,13-dihydrovernodalin (6) (Martin et al., 2009).", "The NMR (1H and 13C) data of 3 and 4–6 are depicted in Tables 1 and 2 respectively.", "The isolated compounds (1–6) were screened through a series of in-vitro cellular assays directed towards the cytotoxic activity and compounds (4–6) towards the antiinflammatory activity.", "Results of the study are summarized in Tables 3 and 4.", "Sesquiterpene lactones (compounds 4, 5 and 6) were cytotoxic towards all the six cell lines.", "Compound 4 was more cytotoxic than 5 and 6.", "This is evident from the previously reported data (Lee et al., 1971; Chadwick et al., 2013) that sesquiterpene lactones have potential cytotoxicity.", "The compounds with α methylene γ lactone moiety are more toxic as compared to other sesquiterpene lactones.", "Since compound 4 is having the same moiety that's why showing highest activity with lowest IC50 values for cytotoxicity followed by 6 and 5.", "It is reported that hydrogenation of congugative α methylene γ lactone as in 6 suppress the activity of compounds.", "Thus the cytotoxicity result of 4 and 6 are in accordance with previously reported cytotoxic activity results of sesquiterpene lactones having similar type of moieties (Lee et al., 1971).", "Compound 4 has also been reported for cytotoxicity against KB cell culture at conc.", "of 2.0 μg/ml (Kupchan et al., 1969) and antibacterial activity (Jisaka et al., 1993).", "Compounds 4, 5 and 6 were selected for screening antiinflammatory activity in terms of inhibition of NF-kB and iNOS in PMA induced chondrocytes and LPS induced macrophages, respectively.", "All three isolates inhibited NF-kB and iNOS activity induced by inflammatory stimuli (PMA and LPS) indicating an activity towards inflammation.", "Interestingly, compounds 4 and 6 were more active than 5 towards both targets (NF-kB and iNOS).", "A control transcription factor Sp-1 was included in the assay to determine if the inhibition of NF-kB transcription factor was specific.", "Inhibition of Sp-1 is also indicative of general cytotoxicity of the test sample.", "It was noticed that compounds 4 and 6 inhibited Sp-1 at 67 and 70 μM, respectively, which is much higher than the concentration responsible for NF-kB inhibition (2.54 and 7.54 μM).", "Parthenolide, positive control, inhibited NF-kB with IC50 of 2.92 μM and Sp-1 at 46.31 μM.", "The inhibition of iNOS by compound 4 and 6 was more potent than compound 5 with IC50 values of 1.83 and 2.62 μM.", "In the same assay the higher concentrations of 4–6 were toxic to the cells (IC50 21.81–35.71 μM).", "The IC50 values of 4 and 6 for iNOS inhibition were comparable to the IC50 values of parthenolide (2.01 μM).", "The inhibition of NF-kB by compound 4 was also similar to parthenolide.", "None of the isolates showed any antioxidant effect and did not decrease oxidative stress in ABAP induced HepG2 cells.", "Apart from this 4 has been revealed as a competitive antagonism against histamine in guinea pig, a biphasic enhancement/inhibition of coaxial stimulation of guinea pig ileum and an antiaggregating and disaggregating activity against rabbit platelet aggregation induced by arachidonic acid without inhibition of cyclooxygenase or lipoxygenase (Laekeman et al., 1983).", "Compound 5 is reported to have antibacterial and antifungal activity (Erasto et al., 2006), antichistosomal (Jisaka et al., 1992) and cytotoxicity against human colon cancer and endothelial cell line (Abay et al., 2015) and 6 has been characterized for antiplasmodial activity (Kraft et al., 2003).", "Compound 3, showing cytotoxicity against KB, SK-OV-3 and LLC-PK1 as shown in Table 3, has been reported in literature for vasodilatory (Correa-Hernández et al., 2008) and antinociceptive and antiinflammatory activities (Kupeli and Yesilada, 2007).", "Compound 1 showed mild cytotoxicity against KB and LLC-PK1 (Table 3).", "In literature pharmacological activities of 1 include antitumor, antiulcerogenic, anticarcinogenic and antiinflammatory activities (Jeong et al., 2010).", "Compound 2, which is found to be non cytotoxic against the tested cell line, has already been reported for a long range of pharmacological activities (Kaur et al., 2011).", "In conclusion compound 4–6 belonging to sesquiterpene lactones exhibited antiinflammatory activity which was also associated with the cytotoxicity.", "Further work is suggested to their structural modification toward enhancing their antiinflammatory activity and decreases their toxicity." ]
[ "Compounds (1–6) isolated for the first time from B. schimperi.", "Isolates were screened for cytotoxic and antiinflammatory actvity.", "Antiinflammatory activity of 4 was comparable to the activity of parthenolide." ]
Metalorganic vapor-phase epitaxial growth simulation to realize high-quality and high-In-content InGaN alloys
We studied chemical reactions in the metalorganic vapor-phase epitaxial growth of InGaN from the TMGa/TMIn/NH 3 /H 2 /N 2 system. It was found that the InGaN growth has three main pathways: pyrolysis of TMGa:NH 3 adducts; pyrolysis of TMIn; and pyrolysis of NH 3 molecules. The InGaN simulations indicated good agreement with the experiments in temperature-dependences of growth rate and composition by considering atomic In re-evaporation. The simulation result showed that our thinner flow channel method could produce more condensed NH 2 and In-atom concentrations compared with conventional technology. Therefore, we could raise InGaN growth temperature with the same In content by the thinner flow channel. Since the higher growth temperature can realize the higher quality of InGaN, we have succeeded to fabricate InGaN LEDs in the range from green to deep red by using the 5-mm flow channel.
[ "CHEM", "MATE", "PHYS" ]
[ "A1. Computer simulation", "A1. Growth models", "A3. Metalorganic vapor phase epitaxy", "B1. Nitrides", "B2. Semiconducting III-V materials", "B3. Light emitting diodes" ]
[ "1604", "2505", "3104" ]
[ "The bandgap of InGaN varies from 0.67 to 3.42 eV [1,2] as a function of In content.", "It means that InGaN–based LEDs have a potential to cover the spectral range from ultraviolet to infrared, not only the visible spectrum.", "InGaN layers for violet and blue LEDs indicate high efficiency, but the efficiency declines with higher In content from the green region [3–5].", "It is the so-called “green gap” which is the missing part in the high-brightness LEDs including phosphide LEDs [4].", "The growth of luminescent and high-In-content InGaN is the crucial point to improve or overcome the green gap.", "It is essential to understand the InGaN growth mechanism to improve its quality.", "Several reports have shown that a thermodynamic analysis is beneficial to understand the growth of GaN and InGaN [6–8].", "One of the powerful tools to study InGaN growth is the computational fluid dynamics (CFD) simulation since chemical reactions in metalorganic vapor-phase epitaxial (MOVPE) growth are vast in numbers and compete fiercely at the high-temperature region.", "We have developed the GaN and AlN MOVPE chemical reaction sets using the thermo-chemical CFD simulation [9–11] by considering thermal effect at high growth temperatures such as 1000 °C [12].", "In this paper, InGaN growth pathways in MOVPE are reported.", "Also, fabrication of yellow and red LEDs and the effect of the thinner flow channel are studied.", "We used a single-wafer horizontal MOVPE reactor [13].", "The height of the flow channel inside was 7.5 mm or 5.0 mm.", "Precursors were Trimethyl-gallium (TMGa), Trimethyl-indium (TMIn), ammonia (NH3), H2-diluted silane (SiH4) and bis-cyclopentadienyl-magnesium (Cp2Mg).", "The carrier gas was hydrogen for GaN growth or nitrogen for InGaN region.", "Substrates were 2-inch c-plane sapphire wafers.", "We kept 100 kPa of the reactor pressure.", "The growth temperature was 1030 °C for GaN.", "The growth temperatures were calibrated by melting points of metals on the substrate surface.", "The value of growth temperature hereafter is that at the center of a substrate because our machine has temperature distribution that the last 5-mm edge area is a little bit cooler.", "A substrate/quartz tray/SiC-coated carbon susceptor could rotate together, but we did not rotate the substrate to compare with computational results.", "To study InGaN growth rate and In content, we grew unintentionally-doped (uid) InGaN (300 nm)/low-temperature (LT = 525 °C) InGaN buffer/sapphire by using the 7.5-mm flow channel.", "We chose an InGaN growth temperature in the range of 671–855 °C.", "The gas flow rates were TMGa = 11.3 μmol/min, TMIn = 20.5 μmol/min, NH3 = 4.5 slm, and N2 = 10.6 slm.", "The V/III ratio was kept at 6320.", "Since the layer thickness of InGaN was set to be approximately 300 nm, InGaN layers are fully relaxed.", "The In contents and thicknesses of samples at the center of wafers were evaluated or confirmed by x-ray reciprocal space mapping and a scanning electron microscope.", "The InGaN five-quantum-wells (5QWs) structure is [InGaN (3.5 nm) well/(In)GaN (10 nm) barrier] × 5/uid-GaN (2 μm)/LT-GaN/sapphire grown by using the 7.5-mm or 5-mm flow channel.", "A small amount of TMIn was introduced for the (In)GaN barriers.", "It was typically 10% of the TMIn flow in the wells.", "The LED structures are InGaN QWs embedded in p- and n-type GaN layers on LT-GaN/c-sapphire substrates.", "Details of the growth conditions of LEDs will appear later.", "We measured electroluminescence (EL) spectra from the InGaN-based LEDs on a quarter wafer by a spectrometer calibrated in power from 300 nm to 1000 nm.", "We used the identical configuration for measurement using an optical fiber from the spectrometer, electrical probes for current injection, and an LED chip on the quarter wafer.", "Therefore, it is possible to evaluate their relative intensities.", "We used the code of CFD-ACE+ (ESI, France) with the Nitride-MOVPE database (Wave Front, Japan) for simulation.", "The database is based on our previous works [9–12].", "We performed two-dimensional (2D) simulations.", "The 2D simulation model is the cross-sectional of the experimental setup at the center of the 2-inch substrate.", "The flow-channels are made of quartz GE124 (GE Quartz, USA).", "We adopted the thermal radiation and absorption spectra from the quartz parts experimentally measured at elevated temperatures [12].", "We considered imperfect two thermal contacts (some spaces) among a sapphire substrate, a quartz tray, and a SiC-coated susceptor.", "In case of a substrate and a tray, we set their space 8.5 μm.", "This is the distance of the maximum and minimum values in the range of 500 μm length of the finely grounded backside of a sapphire substrate measured by the alpha-step (KLA-Tencor, USA).", "Space was filled with nitrogen gas which was the carrier gas during the InGaN growth.", "The nitrogen gas in the space acts as a thermal conductor.", "Also, thermal radiation and absorption in both sides were considered.", "Strains in nitride layers are not included in our simulation.", "In other words, our simulation shows a fully relaxed state.", "We have constructed modeling of reaction pathways in a TMGa/TMIn/NH3/H2/N2 system.", "There are mainly three kinds of reaction groups: pyrolysis of precursors; pyrolysis of adducts such as TMGa:NH3; and polymer formations.", "We adopted the modified Arrhenius expression (i.e., k = ATnexp(−E/RT), where k, A, T, E, and R are the chemical reaction rate constant, the frequency factor, temperature, the activation energy, and the gas constant, respectively.)", "for each chemical reactions [9,10].", "Fig. 1 shows the overall chemical reactions in the TMGa/TMIn/NH3/H2/N2 system.", "Some experiments show that the introduction of trimethyl-aluminum (TMAl) in GaN growth causes a decrease in growth rate [14,15].", "The additional polymer formations among TMGa and TMAl happen quickly at the growth temperature [16].", "However, reactions among TMGa and TMIn are not so reactive compared with that.", "Therefore, the chemical reactions in Fig. 1 are the simple plus of reactions in the TMGa/NH3/H2/N2 system and those in the TMIn/NH3/H2/N2 system.", "We included the thermal re-vaporization of Ga atoms and In atoms.", "Also, hydrogen etching of GaN and InN is considered.", "Fig. 1 indicates that molecules of NH2 and In would contribute the InN growth, and Ga-N molecules contribute the GaN growth [9].", "The simulations of InGaN MOVPE were compared with experiments using the identical growth conditions.", "The in-plane distributions of growth rate and In content are shown in Fig. 2.", "The simulation shows good agreement qualitatively and quantitatively with the experiment.", "The growth rate gradually decreases from the upstream side to the downstream side.", "That corresponds to the reactive molecules being gradually withered.", "Fig. 3 is temperature dependences of InGaN growth rate and In content in experiment and simulation.", "We simulated two kinds of cases with and without re-evaporation of In atoms adsorbed on the growth surface.", "Considering the In re-evaporation, the simulation shows intense temperature dependences of growth rate and In content.", "The simulation considering In re-evaporation indicates good agreements with experimental growth rate and In content.", "It is found that growth temperature strongly influences on In re-evaporation from InGaN.", "Since GaN growth rate increases slightly with temperature in the range from 400 °C to 1000 °C under the mass-transport limit [9], the contribution of GaN in InGaN growth rate should be positive at the temperature range in Fig. 3.", "However, the decomposition of InN becomes significant at around 700 °C because the two simulated In contents with and without In re-evaporation start split at around 650 °C.", "InGaN growth rate decreases with temperature both for experiment and the simulation with In re-evaporation.", "There are some differences in InGaN growth rates between experiment and the simulation with In re-evaporation at 671 °C and 717 °C.", "The experimental growth rate was calculated from the total thickness and the growth time without considering porosity.", "However, InGaN layers grown at low temperature become porous [17].", "We recognized that the same phenomenon on the InGaN layers grown at these temperatures.", "Therefore, the experimental growth rates at the low temperatures were overestimated.", "It is possible to suppress the In re-evaporation if there are more atomic In and decomposed NH2 concentrations just above the growth surface.", "In this work, we introduced the thinner (5.0 mm) flow channel.", "The effect of the 5-mm flow channel on these reactive molecule concentrations are significant as shown in Fig. 4.", "We compared the conventional (7.5 mm) and the 5-mm flow channels under the same flow conditions (TMGa = 11.2 μmol/min, TMIn = 43.4 μmol/min, NH3 = 4.5 slm, N2 = 10.6 slm), experimentally and numerically.", "The growth temperature was 785 °C.", "The simulation result indicates that both of In and NH2 concentrations in the 5-mm flow channel are much higher than those in the conventional one by 27% and 20% on the substrate surface, respectively.", "In experiments, the In contents of InGaN layers grown by using the 5-mm flow and 7.5-mm channels were 29% and 14%, respectively.", "It is found that the higher In content in InGaN is originated from the higher In and NH2 concentrations on the growth surface.", "We think that temperature distribution in the space inside of the flow channel seems to be a key point to realize those higher concentrations.", "Temperatures of a substrate and the upper wall of a flow channel would be similar in both flow-channels because the upper wall is heated by thermal radiation from the substrate side.", "However, gas temperature distributions will be influenced by the different channel heights since gas is heated mainly by contact and diffusion.", "The calculated minimum temperatures along the height direction at the substrate center are 200 °C and 390 °C for the 7.5-mm and 5-mm flow channels, respectively.", "The higher minimum temperature is the case of the thinner flow channel.", "The ratio of a certain higher temperature region in the inside volume of the 5-mm-flow channel is higher than that for the 7.5-mm-flow channel.", "It means that higher numbers of NH2 molecules and In atoms should be generated at the hot region of the 5-mm flow channel.", "We need to study more about the driving force to collect In atoms and NH2 molecules to the growth surface, but Soret diffusion seems to a possible driving force to enrich decomposed (=smaller) molecules to the substrate side and resulting in those higher concentrations.", "The growth of higher-In-content InGaN by the 5-mm flow channel was confirmed in the range of 760–820 °C in our previous work [13].", "Internal quantum efficiencies (IQEs) from InGaN 5QWs structures were measured to compare the effects of the 7.5-mm and 5-mm flow channels.", "We define the ratio of integrated photoluminescence (PL) intensities at room temperature (RT) and 18 K as the IQW value of a sample at RT.", "The excitation for the PL measurements was a 406-nm InGaN laser which can excite only the InGaN region.", "Fig. 5 shows IQE values from InGaN QW structures with different PL peak wavelength at RT.", "If we make the envelope of the best data from the samples grown by the 7.5-mm flow channel at each temperature, it is possible to recognize the typical “green gap”.", "The samples in the blue region until ca. 500 nm keep high IQE values, then IQE goes down abruptly to a few % from the green region.", "In case of the 5-mm flow channel, the IQE value in the blue region is similar with those in the 7.5-mm case.", "However, the IQE values in the 5-mm case are better than those in the 7.5-mm case in the 550–600 nm region.", "Growth temperatures of samples with the PL peak at 600 nm are 710 °C and 770 °C for the 7.5-mm and 5-mm flow channels, respectively.", "We confirmed that the higher growth temperature for InGaN realizes the higher quality [18].", "The 5-mm flow channel MOVPE has a high potential to grow high-quality high-In-content InGaN layers to realize highly efficient green-yellow-red LEDs.", "The higher temperature is better to grow high-quality InGaN [18].", "Since In and NH2 concentrations in the 5-mm flow channel are higher than those in the conventional one, we can increase growth temperature by keeping for the same In content of the InGaN active region by 50–60 °C [13].", "We grew InGaN LED structures by MOVPE.", "The normalized EL spectra from the various-In-content InGaN-based LEDs are shown in Fig. 6.", "LEDs with the peak wavelength of 410–650 nm have coherent structures (SQW-5QWs) except for the 740-nm LED.", "The 740-nm LED adopts a strain-relaxed structure (16QWs) to introduce more In [13].", "We varied InGaN growth temperatures as the primary parameter to control the EL wavelength until 490 nm.", "InGaN layers for LEDs with peaks at 410–490 nm were grown by using the 7.5-mm flow channel at 820–760 °C, respectively.", "We adopted the 5-mm flow channel to grow LED structures emitted at the range of 520–740 nm.", "The growth temperature for those LEDs was kept at 785 °C.", "We changed the V/III ratio by using various NH3 flow rates to adjust In content in InGaN.", "Flow rates of TMGa and TMIn were kept at 12 μmol/min and 24.4 μmol/min, respectively.", "V/III ratios in the range of 5500–18,000 were adopted to realize higher In contents with the EL peaks of 520–740 nm, respectively.", "It is noted that the EL tail of the 740-nm LED reaches around 900 nm, and In0.5Ga0.5N can emit the light around 900 nm.", "The result encouraged a possibility of longer wavelength optical device application.", "The 5-mm flow channel MOVPE has the potential to grow infrared InGaN-based LED structures.", "Fig. 7 shows integrated intensities and full widths at half maximum (FWHMs) of EL spectra from the LEDs in Fig. 6.", "These intensity and FWHM change gradually and continuously from 410 nm to 740 nm.", "We used the 5-mm flow channel for the LEDs with peaks at 520–740 nm, but there is no obvious step in both cases around 520 nm.", "It means that the effect of the 5-mm flow channel is small at around 520 nm.", "That is correspond with the IQE result.", "Therefore, we can expect the effect of the 5-mm flow channel at the longer wavelength side.", "The decline of the EL intensity will be originated from the degradation of InGaN crystallinity and enhancement of the quantum confined Stark effect in the QW region with higher In content.", "The ratio of EL intensities at 500 nm (grown by the 7.5-mm flow channel) and 600 nm (by the 5-mm flow channel) is approximately 7:1.", "On the one hand, the IQE values of samples grown by the 7.5-mm-flow channel at 500 nm, 600 nm are 83% and 4%, respectively, and resulting in the ratio of 20:1.", "When we compare with 18% of IQE from a 600-nm sample grown by the 5-mm flow channel, the ratio becomes approximately 5:1.", "Therefore, the ratio from 500 nm (7.5-mm-flow channel) and 600 nm (the 5-mm flow channel) is similar both in EL and IQE.", "We think that the quality of high-In-content InGaN samples grown by the 5-mm flow channel is higher rather than those by the 7.5-mm-flow channel.", "The FWHM value is getting broaden continuously with In content as shown in Fig. 7(b).", "It is found that the 5-mm flow channel cannot suppress the fluctuation of In content in InGaN.", "We studied InGaN MOVPE through simulations and experiments.", "It was found that atomic In from TMIn and NH2 molecules decomposed from ammonia are the key molecules in In incorporation into InGaN.", "The In re-evaporation from growth surface determines the In content in InGaN.", "The 5-mm flow channel can increase the atomic In and decomposed NH2 on the growth surface, and resulting in the growth of high-In-content InGaN or the same In content at the raised growth temperature compared with InGaN by the 7.5-mm flow channel.", "We have grown green-yellow–red InGaN-based LED structures by the 5-mm flow channel MOVPE.", "This method has the potential to realize InGaN-based LEDs until infrared." ]
[ "Successful InGaN-MOVPE simulation in growth rate and alloy composition.", "Numerical analysis of growth of InGaN layers by the 5-mm flow channel.", "Experimental analysis of the quality of InGaN QWs grown by using different channels.", "Successful growth of yellow-red LED structures by using the 5-mm flow channel." ]
Substrate-Assisted Catalysis in Polyketide Reduction Proceeds via a Phenolate Intermediate
SimC7 is a polyketide ketoreductase involved in biosynthesis of the angucyclinone moiety of the gyrase inhibitor simocyclinone D8 (SD8). SimC7, which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, catalyzes reduction of the C-7 carbonyl of the angucyclinone, and the resulting hydroxyl is essential for antibiotic activity. SimC7 shares little sequence similarity with characterized ketoreductases, suggesting it might have a distinct mechanism. To investigate this possibility, we determined the structures of SimC7 alone, with NADP+, and with NADP+ and the substrate 7-oxo-SD8. These structures show that SimC7 is distinct from previously characterized polyketide ketoreductases, lacking the conserved catalytic triad, including the active-site tyrosine that acts as central acid-base catalyst in canonical SDR proteins. Taken together with functional analyses of active-site mutants, our data suggest that SimC7 catalyzes a substrate-assisted, two-step reaction for reduction of the C-7 carbonyl group involving intramolecular transfer of a substrate-derived proton to generate a phenolate intermediate.
[ "BIOC", "PHAR" ]
[]
[ "1303", "1308", "1312", "1313", "3002", "3004" ]
[ "Angucyclin(on)es form the largest group of polycyclic aromatic polyketides, many with anticancer and antibacterial activities (Kharel et al., 2012).", "They share a polyketide-derived tetracyclic benz[a]anthracene carbon skeleton, but numerous structures are generated by a range of tailoring reactions, including O- or C-linked deoxysugar glycosylation to form angucyclines.", "In general, these tailoring enzymes are not well understood.", "An important challenge, therefore, is to define the step catalyzed by each enzyme and to determine their reaction mechanisms.", "This knowledge is particularly relevant to the rational engineering of angucyclin(on)e biosynthetic pathways for novel therapeutics.", "Simocyclinone D8 (SD8) is a potent DNA gyrase inhibitor isolated from Streptomyces antibioticus that consists of an angucyclinone connected to a chlorinated aminocoumarin via a D-olivose deoxysugar and a tetraene diester linker (Figure 1) (Schimana et al., 2000; Edwards et al., 2009; Hearnshaw et al., 2014).", "SD8 is bifunctional, with the angucyclinone and the aminocoumarin at opposite ends of the molecule binding to two distinct pockets on the DNA binding surface of the GyrA subunit of gyrase (Edwards et al., 2009; Hearnshaw et al., 2014), thereby inhibiting DNA supercoiling at submicromolar concentrations (Edwards et al., 2011).", "Because gyrase is essential in bacteria but absent from humans, it is an attractive target for antimicrobial drugs, as exemplified by the clinically successful fluoroquinolones (Collin et al., 2011).", "SimC7 was originally annotated as a dehydratase and predicted to be involved in the biosynthesis of the tetraene linker of SD8 (Trefzer et al., 2002).", "However, we recently showed that SimC7 is in fact an NAD(P)H-dependent ketoreductase that catalyzes the reduction of a carbonyl to a hydroxyl group at the C-7 position of the angucyclinone (Schäfer et al., 2015).", "This enzymatic step is essential for antibiotic activity, converting the almost inactive 7-oxo-simocyclinone D8 (7-oxo-SD8; half maximal inhibitory concentration [IC50] ∼50–100 μM) into the potent gyrase inhibitor SD8 (IC50 ∼0.1–0.6 μM) (Schäfer et al., 2015).", "Based on the intermediates produced by S. antibioticus, it seems that the biosynthesis of SD8 starts with assembly of the angucyclinone, followed by the attachment of the deoxysugar, then the tetraene linker, and finally the aminocoumarin (i.e., SD8 is assembled from right to left in Figure 1) (Schimana et al., 2001).", "Therefore, the physiological substrate of SimC7 is most likely a 7-oxo angucyclinone intermediate lacking the attached deoxysugar, tetraene linker, and aminocoumarin, an intermediate that is detectable only in ΔsimC7 mutants (Schäfer et al., 2015).", "Despite this, the enzyme readily accepts as a substrate the full-length intermediate 7-oxo-SD8, the major product made by ΔsimC7 mutants (Schäfer et al., 2015).", "The angucyclinone moiety of SD8 is synthesized by a type II polyketide synthase (SimA1-3) and multiple tailoring enzymes (SimA4-13, SimC7) that catalyze cyclization, aromatization, oxidation, and reduction reactions.", "Several ketoreductases of the short-chain dehydrogenase/reductase (SDR) family that act on angucyclinones or related polyketides have been characterized.", "The reduction of carbonyl groups at the C-6 and C-9 positions of polyketides has been functionally characterized, and the structures of the corresponding SDR enzymes have elucidated their reaction mechanisms and factors determining their stereoselectivity.", "The ketoreductases LanV and UrdMred act on the C-6 carbonyl group of angucyclic polyketides from the landomycin and urdamycin pathways (Paananen et al., 2013; Patrikainen et al., 2014).", "In contrast, the ketoreductases ActKR and HedKR act on the C-9 carbonyl group of early intermediates in the actinorhodin and hedamycin polyketide pathways (Javidpour et al., 2011a, 2011b, 2013; Korman et al., 2004, 2008).", "The LanV, UrdMred, ActKR, and HedKR structures revealed the catalytic Ser-Tyr-Lys triad characteristic of SDR enzymes, where the latter two residues form a YxxxK motif.", "In these classical SDR proteins, the conserved active-site tyrosine serves as central acid-base catalyst that donates a proton to the substrate.", "The adjacent lysine residue lowers the pKa of the tyrosine hydroxyl group and often contributes directly to a proton relay mechanism, and the hydroxyl group of the serine stabilizes and polarizes the carbonyl group of the substrate (Kavanagh et al., 2008).", "At the sequence level, SimC7 shares little similarity with any characterized ketoreductase, even with functionally analogous polyketide ketoreductases.", "The striking differences between the amino acid sequence of SimC7 and those of HedKR, ActKR, LanV, and UrdMred suggested that SimC7 might have a novel catalytic mechanism.", "To investigate this possibility, we determined the structures of SimC7 alone (apo; 1.6 Å resolution), the binary complex with NADP+ (1.95 Å), and the ternary complex with both NADP+ and 7-oxo-SD8 (1.2 Å) (Tables S1 and S2).", "Our results reveal that SimC7 is structurally distinct from previously characterized polyketide ketoreductases and, importantly, lacks the canonical SDR Ser-Tyr-Lys catalytic triad (Kavanagh et al., 2008; Kallberg et al., 2010; Persson and Kallberg, 2013).", "Instead, our data suggest that SimC7 catalyzes a substrate-assisted, two-step reaction for the reduction of the C-7 carbonyl group involving an unusual phenolate intermediate.", "SimC7 is made up of two domains, the larger of which is the nucleotide binding domain that adopts a Rossmann fold (Figures 2A, 2B, and S1); the smaller substrate binding domain, characteristic of the so-called extended SDR subfamily (Kavanagh et al., 2008), is mainly α-helical and is largely formed by two insertions in the nucleotide binding domain (between β6 and α6, and between β9 and α10).", "Notably, the latter insertion contains a “lid” motif consisting of two antiparallel α helices (α8 and α9) that folds over the active site (Figures 2A, 2B, and S1).", "The substrate binding domain is completed by a short helical segment at the C-terminus of the polypeptide chain.", "Overall the apo, binary, and ternary SimC7 structures are very similar (Table S3), with the notable exception of the lid motif, which displays a number of different conformations (Figure 2C).", "Although the changes are not large (maximum Cα-Cα shift 5.35 Å; Table S3), there is a clear closure of the lid over the bound substrate (Figure 2C), suggesting a role in gating access to the active site and/or substrate capture.", "Moreover, in the ternary complex the underside of the lid contributes to the tight, highly hydrophobic substrate binding pocket (Figure 3) that provides the necessary environment for catalysis (see below).", "Structures annotated as SDR proteins (PFAM family PF00106) are prevalent in the PDB, with more than 600 entries.", "To look for structural homologs of SimC7, we carried out a structure-based similarity search using the DALI server (Table S4).", "Strikingly, characterized angucyclinone ketoreductases ranked very low in the search, the closest match being LanV (Figure S2A), which was the 166th ranked hit after filtering for sequence redundancy (Table S4).", "Instead, the two most structurally similar proteins to SimC7 were quinone oxidoreductase (QOR2) from Escherichia coli (PDB: 2ZCV) (Kim et al., 2008a) (Figure S2A) and triphenylmethane reductase (TMR) from Citrobacter sp.", "KCTC 18061P (PDB: 2VRB) (Kim et al., 2008b).", "QOR2 and TMR share with SimC7 the ability to reduce substrates with extensively conjugated pi systems but have roles in the detoxification of xenobiotics rather than the biosynthesis of natural products.", "The majority of the closest structural homologs of known function are involved in sugar biosynthesis, many of them epimerases.", "SimC7 and its closest structural homologs all fall into the extended SDR subfamily of proteins, characterized by having two distinct domains that together form a partially occluded active-site pocket at their junction (Figure S2A).", "In contrast, the other structurally characterized polyketide ketoreductases such as LanV are more distantly related and belong to the classical SDR subfamily (Kavanagh et al., 2008), in which three insertions within the core Rossmann fold motif delineate a more accessible active-site cavity but do not constitute a well-defined substrate binding domain (Figure S2A).", "In the ternary complex with substrate, determined at 1.2-Å resolution, the angucyclic ring system of 7-oxo-SD8 binds adjacent and parallel to the nicotinamide ring of the cofactor (Figures 2D, 2E, and S3), where it adopts a relatively planar conformation differing from the conformations seen in the DNA gyrase-SD8 and SimR-SD8 complexes, in which the A ring of the angucyclinone in SD8 is tilted upward toward the epoxide (Hearnshaw et al., 2014; Le et al., 2011) (Figure S2B).", "This planar conformation is most likely enforced by the shape of the very constricted and highly hydrophobic substrate pocket (Figure 3).", "Within this hydrophobic pocket, 7-oxo-SD8 is bound only by a single direct hydrogen bond between the side chain of Ser95 and the C-7 carbonyl oxygen of the angucyclinone moiety, which may help to position the latter exactly above the C-4 position of the nicotinamide ring, where it is poised for direct hydride transfer (highlighted by black spheres in Figures 2C–2E, 3B, and S3).", "As mentioned above, the natural substrate for SimC7 is likely to be a 7-oxo angucyclinone intermediate lacking the deoxysugar, the tetraene linker, and the aminocoumarin.", "Consistent with this, only the angucyclinone moiety is buried in the active site of SimC7.", "Roughly half of the tetraene linker is visible in the electron density, projecting away from the protein surface, and the aminocoumarin ring is not resolved at all (Figure S3).", "SimC7 lacks the Ser-Tyr-Lys catalytic triad characteristic of canonical SDR proteins (Figure 4 and Table S4).", "While the serine is conserved (Ser95), the other two residues (i.e., the YxxxK motif) are not, being instead replaced by Ile108 and His112, respectively.", "This Ser-Ile-His triad is unlike any described for the five subfamilies of SDRs defined by Kavanagh et al. (2008).", "Particularly surprising is the absence of the tyrosine residue that acts as the acid-base catalyst in the classical SDR mechanism (Figure 4A).", "Inspection of the structure of the ternary complex shows that none of the five tyrosine residues in SimC7 is sufficiently close to C-7 of the angucyclinone ring system of the substrate to play a direct role in catalysis.", "Furthermore, the structure also shows that there is no alternative residue that could act as an acid-base catalyst.", "Consequently, SimC7 must perform ketoreduction of 7-oxo-SD8 via a novel mechanism.", "Based on the structure of the ternary complex of the enzyme with NADP+ and 7-oxo-SD8, we propose a simple two-step mechanism for SimC7 that does not depend on catalytic residues in the protein, but rather takes advantage of the specific properties of the substrate itself, and is thus a novel example of substrate-assisted catalysis (Dall'Acqua and Carter, 2000).", "In the first step, the hydrophobic environment of the substrate binding pocket and the juxtaposition of the quinone-like C ring and the phenyl-like D ring of the angucyclinone favor the formation of an intramolecular hydrogen bond between the proton on the C-8 hydroxyl group and the oxygen of the neighboring C-7 carbonyl group (Figure 4B).", "This enhances the polarization of the latter such that the electrophilicity of C-7 is increased, making it a good acceptor for direct hydride transfer from the 4-pro-S position of the nicotinamide ring.", "Crucially, the hydride donor and acceptor carbon atoms are only 3.0 Å apart in the crystal structure.", "The C-7 hydroxyl group is then formed by internal proton transfer from the neighboring C-8 hydroxyl group, generating a phenolate intermediate in which the negative charge on the C-8 oxygen atom is stabilized by the aromatic D ring.", "In the second step of the reaction, the phenolate intermediate leaves the substrate binding pocket and the proton required to reinstate the C-8 hydroxyl group is recovered by abstraction from bulk water, which is not possible within the confines of the active site (Figure 4B).", "Exchange of the negatively charged reaction intermediate is most likely accelerated by repulsion from the hydrophobic active-site cavity.", "Finally, the direct hydride attack from below the angucyclic polyketide unambiguously explains the 7S stereochemistry of simocyclinones.", "In support of this proposed mechanism, molecular modeling predicts the existence of the key intramolecular hydrogen bond between the C-8 hydroxyl group and the C-7 carbonyl group of 7-oxo-SD8, and an equivalent intramolecular hydrogen bond is observed in the small-molecule crystal structure of panglimycin, a closely related polyketide (Fotso et al., 2008).", "Furthermore, molecular modeling also predicts that the C-8 hydroxyl group will have the most acidic and exchangeable proton in the angucyclic polyketide, with an estimated pKa of 6.9–7.7, and therefore could readily transfer to the neighboring C-7 oxygen at the end of step 1.", "Indeed, there is no other possible proton donor (neither protein nor water derived) sufficiently close to O-7 to perform this role.", "Based on their structures (Figure S4A), there are four other angucyclinones in which a SimC7-like mechanism might generate a C-7 hydroxyl group: panglimycin, elmycin, grisemycin, and kiamycin (Fotso et al., 2008; Xie et al., 2012, 2016).", "However, the biosynthetic gene clusters for these molecules have yet to be reported, and it is not known whether a SimC7-like enzyme is involved in their synthesis.", "Most other angucyclin(on)es have a carbonyl group at C-7.", "To investigate the potential roles of the SimC7 “catalytic triad” residues in the proposed reaction mechanism, we mutagenized Ser95, Ile108, and His112 (Figure 4B).", "In the wild-type enzyme, the hydroxyl group of Ser95 could aid catalysis by helping to bind and correctly orient the substrate, and by providing additional polarization to the C-7 carbonyl group via a hydrogen bond, the latter role being consistent with the function proposed for the structurally equivalent Ser/Thr residues in the classical SDR mechanism (Figure 4A) (Kavanagh et al., 2008; Kallberg et al., 2010; Persson and Kallberg, 2013).", "However, neither effect would appear to be crucial for activity, as a S95A mutant showed 86% substrate conversion relative to the wild-type (Figure 4C).", "Therefore, SimC7 must be able to orient 7-oxo-SD8 for catalysis without the necessity for the hydrogen bond from Ser95, and this likely arises because the active-site cavity of the binary complex closely matches the shape of the substrate (Figure 3B).", "Ile108 contributes to the hydrophobic surface that forms one side of the active-site cavity and has a nonspecific role in helping to trap the angucyclinone group against the nicotinamide ring of the cofactor (Figure 3B).", "Unsurprisingly, an I108A substitution had almost no effect, whereas changing it to aspartate (I108D) abolished activity (Figure 4C).", "In the latter case, the introduction of a negative charge would interfere with the hydrophobic environment and could disrupt the intramolecular hydrogen bond in the substrate, both being necessary for catalysis.", "Finally, mutation of His112 (H112A, H112N, and H112Q) strongly reduced or abolished enzyme activity (Figure 4C), consistent with its key role in binding and positioning the cofactor via a hydrogen bond to the 2′-hydroxyl of the nicotinamide ribosyl moiety (Figures 2D, 2E, and 4B).", "The sequence of SimC7 is distinct from previously characterized polyketide ketoreductases, and the structural data reported here suggest that it catalyzes a novel substrate-assisted, two-step reaction for the reduction of the C-7 carbonyl group.", "This mechanism involves the intramolecular transfer of a substrate-derived proton to generate a phenolate intermediate, negating the need for proton transfer from a canonical SDR active-site tyrosine.", "Like SimC7 (Ser-Ile-His), the two closest structural homologs, TMR (Tyr-Leu-His) and QOR2 (Leu-Leu-His), also have unusual active-site triads.", "Thus SimC7, TMR, and QOR2 share an I/LxxxH motif and have substrates with extensively conjugated pi systems.", "No enzyme-substrate complex crystal structures have been described for TMR or QOR2 and no firm proposals exist for their catalytic mechanisms, but their substrate structures and the data provided here suggest they are also likely to employ noncanonical mechanisms.", "Our data therefore point to members of the extended SDR sub-family having I/LxxxH active site motifs as a source of new biochemistry.", "For a full explanation of the experimental protocols, see Supplemental Experimental Procedures.", "Point mutants of simC7 were generated by PCR-based site-directed mutagenesis.", "All constructs were verified by sequencing.", "Proteins were expressed in E. coli as N-terminally His-tagged fusions, purified by nickel-affinity chromatography and assayed by high-performance liquid chromatography as described previously (Schäfer et al., 2015).", "The structural integrity of purified proteins was verified using circular dichroism.", "SimC7 was labeled with selenomethionine (SeMet) by metabolic inhibition, and crystals of both native and SeMet proteins were grown by vapor diffusion (crystals of binary and ternary complexes were obtained by cocrystallization).", "Crystals were harvested and flash-cooled in liquid nitrogen.", "The native crystals did not require further cryoprotection, while the SeMet-labeled crystals were cryoprotected by supplementing the crystallization solution with 25% (v/v) glycerol.", "All X-ray data were collected at the Diamond Light Source.", "For de novo structure determination, a single-wavelength anomalous dispersion dataset was collected at the Se K X-ray absorption edge for an SeMet-labeled SimC7 crystal.", "Native and SeMet data were combined to solve the structure of SimC7 in complex with NADP+.", "All other structures were determined by molecular replacement using the latter as a template.", "All authors designed the experiments.", "M.S. and C.E.M.S. performed the experiments.", "All authors analyzed the data.", "M.S., D.M.L., and M.J.B. wrote the paper, and all authors made revisions." ]
[ "SimC7 catalyzes reduction of the C-7 carbonyl of the angucyclinone of simocyclinone", "SimC7 lacks the conserved catalytic triad characteristic of canonical ketoreductases", "SimC7 reduces the C-7 carbonyl via a substrate-assisted, two-step reaction", "This involves intramolecular transfer of a substrate proton to generate a phenolate" ]
Identifying Potential Ageing-Modulating Drugs In Silico
Increasing human life expectancy has posed increasing challenges for healthcare systems. As people age, they become more susceptible to chronic diseases, with an increasing burden of multimorbidity, and the associated polypharmacy. Accumulating evidence from work with laboratory animals has shown that ageing is a malleable process that can be ameliorated by genetic and environmental interventions. Drugs that modulate the ageing process may delay or even prevent the incidence of multiple diseases of ageing. To identify novel, anti-ageing drugs, several studies have developed computational drug-repurposing strategies. We review published studies showing the potential of current drugs to modulate ageing. Future studies should integrate current knowledge with multi-omics, health records, and drug safety data to predict drugs that can improve health in late life.
[ "BIOC", "MEDI" ]
[ "ageing", "computational biology", "drug repurposing", "longevity" ]
[ "1310", "2712" ]
[ "Human life expectancy (see Glossary) has steadily increased since the middle of the 19th century in many parts of the world [1] and is projected to continue to do so [2].", "Longer lives are a result of improved living conditions and medical care, which have made people healthier at most ages [3,4].", "However, the years of healthy life (healthspan) have not kept up with the overall increase in life expectancy, and there is a large and growing period of poor function and ill health at the end of life [5].", "Ageing is the major risk factor for illness, including the major chronic and killer diseases: cancer, metabolic and cardiovascular disease, and dementia [6,7].", "The increasing proportion of unhealthy old people, often with two or more diseases (multimorbidity) [8–10], and associated problems from the use of multiple drugs for their treatment (polypharmacy) [11–14], is posing an increasing burden on elderly people, their carers and social networks, and healthcare systems.", "Reducing the impact of ill health at the end of life is, therefore, a high priority for national governments and international health organisations [15].", "Since ageing is the major risk factor for poor functioning and disease, intervening to ameliorate its effects could also prevent multiple age-related conditions simultaneously.", "There is growing evidence for the feasibility of this approach.", "People who die when they are very old (100, 105, 110) show progressively less multimorbidity at the end of their lives [16,17].", "Thus, a healthy ageing phenotype in humans can be achieved, and if we could understand the mechanisms leading to it, we might be able to extend it to the general population.", "Additionally, work over the past ∼20 years has shown that environmental, genetic, and pharmacological interventions in animals can extend both their lifespan and their healthspan [18,19].", "Dietary restriction (DR), a reduction in food intake that avoids malnutrition, can extend lifespan and induce a marked improvement in health during ageing in diverse organisms [20,21] including rodents.", "Two studies of rhesus monkeys subjected to DR found that the animals had lowered plasma triglycerides, diabetes, cardiovascular disease, sarcopenia, incidence of neoplasms, and brain atrophy, all features of ageing in humans [22–24].", "However, compliance with DR regimes in humans is low, and for this reason, it is not a practical public health intervention.", "Changes in diet are monitored by many nutrient-sensing systems, including the insulin/insulin-like growth factor and target of rapamycin (mTOR) signalling network.", "This highly conserved network senses nutrients, growth factors, and stress status, and modulates the costly activities of the organism, such as metabolism, growth, and reproduction, accordingly.", "Genetic interventions that reduce the activity of the network have proved to extend lifespan in nematode worms, fruit flies, and mice [18,19,25].", "These long-lived mutants are protected against many natural pathologies of old age and also those associated with genetic models of age-related diseases.", "Genetic variants in orthologous genes in humans are associated with survival to advanced ages [26–28].", "Mechanisms of ageing are highly conserved during evolution, and the process shows a set of characteristic hallmarks of ageing [29], which are also present in the aetiology of age-related diseases [7,29].", "Interventions that improve health during ageing and increase lifespan in laboratory animals do so by reducing the impact of one or more of these hallmarks.", "Increasingly, attention is turning to the possibility of pharmacological intervention into the ageing process, with a view to preventing age-related diseases.", "Although, ageing is becoming popular amongst the conditions under study in clinical trials, which aim, for example, to find drugs that restore gene expression levels to that of young healthy subjects (ClinicalTrials.gov identifier: NCT02432287) or reduce ageing-related biochemical parameters (NCT03451006), de novo drug development for ageing poses major challenges.", "We lack good biomarkers of ageing that could give a rapid prediction of the outcome of drug treatment, and a clinical trial with a potentially long-term treatment of an initially healthy population would be both prohibitively costly and would require drugs that are almost completely safe and free of side-effects.", "However, unsurprisingly, many of the gene products identified as potential targets to reduce the impact of ageing are already the targets of drugs licensed to treat specific age-related conditions.", "For instance, the licensed drug sirolimus (rapamycin), an inhibitor of the mTOR Complex 1 (TORC1), is used to prevent rejection of tissues after transplant, restenosis after cardiac surgery, and to treat cancer.", "This drug also extends lifespan in yeast, nematode worms, flies, and mice, and prevents many age-related conditions in ageing mice [30–32].", "Inhibitors of mTOR can also potentiate the weak response to immunisation against influenza in ageing mice and humans [33,34].", "Metformin, a drug used to treat type 2 diabetes, also targets the nutrient-sensing network.", "This drug has been shown to reduce all-cause mortality in diabetic patients when compared with those receiving nonmetformin therapies, and even non-diabetics in multiple studies [35], and is under trial for protection against the effects of ageing [36,37].", "Repurposing of drugs, to protect against the effects of ageing and hence delay or prevent age-related diseases, is thus an increasingly realistic prospect.", "Indeed, some existing drugs could already be viewed as being used in this way.", "Both statins and drugs that lower blood pressure are widely used to prevent cardiovascular disease [38,39], for which age is the major risk factor.", "Non-compliance with preventive drug regimens will always limit their efficacy, but there is at least the prospect that many age-related conditions could be warded off pharmacologically by those who wish to do so.", "There is, therefore, growing interest in identifying and prioritising such potentially geroprotective drugs.", "In this short review, we discuss the available data and some of the bioinformatics methods that are being used to this end.", "It is important to stress that these are methods to prioritise drugs for experimental testing.", "They provide the first step of many required for successful drug repurposing.", "Here we review 12 recent publications, summarised in Table 1 and Figure 1, aiming to identify and prioritise prolongevity drugs for animal models and humans.", "All such studies have been enabled by the development of powerful databases for the annotation and curation of genes/proteins (Ensembl [40], UniProt [41]), their associated functions and pathways (Gene Ontology [42], KEGG [43], Reactome [44]) and chemical ligands and drugs interacting with them (ChEMBL [45], DrugBank [46], STITCH [47], drug gene interaction database; DGIdb [48], protein data bank; PDB [49]) or affecting their expression (Connectivity Map [50], CREEDS [51]), as well as drugs (DrugAge [52], Geroprotectors.org [53]) and targets (GenAge [54], Aging Clusters [55]) implicated in ageing and age-related disease mechanisms (Figure 1).", "Although the published studies of drug repurposing to target ageing use different strategies and sources of data, they can be classified into two main categories: methods employing the structural information to predict drugs potentially interacting with proteins already identified as being involved in ageing, and methods based on the similarity between ageing-related drugs or genes based on molecular structure, interactions, pathways, or networks (Table 1).", "The concept here is to find drugs which are known to target those genes that have been implicated in ageing.", "Two studies adopted methods based on the hypothesis that proteins or ligands with similar structures are likely to bind similar ligands or proteins, respectively, to predict drug-target interactions.", "The first of these (Study #1) [56] aimed to identify novel drugs targeting three specific temperature-sensing proteins implicated in ageing in the rotifer Brachionus manjavacas (TRP7, S6P, FhBC).", "The authors used a virtual screening (Box 1) software called FINDSITEcomb that combines protein modelling with sophisticated threading approaches to model the target.", "The pockets in the model are then compared with the pockets in experimentally determined structures of proteins with ligands or modelled structures with known binders.", "The ligands of the top 100 ranked pockets are then compared with a library of screened ligands and ranked by ligand similarity.", "The authors screened 1347 FDA approved drugs in silico and tested four drugs for each target experimentally in the rotifers for their effects on lifespan and healthspan.", "Out of the 12 compounds tested, 5 significantly increased the rotifers’ lifespan.", "Changes in healthspan, approximated by swimming speed, reproduction, and mitochondrial activity, were also observed.", "In a subsequent study by the same authors (Study #2) [57], the number of proteins analysed was expanded to include a set of ageing-related genes found in other animal models that are orthologous to genes in rotifers.", "This time a total of 94 targets were screened in silico using the FINDSITEcomb software.", "The top 1% binding compounds for each target were further ranked by their cumulative lifespan extension achieved by genetic interventions into their targets as taken from experimental model organism data, and filtered according to availability and previously predicted side effects [58].", "From the 31 drugs experimentally tested in rotifers by two 10-day survival screens, seven drugs were further tested in two whole-life survival analyses, two of which resulted in a median lifespan extension of 13–42%.", "The prolongevity effect of these drugs was observed even when drug treatment was initiated in middle age.", "Another in silico screening study (Study #3) [59] was restricted to a single gene, AMP-activated protein kinase (AMPK), activation of which partially mediates the effects of DR on ageing.", "To find new molecules to activate AMPK and theoretically mimic DR, Mofidifar et al. [59] performed virtual screening (Box 1) using molecular docking of 1908 FDA approved drugs.", "The interaction between the top-ranked compounds and their targets was then further checked by more detailed molecular dynamics (Box 1).", "The study reported four compounds with predicted high affinity for AMPK, but these were not tested experimentally.", "Using a priori information on known ageing-related genes, prolongevity drugs, or gene expression profiles, several studies have implemented a series of similarity-based approaches to identify novel anti-ageing drugs.", "Given that drugs targeting ageing-related gene products are expected to affect the ageing process, Fernandes et al. (Study #4) [60] focused on finding drugs that target human genes which have orthologues associated with longevity in animal models.", "The drugs were ranked by the likelihood of targeting ageing-related genes among all targets.", "For this calculation, only inhibitory drugs interacting with antilongevity genes and activators targeting prolongevity genes were considered.", "In total, 376 drugs were obtained, of which, 20 were considered to be statistically significant.", "Thirteen targeted histone deacetylases, and three were previously associated with lifespan extensions in animal models.", "Recently, Fuentealba et al. (Study #5) [61] used a composite set of ageing-related genes with direct evidence for influencing human ageing, together with physical and functional drug–protein interactions, to implement a similar gene-set overlap analysis (Box 1).", "Study #5 also considered other levels of biological actions, including pathways, functions, and protein–protein interactions.", "Three of the top 10 compounds that ranked highest on an aggregate score were previously shown to increase lifespan in animal models, and seven had been proposed to affect longevity by other drug-repurposing methods.", "The prolongevity effects of the top-ranked compound (tanespimycin) was experimentally validated in Caenorhabditis elegans.", "An alternative approach is to find drugs similar to known prolongevity drugs using machine learning (Box 1), which is a strategy well-suited for prediction tasks.", "Liu et al. (Study #6) [62] attempted to predict new prolongevity drugs for C. elegans.", "They adopted a semisupervised algorithm trained with high-confidence prolongevity drugs derived from an experimental screen for C. elegans [63], together with their associated ageing-related genes curated from the literature and the GenAge database [54].", "They produced a rank-ordered list of 785 drugs with a potential to increase lifespan in worms, with experimental validation for one drug in their list, using a lifespan assay.", "A separate machine learning approach (Study #7) [64] was trained with chemical descriptors of known prolongevity drugs and functional annotation of their targets.", "Using a supervised algorithm (i.e., random forest), they generated a ranked list of drugs predicted as lifespan-extending compounds, although no validation was performed.", "The Connectivity Map Resource provides drug-induced expression profiles for 1309 compounds.", "Comparing these profiles with ageing-related gene expression signatures using a gene-set enrichment analysis (Box 1) can reveal drugs that generate changes in expression correlated (positively or negatively) to those seen in ageing (or any other biological process or disease).", "This approach requires no a priori list of ‘ageing genes’ and can therefore potentially identify new targets, based solely on expression profile similarities.", "The first study (Study #8) [65] used DR expression profiles in rats and rhesus monkeys to find DR mimetics.", "They identified 11 drugs that could potentially increase lifespan by mimicking DR. They experimentally tested several of the drugs in C. elegans and found that most extended lifespan.", "Another study (Study #9) [66] used a meta-analysis of gene expression changes in the ageing human brain to identify robust gene expression changes in ageing and find drugs targeting those genes.", "Using the Connectivity Map data, the authors identified 24 drugs and provided in silico validation by showing significant enrichment of known prolongevity drugs in their list.", "Importantly this data-based approach can identify novel drugs and genes, not previously associated with ageing.", "Yang et al. (Study #10) [67] used a network-based methodology, called ANDRU (ageing network-based drug discovery).", "Instead of relying on model organisms, this approach was also driven by human transcriptome data (GTEx) from young and old adipose and artery tissues and signatures from the CREEDS database [51] to identify differentially expressed genes within the ageing-related networks and drugs reversing these changes.", "They report three distinct drugs ranking in the top five.", "Although none is previously reported as a lifespan modulator, these drugs target pathways that change in expression with age, such as metabolic enzymes and lipid metabolism.", "One of the major challenges to developing anti-ageing drugs is experimental validation.", "Since clinical trials involve many ethical considerations and are very expensive, such drugs are pretested in model organisms.", "In this spirit, Aliper et al. (Study #11) [68], aimed to predict which prolongevity drugs previously tested in C. elegans could work in humans.", "Using young and old human stem cell expression profiles and an algorithm called Geroscope that maps the gene expression changes with age to ageing-related signalling pathways, they ranked a set of candidate drugs by their likelihood of targeting these pathways.", "To do this they calculated the pathway activation strength for each drug.", "They shortlisted ten compounds with prolongevity effects in C. elegans, and tested six of them for geroprotective effects in senescent human fibroblast cultures.", "While the majority of tested drugs improved senescence-associated phenotypes, one drug (PD-98059), a highly selective MEK1 inhibitor, also showed life-prolonging and rejuvenating effects.", "Comparably, to assess which ‘human’ drugs and chemicals are likely to modulate the C. elegans and Drosophila melanogaster orthologue of the target, Ziehm et al. (Study #12) [69] developed a method to rank chemicals binding to genes implicated in human ageing.", "They generated an empirical scoring function that considers the conservation of the domain and binding site at the sequence level between the animal and the human protein, and predicted binding energy for the compounds for the human targets and experimental bioavailability, in addition to scores for drug-likeness, promiscuity, purchasability, and development status.", "Although the authors provided no experimental validation, they conducted a comprehensive literature-mining and molecular-docking procedure to validate their results.", "The studies described above had different aims, methods, and data sources.", "To facilitate their comparison, we have summarised each study in terms of: (i) the drugs identified, (ii) the genes targeted by these drugs, and (iii) all biological pathways (KEGG) known to be targeted by drugs (Figure 2, Key Figure).", "Additionally, we compared the results with the manually curated databases of ageing-related genes (GenAge) and drugs (DrugAge).", "Overall only 12% of all DrugAge drugs are prioritised by at least one study (41 of 346 drugs in DrugAge), with one in every four drugs discovered already present in DrugAge, reflecting the prioritisation process and the low number of drugs reported as significant by each study (15 drugs on average).", "In addition, the 163 drugs identified usually differ between studies, with 91% (149 drugs) of them identified just by one study.", "From the remaining 14 drugs present in more than one study, trichostatin, geldanamycin, tanespimycin, and vorinostat were identified by three studies (Figure 3A) and, while only the first two are present in the DrugAge database, the remaining two have been experimentally validated for prolongevity effects in animal models [61,70].", "Most studies resulted in a list of drugs containing mainly novel candidates not present in DrugAge (122 drugs were classified as novel discovery), the only exception being Aliper et al. [68], which focused only on a set of known prolongevity drugs.", "We also note that 66% of the 122 drugs (i.e., 81 drugs) known to target ageing-related proteins were prioritised by the computational studies reviewed above, as expected considering that these drugs are included in some of the databases used by some of the methods during the prioritisation process.", "Overall, 34% of the GenAge human genes (103 genes) and 10% of the GenAge model organism genes (94 genes) were identified in at least one study, reflecting at least in part the different sizes of the datasets, with more than three times the number of model organism genes in GenAge.", "For clarification, the computational methods identified candidate drugs (which are predicted to modulate ageing) amongst the known drugs, most of which are currently used as therapy for a specific disease.", "Based on the DGIdb database [64], 27% of the druggable genome (i.e., 796 genes) is targeted by at least one of the drugs identified in the computational studies (Figure 3B) and, while few genes were identified in multiple studies, some of them were present in the GenAge database [54].", "Two of these genes, DDIT3 (DNA Damage Inducible Transcript 3) and ERBB2 (Erb-B2 Receptor Tyrosine Kinase 2), were targeted by the drugs prioritised in eight studies.", "However, nine studies also identified drugs targeting BIRC5 (Baculoviral IAP Repeat Containing 5) and KRAS (KRAS Proto-Oncogene, GTPase), and ten studies predicted drugs modulating ABCB1 (ATP Binding Cassette Subfamily B Member 1), which have not previously been related to human ageing.", "Despite this, genes discovered by multiple studies do not necessarily suggest higher relevance to ageing, and may instead reflect research bias (e.g., genes targeted by many drugs because of a role in prevalent disease such as cancer).", "We also observed that 80% of known prolongevity drugs (i.e., 122 of 152 drugs with known targets) target at least one gene that is targeted by the candidate geroprotective drugs identified by these 12 computational studies.", "Intriguingly, among the 319 druggable KEGG pathways, 92% include at least one gene that is targeted by the drugs identified in the studies in Table 1.", "The same tendency was observed for genes in GenAge (83% Model GenAge and 74% Human GenAge), or genes targeted by the DrugAge drugs (88%).", "While this may suggest ageing is ubiquitous and affects all pathways, another possibility is that genes present in many pathways could be discovered repeatedly because they play a central role in diseases and regulatory mechanisms.", "Although this may not conclusively prove that ageing is ubiquitous, the prioritised candidate drugs clearly have a genome-wide effect.", "Although many caveats need to be considered (Box 2), these 12 computational studies have revealed many currently used drugs with a high potential to modulate lifespan in humans.", "However, the challenge today is not to extend lifespan (which is happening anyway), but rather to improve late life health by reducing the multimorbidity associated with old age.", "This challenge has not yet been adequately addressed, either in model organism research or in clinical studies.", "Many questions remain unanswered (see Outstanding Questions).", "Although some of the potential candidate drugs have been tested for effects on longevity in model organisms, the effects on late life health in humans are difficult to assess experimentally.", "To do this, access to human health data in old age, combining studies, and allowing in-depth statistical analyses, including consideration of side effects, will be critical.", "This area is opening up, but a challenge still remaining is gaining access to the clinical data, whilst of course retaining patient confidentiality.", "The new UK Biobank [71] holds great promise, with data from about half a million individuals, including clinical phenotype data and genotyping, with exome sequencing in progress.", "These data will be even more powerful when combined with molecular information and data from longitudinal cohort studies.", "An important initiative for testing drugs in model organisms is the National Institute on Aging Interventions Testing Program [72], a multi-institutional program where researchers test the effect of chemical or environmental perturbations on the lifespan of a genetically heterogeneous mouse model.", "The workflow is designed to increase the reproducibility and find reliable candidates to modulate ageing.", "However, although we have begun to document the effect of specific pathways on the ageing process and lifespan, we still lack an overall understanding of ageing processes, limiting our ability to use well-established drug discovery methods, such as genome-wide association studies, which to date have only provided limited insights [7].", "Although conservation of the ageing-related processes and genes amongst organisms is widely accepted, the ability to translate the effects in model organisms to reliable predictions for humans remains a challenge.", "The need for biomarkers of ageing is becoming ever more important to allow a rapid assessment of the effects of a drug on an organism other than by conducting lengthy, expensive lifespan experiments.", "The emergence of several ‘epigenetic ageing clocks’ [73–75] provides opportunities to actively monitor ageing in both cells and organisms.", "In parallel, high-throughput cellular imaging will allow improved screening and testing.", "Clearly, this area of research is expanding rapidly, and the studies described above are just the beginning.", "Obviously, all this computational work just prioritises candidates before experimental testing, but the methods provide an overview of the current genomic, health, and drug landscape, which can better inform the choice of the best candidates.", "We can expect a flood of both experimental ageing data on model organisms and clinical datasets for human ageing in the near future.", "A common workflow will be the integration of many types of data from many studies using computational approaches, which will become more sophisticated and complex.", "Machine learning approaches, which are increasingly prevalent, will also have an impact here, if sufficiently robust, reliable data can be garnered.", "Indeed, such analyses will allow us to process relatively noisy, unstructured data more effectively.", "The hope is that these in silico approaches will not only increase our understanding of the processes of ageing but also provide a route towards tackling multimorbidity and improving health in late life.", "Will drugs that extend lifespan in model organisms translate effectively to improve late life health in humans?", "Can we predict the effects of a drug considering its mechanism of action and evolutionary relationships?", "What are the best approaches to test such drugs experimentally in humans and avoid serious side effects?", "There is a need to develop both in silico and in vitro schemes to facilitate this translation process.", "Can we predict the side effects of drugs that will be used in primary prevention by healthy individuals for a long term?", "Will they be suitable for elderly frail patients?", "Will a combination of molecular and clinical data reveal the causes and effects of ageing and allow us to differentiate cause and effect?", "Will the most appropriate drugs replicate our responses to ageing or target its causes?", "What are the robust biomarkers of ageing and what are the biological mechanisms making them good biomarkers?", "Can we utilise epidemiological studies and clinical data to study the effects of drugs on healthspan?", "How will we improve access and integration of clinical data across studies and countries?", "Molecular data have traditionally been stored in open access data resources available to all.", "By contrast, personal data is under controlled access depending on patient consent and is often difficult or impossible to access.", "Can we evolve a robust system to allow optimal use of available data globally, whilst retaining patient confidentiality, to address the medical challenges of an ageing population?" ]
[ "The surge of high-throughput technologies over the last decade has promoted the use of computational biology as a method to integrate and interpret the multi-omics information of ageing, diseases, and drugs.", "Ageing-related pathways in animal models are also involved in human diseases.", "Thus, drugs employed to treat human diseases potentially could be repurposed to ameliorate aspects of the ageing process and hence prevent more than one age-related condition in humans.", "Recently, several approaches using different data sources have been developed to prioritise prolongevity compounds for humans and for testing in animal models.", "The development of biorepositories, longitudinal cohort studies, and electronic health records is strengthening the bridge between the clinical and molecular data, thus enabling future research on human ageing." ]
Pompe disease treatment with twice a week high dose alglucoside alfa in a patient with severe dilated cardiomyopathy
There is limited information regarding ideal dosage of alglucoside alfa in patients with Infantile Onset Pompe Disease (IOPD). The U.S. Food and Drug Administration approved alglucoside alfa at dosing of 20 mg/kg every other week, but there are small studies and case reports suggesting that dosing higher than this leads to improved ventilator free survival and development without adverse events. We review the clinical course and short term clinical outcomes one year following late diagnosis of IOPD in a 3 month old who presented severely affected and was treated with 40 mg/kg twice a week for 21 infusions until six months of age then transitioned to 40 mg/kg/week. The patient responded well to 40 mg/kg twice a week treatment without adverse reactions and significant clinical improvement.
[ "BIOC" ]
[ "Alglucoside alfa", "Cardiomyopathy", "Enzyme replacement therapy", "Infantile onset Pompe disease", "Pompe disease" ]
[ "1310", "1311", "1312" ]
[ "Since enzyme replacement therapy (ERT) with alglucosidase alfa was approved by the U.S. Food and Drug Administration (FDA) in 2006, the outcome for children with Infantile Onset Pompe Disease (IOPD) (OMIM #232300) has changed dramatically and is constantly evolving.", "ERT has improved IOPD clinical outcomes by prolonging survival, increasing length of ventilator-free survival, improving cardiac function, and allowing patients to achieve previously unachievable motor milestones [1].", "The FDA approved dose of alglucosidase alfa is 20 mg/kg every other week (eow).", "Even with FDA approved dosing, 50% of treated infants require ventilator support by age three years [1].", "Limited research on small cohorts suggests that there are improved outcomes when ERT is administered with increased frequency at higher doses [1–3].", "Multiple studies in both humans and mice support potential benefits and safety of higher dosing [1,3–8].", "The data suggests that increasing the dose of ERT decreases the respiratory, cardiovascular, and motor decline [1,3].", "A higher initial dose of ERT has been shown to yield better outcomes [1,3].", "Dosing remains controversial with many centers varying dosing from between 20 mg/kg/eow up to 40 mg/kg/week.", "At our center all patients with IOPD are treated with 40 mg/kg/week.", "Patient was a 3 month, 3 week old male born at 35.2 weeks gestation with a past medical history significant for hypotonia, poor feeding and failure to thrive attributed to his prematurity.", "Prior to admission, he presented to his primary care physician for congestion, cough and rhinorrhea.", "This was the third presentation to the primary care doctor for these symptoms, which were attributed to recurrent upper respiratory infections.", "A chest radiograph was obtained at this time demonstrating an enlarged cardiac silhouette.", "An echocardiogram demonstrated ejection fraction of 20% and the patient was transferred to Children's Hospital of Wisconsin (CHW) for further care.", "Upon arrival to CHW, he was admitted to the cardiac intensive care unit and began treatment with diuretics (Aldactazide and Furosemide) and an Angiotensin Converting Enzyme (ACE) inhibitor (Enalapril).", "The suspicion for IOPD was high based on echocardiographic appearance and clinical examination.", "Alpha glucosidase enzyme activity was found to be low, consistent with a diagnosis of IOPD.", "The patient's heart failure worsened and milrinone was started on day six of hospitalization.", "ERT was also initiated on day seven of his hospitalization at 40 mg/kg/twice a week.", "ERT infusions were pretreated with 15 mg/kg Acetaminophen PO and 1 mg/kg Diphenhydramine IV due to concern that he did not have enough cardiac reserve to tolerate an infusion reaction.", "At this time, the Cross Reactive Immunological Material (CRIM) status was unknown and 0.4 mg/kg Methotrexate was given subcutaneously daily for three days starting immediately prior to the first ERT infusion to prevent antibody formation.", "Mild thrush and lymphopenia were noted and Leucovorin rescue was initiated on day 5 of treatment.", "Another dose of 0.4 mg/kg methotrexate was administered subcutaneously with the third ERT infusion (day 7 of treatment).", "Milrinone was discontinued on day 9 of treatment.", "He was found to be CRIM positive by genotype (c.525delT (p.Gly176Argfs*45)/c.2481 + 102_2646 + 31del (p.gly828_Asn882del)) on day 11 of treatment.", "He was started on a beta blocker (Carvedilol) on day 12 of treatment in addition to an ACE inhibitor (Enalapril) and diuretics (Aldactazide and furosemide after ERT).", "Leucovorin was discontinued on day 12 of treatment.", "The patient was transferred from the cardiac intensive care unit to a lower acuity medical unit on day 19 of treatment.", "He continued to progress well and the remainder of his hospitalization was uneventful.", "He never required respiratory support or supplemental oxygen.", "He was discharged day 32 of treatment.", "Urine Hex4 and ERT antibody levels were monitored monthly and are demonstrated in Fig. 1 [9].", "He was found to have silent aspiration and was reliant on nasogastric tube feeding until a gastrostomy tube was placed.", "The patient underwent intravenous catheter placement and gastrostomy tube placement at eight months of age.", "The changes in the patient's echocardiograms over time of treatment are illustrated in Fig. 2 and Fig. 3.", "At presentation he had a severely dilated left ventricle (Fig. 2B) with mild to moderate hypertrophy (Fig. 3B).", "Left ventricular mass index (LVMI) was 251 g/ht2.7.", "There was severely diminished left ventricular systolic function with an ejection fraction of 20% (Normal 55–70%).", "While right ventricular systolic function was normal, it was impinged upon by the left ventricular dilation.", "NT Pro Brain Natriuretic Peptide (NT Pro-BNP) at presentation was 19,800 pg/mL.", "After 16 ERT infusions (54 days of treatment), the echocardiogram demonstrated LVMI was 165 g/ht.", "2.7.", "There was similar levels of left ventricular dilation (Fig. 2C) and a mild decrease in left ventricular hypertrophy (Fig. 3C).", "NT Pro-BNP after 16 ERT infusions (57 days of treatment) was 2240 pg/mL.", "After four months of treatment LVMI was 121 g/ht2.7, left ventricular ejection fraction was 26%, and NT Pro-BNP was 207 pg/mL.", "The improvement in left ventricular dilatation is visible (Fig. 2D) and there is continued improvement of left ventricular hypertrophy (Fig. 3D).", "After one year and two weeks of treatment LVMI was 64 g/ht2.7 and left ventricular ejection fraction had improved up to 47% (normal 55–70%).", "The left ventricular dilation has significantly improved (Fig. 2E) and the left ventricular hypertrophy is not appreciable (Fig. 3E).", "At time of initiation of ERT patient had very little movement, no head control, was unable to move his limbs against gravity, and unable to make facial expressions to the point he was unable to keep his eyes closed while sleeping.", "The day after his first ERT infusion minimal facial expression was noted.", "Patient had a physical therapy evaluation after two infusions (5 days of treatment).", "In supine, he was unable to bring his hands together, bring his hands to his face, or move his low extremities against gravity.", "In prone, he was unable to lift or turn his head.", "In supported upright sitting, he demonstrated no head control.", "Patient was able to intermittently activate neck flexors and extensors in supported upright sitting, rotate head 45 degrees towards R and L in supine, flex shoulders to 25 degrees in supine, and demonstrated active palmar and plantar grasp.", "Following 3 infusions (8 days), patient had an occupational therapy evaluation.", "Patient able to actively grasp toy placed in hand, and actively flex shoulders to 45 degrees and maintain this position for 3 s in reclined sitting.", "Following five infusions (15 days), in supine, patient was able to actively flex and extend bilateral knees and hips, actively dorsiflex and plantarflex bilateral ankles, and bring bilateral hands to mouth.", "Patient also demonstrated active thumb opposition with grasp.", "After seven infusions (22 days), patient was able to lift and turn head when being held upright at caregiver's chest, and demonstrated active chin tuck in supported upright sitting.", "After eight ERT infusions (26 days), he demonstrated midline head control for up to 2 s in supported upright sitting, was able to turn his head from left to/from right in prone on surface, was able to hold a lightweight rattle for 2–3 s, and was able to maintain hands to mouth for 6–8 s.", "After nine ERT infusions (29 days), he was able to actively flex his lower trunk off surface in supine and maintain UE reach for 10 s in supine.", "After 10 ERT infusions (33 days) he was able to maintain midline head control for up to 3 s in supported upright sitting and was able to actively adduct hips against gravity in supine.", "There was a one week gap between infusion 10 and 11 due to a holiday.", "After 13 ERT infusions (47 days), patient was able to reach right hand to right upper leg in supine, and roll from supine to right sidelyinig independently.", "After 14 ERT infusions (47 days), patient was able to transfer toys between hands and began to inconsistently activate lateral neck muscles for lateral head righting.", "After 21 ERT infusions (75 days) he demonstrated “bobbing” head control for two minutes and five seconds.", "After 21 ERT infusions (75 days) he was taken to 40 mg/kg/weekly infusions.", "He continued to progress developmentally.", "He currently (16 months of age) can roll, sit independently, and propel self in an infant wheelchair.", "Reports by Chen Lei-Ru et al. demonstrate that systolic dysfunction in IOPD usually occurs after 5 months of age [10].", "They also note that cardiac remodeling can be unpredictable after 5 months of age particularly in the presence of systolic dysfunction and these patients seem to have a less favorable prognosis.", "They speculate this is due to a loss of myofibrils caused by accumulation of glycogen in the cytosol resulting in systolic dysfunction [10].", "There was concern that the driver of the poor response was the process creating the systolic dysfunction as compared to the age as reported in the study [10].", "Given the severity of systolic dysfunction noted in our patient at only 3 months of age we treated aggressively with a dose of 40 mg/kg/twice weekly to try to promote maximal cardiac remodeling to occur.", "In addition to significant cardiac dysfunction the patient had severe hypotonia, and while at time of presentation he had adequate respiratory function it was not clear if respiratory dysfunction would evolve.", "A study comparing 20 mg/kg/eow to 40 mg/kg/week found that patients on 40 mg/kg/week had fewer respiratory infections and hospitalizations, and that patients whose dose was increased had decreases in respiratory infection following dose increase [1].", "This study also found suggestion that higher dosing may be more beneficial in patients with more severe IOPD [1].", "Studies looking at the level of alpha glucosidase activity within skeletal muscle also support using higher doses of alglucosidase alfa suggesting skeletal muscle activity only normalized with 40 mg/kg/week dosing [7,8,11].", "Given this patient's level of decompensation and cardiac dysfunction, preservation of respiratory function and reduction of infection risk was critical until his cardiac function improved to a more stable level and contributed to the rationale of a dose of 40 mg/kg/kg twice a week.", "When therapy was initiated the initial plan had been to give 40 mg/kg twice a week for two weeks and then decrease the dose to weekly.", "The duration of 40 mg/kg twice a week treatment was extended based on the significant response and significant improvements the patient had in a rapid timeframe.", "We speculate that this increased dose may have decreased the length of stay the patient required in the cardiac intensive care unit (19 days after starting treatment) and his total hospital length of stay (discharged 32 days after starting treatment).", "There were no adverse events in our patient, it should be noted that the drug was maximally concentrated and diuretic dosing was given after the infusion to minimize risk for volume overload.", "We suggest that administering 40 mg/kg twice a week alglucosidase alfa in severely affected and decompensated patients with IOPD is a reasonable consideration to aid in recovery.", "Our patient demonstrated increased levels of Hex4 following transition to 40 mg/kg/week and it is possible that 40 mg/kg twice a week suggesting twice a week dosing kept him under better control.", "While this was true for our patient there is substantial clinical variation among IOPD patients and there have been patients with documented cardiac improvement on lower dosing [10]." ]
[]
Vascular Fibrosis in Aging and Hypertension: Molecular Mechanisms and Clinical Implications
Aging is the primary risk factor underlying hypertension and incident cardiovascular disease. With aging, the vasculature undergoes structural and functional changes characterized by endothelial dysfunction, wall thickening, reduced distensibility, and arterial stiffening. Vascular stiffness results from fibrosis and extracellular matrix (ECM) remodelling, processes that are associated with aging and are amplified by hypertension. Some recently characterized molecular mechanisms underlying these processes include increased expression and activation of matrix metalloproteinases, activation of transforming growth factor-β1/SMAD signalling, upregulation of galectin-3, and activation of proinflammatory and profibrotic signalling pathways. These events can be induced by vasoactive agents, such as angiotensin II, endothelin-1, and aldosterone, which are increased in the vasculature during aging and hypertension. Complex interplay between the "aging process" and prohypertensive factors results in accelerated vascular remodelling and fibrosis and increased arterial stiffness, which is typically observed in hypertension. Because the vascular phenotype in a young hypertensive individual resembles that of an elderly otherwise healthy individual, the notion of "early" or "premature" vascular aging is now often used to describe hypertension-associated vascular disease. We review the vascular phenotype in aging and hypertension, focusing on arterial stiffness and vascular remodelling. We also highlight the clinical implications of these processes and discuss some novel molecular mechanisms of fibrosis and ECM reorganization.
[ "MEDI" ]
[]
[ "2705" ]
[ "With aging, the vasculature undergoes functional, structural, and mechanical changes characterized by endothelial dysfunction, thickening (remodelling) of the vascular wall, and increased stiffening, respectively (Fig. 1).", "These changes result in a reduced capacity of arteries to adapt to tissue demands and accordingly may lead to ischemic injury.", "Preclinical and clinical studies have clearly demonstrated that with aging, there is impaired endothelium-dependent vasorelaxation with associated increased permeability and vascular inflammation.", "Epidemiologic, cross-sectional, clinical, and postmortem studies in healthy individuals of variable ages have clearly demonstrated that intimal wall thickening and dilatation are noticeable structural changes that occur in conduit arteries with advanced age.", "Findings from noninvasive vascular phenotyping studies in healthy individuals have demonstrated that intima-media thickness increases 2- to 3-fold between 20 and 90 years of age.4", "Studies in aging nonhuman primates also showed a relationship between intimal thickness in the thoracic aorta and aging.5", "Exact factors causing progressive intimal thickening with aging in otherwise healthy individuals remain elusive, but a number of distinctive changes at the cellular and morphologic levels have been identified, including fracture of elastin fibres within the tunica media, increased collagen deposition, cellular senescence, and dysregulated cell proliferation.", "Associated with these events is remodelling of the ECM, which is an essential component of the connective tissue surrounding the vascular wall.", "The ECM is composed of basic structural elements (collagen and elastin) and more specialized proteins including fibronectin and proteoglycans.", "The ECM is a dynamic structure and its components are continuously being turned over through highly regulated systems involving activation of MMPs and TIMPs.", "Dysregulation of these processes, together with alterations in profibrotic and proinflammatory signalling pathways, likely contribute to aging-associated vascular structural changes.", "The authors have no conflicts of interest to disclose.", "The overall vascular phenotype of an individual at any 1 time depends not only on “aging” but also on a combination of multiple interacting factors, such as genetic factors, diet, smoking, diabetes, dyslipidemia, oxidative stress, and obesity.6,7", "Moreover, in the presence of prohypertensive factors, there is acceleration of aging-associated vascular changes that leads to exaggerated vascular injury and arterial stiffening.", "In susceptible individuals, the interplay between aging and hypertension leads to “early vascular aging” and arterial stiffness, in which the vascular phenotype in young hypertensive individuals resembles that of elderly otherwise healthy individuals (Fig. 1).", "Normally, conduit arteries distend to accommodate large pressure ejections from the heart during systole to facilitate perfusion to tissues during diastole.", "This is determined in large part by the elasticity, distensibility, and compliance of the arterial system.", "Loss of elasticity and increased stiffness demand greater force to accommodate blood flow, leading to increased systolic blood pressure, increased cardiac work load, and consequent cardiac hypertrophy and risk of cardiovascular events.", "Aortic stiffness also affects the microcirculation and vice versa.7,8", "Aortic wall stiffening causes increased pulse wave velocity (PWV) and premature reflected waves with elevated central hemodynamic load leading to damage of peripheral small arteries.9", "Remodelling of small arteries in turn leads to increased peripheral vascular and pulse wave reflection, which can further contribute to aortic stiffness.10", "Arterial stiffness can be assessed by measuring PWV, pulse wave analysis, ambulatory arterial stiffness (using 24-hour ambulatory blood pressure monitoring) and evaluating endothelial function (flow-mediated dilation).", "PWV is the most commonly used approach and measures the speed of the pressure pulse from the heart as it is propagated through the arteries; it is calculated by dividing the distance travelled by the time taken to travel the defined distance.", "Stiffer arteries result in a more rapid travel time and hence a higher PWV.", "Various approaches can be used to measure PWV, including applanation tonometry, oscillometry, Doppler echocardiography, and magnetic resonance imaging.", "Although the measurement of PWV is considered to be the most simple, noninvasive, robust, and reproducible method to determine arterial stiffness,11 it is not yet used in routine clinical practice.", "Carotid-femoral PWV is a direct measure of aortic stiffness and is now considered the gold standard for its evaluation in clinical and epidemiologic studies.12", "Arterial stiffness is a natural consequence of advancing age and is accelerated in hypertension.", "It is also an independent predictive risk factor for cardiovascular events and, as such, aortic PWV is now recognized as an important biomarker in the determination of cardiovascular risk.", "Arterial stiffness has a bidirectional causal relationship with blood pressure, because high blood pressure causes arterial wall injury, which promotes stiffening, whereas arterial stiffening itself is the major cause of increased systolic blood pressure, especially in the elderly,8,13 Multiple interacting factors at the systemic (blood pressure, hemodynamics), vascular (vascular contraction/dilatation, ECM remodelling), cellular (cytoskeletal organization, inflammatory responses), and molecular (oxidative stress, intracellular signalling, mechanotransduction) levels contribute to arterial stiffness in aging and hypertension.", "Dysregulation of endothelial cells, vascular smooth muscle cells, and adaptive immune responses has also been implicated in arterial aging and vascular damage in hypertension.", "A detailed discussion of all these mechanisms is beyond the scope of this review and is addressed elsewhere this issue of the Canadian Journal of Cardiology.14", "Here we focus on some molecular and cellular events that contribute to vascular fibrosis and ECM remodelling.", "The ECM is an essential component of the connective tissue that surrounds cells.", "In addition to maintaining cellular and vascular integrity, it plays a fundamental role in cell signalling and regulation of cell-cell interactions.", "The ECM comprises multiple structural proteins, including collagens, elastin, fibronectin, and proteoglycans.", "Composition of the ECM varies from organ to organ, with collagen types I and III representing the predominant isoforms in the vascular ECM.15", "The absolute and relative quantities of collagen and elastin determine biomechanical properties of vessels, in which an elastin deficiency/collagen excess leads to vascular fibrosis and increased stiffness.4,15", "In healthy individuals, collagen deposition and turnover are tightly regulated, and the ratio of collagen to elastin remains relatively constant.", "However, an imbalance in these processes leads to excessive ECM protein deposition, particularly collagen and fibronectin, contributing to vascular fibrosis and stiffening in aging and during the development of hypertension.15", "Collagens are particularly important in these processes because they are the most abundant and stiffest of the ECM proteins.", "Increased collagen content and destruction of the elastin fiber network together with a proinflammatory microenvironment contribute to ECM remodelling and increased intima-media thickening and vascular stiffness in small and large arteries in human and experimental hypertension.", "Contributing to the profibrotic process is transglutaminase (TG2), which is secreted into the ECM, where it catalyzes formation of ε-(γ-glutamyl)lysine isopeptide, in a Ca2+-dependent manner.16", "TG2 acts as an extracellular scaffold protein as well as a cross-linking enzyme.", "Numerous ECM proteins are TG2 substrates, such as fibronectin, collagen, and laminin.16", "Under physiological conditions, TG2 regulates fibroblast activity and ECM organization, with little protein cross-linking.", "However, in pathologic conditions, increased TG2/ECM protein crosslinking and altered TG2 activity cause increased rigidity and stiffening of the vascular wall, processes that may contribute to remodelling in aging and cardiovascular disease.", "Recent evidence indicates altered TG2 activity and functionality in large arteries of hypertensive rats.17", "TG2 dysregulation has also been implicated in small-vessel changes and inward remodelling in hypertension.18", "Fundamental to many of the processes underlying ECM reorganization and fibrosis in aging and hypertension is activation of MMPs and TIMPs.", "ECM proteins, including collagen and elastin, are regulated by MMPs, a family of endopeptidases, which are activated by many factors associated with aging and hypertension, such as proinflammatory signalling molecules (cytokines, interleukins), growth factors, vasoactive agents (Ang II, ET-1, aldosterone) and ROS.", "MMP activity is controlled at 3 levels: gene transcription, proenzyme activation, and activity inhibition.18", "Signalling pathways involved in regulating MMP transcription include p38 MAPK, which can enhance or repress MMP expression in a cell type–dependent manner (Fig. 2).", "Commonly, MMPs are activated in the pericellular space by other MMPs, including membrane-type MMPs and MMP-3, or by serine proteases like plasmin and chymase.", "Activated MMPs degrade collagen, elastin, and other ECM proteins, resulting in a modified ECM, often associated with a proinflammatory microenvironment that triggers a shift of endothelial and vascular smooth muscle cells to a more secretory, migratory, proliferative, and senescent phenotype, which contributes to fibrosis, calcification, endothelial dysfunction, and increased intima-media thickness, further impacting on vascular remodelling and arterial stiffness.", "The effect that MMPs have on vascular fibrosis in hypertension is not completely elucidated, with both inhibitory and stimulatory modulation observed.19", "This probably relates to activation of different MMP isoforms and downstream signalling pathways.", "For instance, MMP-1 overexpression attenuates fibrosis,20 whereas MMP-9 activation potentiates fibrosis and DNA damage.21", "MMP2 activation leads to stimulation of TGF-β1 signalling; increased vascular smooth muscle cell production of collagens I, II, and III; and increased fibronectin secretion, processes that lead to collagen accumulation in the vascular wall.", "Although activation of vascular MMP2 and MMP9 in hypertension is associated with collagen accumulation, activation of MMP8 and MMP13 is associated with collagen degradation, processes especially important in arterial wall plaque and plaque rupture.22,23", "MMP2/MMP9 activation through TGF-β1/SMAD signalling also induces activation of myofibroblasts and increased infiltration of monocytes/macrophages, leading to oxidative stress, inflammation, and vascular wall injury.", "Vascular MMP2 and MMP9 are activated by numerous prohypertensive factors, including Ang II, ET-1, and salt, as well as mechanical and physical factors, such as shear stress and pressure.", "MMP2, MMP7, MMP9, and MMP14 are upregulated by aging.", "MMP2 activation is increased in aged rat aorta, leading to increased TGF-β1 and SMAD activation.24", "Young rats infused with Ang II exhibit increased MMP2 activation with intima-media thickness and vascular fibrosis changes that are typical in old untreated rats.24", "The importance of MMPs in vascular fibrosis in aging and hypertension is further evidenced by MMP inhibitors, such as PD166793, which blunted age-associated vascular fibrosis and remodelling in experimental models.25,26", "MMPs are normally inhibited by endogenous inhibitors called TIMPs, of which there are multiple isoforms.", "Alterations in the balance between ECM MMPs and TIMPs may contribute to the profibrotic phenotype in aging and hypertension.19,24", "The 4 TIMP isoforms—TIMP1, TIMP2, TIMP3, and TIMP4—are responsible for the inhibition of > 20 MMPs, and the relationship between MMPs and TIMPs changes with age.", "For instance, increased MMP2 expression and activity is observed in vessels of old rats and nonhuman primates compared with young counterparts.5,27", "Furthermore, TIMPs are downregulated in aged animals with heart failure but not in young animals.28", "The TGF-β superfamily consists of > 40 members that share common sequence elements and structural motifs and includes TGF-β, bone morphogenetic protein, activin, inhibin, and growth differentiation factors.29-32", "Disruption of the TGF-β pathway has been implicated in arterial aging and vascular fibrosis.29-32", "Three isoforms (TGF-β1, TGF-β2, and TGF-β3) exist; TGF-β1 is most frequently upregulated in ECM remodelling and fibrosis and is consequently regarded as an important regulator of the ECM.", "In the vascular system, TGF-β1 is expressed in endothelial cells, vascular smooth muscle cells, myofibroblasts, and adventitial macrophages.", "Activation of vascular TGF-β1, and its downstream signalling effector SMAD, increases the synthesis of ECM proteins such as fibronectin, collagen, and plasminogen activator inhibitor-1 (PAI-1).33,34", "TGF-β reduces collagenase production and stimulates expression of TIMPS, resulting in excessive matrix accumulation, in part resulting from inhibition of ECM degradation.35", "TGF-β signalling predominantly occurs through the cytoplasmic proteins, SMADs, which translocate to the nucleus and act as transcription factors.", "The SMAD family comprises receptor-activated SMADs (SMAD2, SMAD3, SMAD5, and SMAD8), inhibitory SMADs (SMAD6, SMAD 7) and common-partner SMADs (SMAD4).", "SMAD2 and SMAD3 are specific mediators of TGFβ/activin pathways, whereas SMAD7 inhibits both BMP and TGF-β/activin signalling.", "SMAD activation results in increased transcription of many genes involved in ECM formation, including fibronectin, procollagens, PAI-1, and connective tissue growth factor (CTGF).32", "In vascular smooth muscle cells, overexpression of SMAD7 inhibits TGF-β–induced fibronectin, collagen, and CTGF production.36", "Important non-SMAD pathways implicated in TGF-β profibrotic signalling include extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 MAPK, and phosphoinositide 3-kinase/Akt.37", "SMAD translocation to the nucleus can be modulated by Ras-activated ERK1/2.", "ERK inhibition reduces TGF-β–stimulated SMAD phosphorylation as well as collagen production, suggesting that ERK activation is necessary for an optimal response to TGF-β1.36", "Activation of TGF-β1 and receptor-mediated signalling are increased in the aortic wall with aging and during development of hypertension.24", "Important in the context of these conditions, Ang II,38,39 mechanical stress,34,40 ET-1,36and ROS41 are all elevated and are known to mediate TGF-β activation, with resultant vascular fibrosis.", "Additionally, MMPs (particularly MMP2 and MMP9) enhance release of TGF-β1, whereas TGF-β1 stimulates TIMP, resulting in inhibition of ECM degradation, which further induces ECM accumulation and vascular remodelling and fibrosis.", "Ang II can activate the SMAD pathway independent of TGF-β1, with implications for fibrosis.36,42", "Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitor (serpin) gene family and functions as an inhibitor of the serine proteases, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA).", "PAI-1 inhibits fibrinolysis and hence regulates dissolution of fibrin and inhibits degradation of the ECM by reducing plasmin generation.", "PAI-1 normally maintains tissue homeostasis through regulating the activities of uPA, tPA, plasmin, and MMPs.", "In pathophysiological conditions, PAI-1 upregulation contributes to accumulation of ECM proteins and tissue fibrosis by preventing tissue proteolytic activity and reducing collagen degradation.", "Together with increased TGF-β1 activity, PAI-1 activity and expression are increased in experimental models of aging and in aged individuals.43,44", "PAI-1 is upregulated in aging-associated pathologic conditions, including hypertension.45", "Increased PAI-1 is also recognized as a biomarker of cellular senescence in aging and hypertension.46", "CTGF is a 38-kDa cysteine-rich secreted potent profibrotic factor implicated in fibroblast proliferation, cellular adhesion, and ECM synthesis.", "CTGF expression in the vasculature is enhanced by several stimuli, including TGF-β1, tumor necrosis factor-α, and mechanical stress.47", "Ang II–induced vascular fibrosis is mediated by CTGF, and vascular smooth muscle cells treated with CTGF antisense oligonucleotides are protected against agonist-induced ECM protein expression.36,48", "CTGF may play an important role in arterial aging and vascular fibrosis; a number of experimental models have demonstrated increased levels of CTGF and associated vascular fibrosis with increasing age.49,50", "Galectin-3 (LGALS3) is a 29- to 35-kDa carbohydrate-binding lectin expressed on the cell surface of many cell types, including fibroblasts and endothelial and inflammatory cells.", "It is secreted mainly by activated macrophages, and it is ligand activated by oligosaccharides.", "Galectin-3 is also activated by other ligands, including glycosylated matrix proteins such as laminin, collagen, elastin, fibronectin, and integrin.", "The cellular actions of galectin-3 lead to cell proliferation, adhesion, and fibrosis.", "Galectin-3 has been shown to play an important role in fibrosis and tissue remodelling.", "In heart failure, plasma galectin-3 levels are increased.51", "In the recent Prevention of Renal and Vascular End-Stage Disease (PREVEND) study in which plasma galectin-3 levels were measured in 7968 individuals, plasma levels correlated positively with increasing age and cardiovascular risk factors, including hypertension.52", "Because of its role in fibrosis, galectin-3 is now considered by many to be an important biomarker of cardiovascular fibrosis.", "The precise mechanisms through which galectin-3 influences ECM remodelling and fibrosis are still unclear, although activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and protein kinase C (PKC) pathways,53,54 as well as oxidative stress and inflammation, have been suggested.", "In addition, galectin-3 may directly increase production of ECM proteins.", "In rat vascular smooth muscle cells, overexpression of galectin-3 enhanced aldosterone-induced collagen 1 synthesis, whereas spironolactone or modified citrus pectin (galectin-3 inhibitor) reversed these effects.55", "Galectin-3 inhibition also attenuated cardiovascular fibrosis and left ventricular dysfunction in a mouse model of heart failure.56", "Many vasoactive factors activate profibrotic pathways, including Ang II, ET-1, and aldosterone (Figs. 2 and 3).", "Downstream signalling involves activation of redox-sensitive genes and transcription factors, early growth response factor-1, and activation of TGF-β1, MMPs, galectin-3, and MAP kinases.57-61", "The aging vasculature is characterized by increased levels of Ang II,5 angiotensin-converting enzyme,17,31,61 mineralocorticoid receptors,62 and endothelin-converting enzyme-1.63,64", "As such, increased levels of these factors, their receptors, and downstream targets could represent an important event during aging that leads to vascular stiffness.", "The renin-angiotensin-aldosterone system plays a central role in structural and mechanical changes in the vasculature.", "Ang II acts through activation of 2 receptors—AT1 and AT2—in which AT1 plays a major role in the production of ECM proteins.65-68", "This is highlighted by studies demonstrating that antagonism of Ang II receptors results in decreased fibrosis.69,70", "The precise signalling events involved in Ang II-induced vascular fibrosis are incompletely determined; however, in mesangial cells, TGF-β1 activity is increased by Ang II, an effect not observed when activator protein 1 binding sites or PKC- and p38 MAPK–dependent pathways are inhibited.65", "In addition, galectin-3 seems to be associated with Ang II–induced fibrosis, and its expression is related to the severity of renal dysfunction in aging; mice subjected to Ang II infusion develop cardiac fibrosis,71 an effect not observed in galectin-3 knockout animals.", "Furthermore, cultured fibroblasts exposed to galectin-3 have reduced collagen production and deposition.60", "Ang II–induced activation of p38 MAPK is also associated with the development and progression of fibrosis, commonly observed in aging and hypertension.72-74", "It has been suggested that Ang II induces activity of MMPs and TIMPs and upregulation of CTGF during aging.75-80", "Accumulating evidence implicates aldosterone as an important pathophysiological mediator in cardiovascular remodelling by promoting vascular hypertrophy, fibrosis, inflammation, and oxidative stress.81-83", "Evidence from animal models and clinical trials of heart failure and hypertension demonstrate that chronic blockade of mineralocorticoid receptors, through which aldosterone signals, reduces cardiovascular fibrosis.", "In rats, aldosterone infusion increases aortic media cross-sectional area associated with elevated collagen levels, particularly increased collagen I synthesis.84,85", "In the context of aging, aldosterone levels have been shown to decline in older age.86,87", "This is associated with increased expression of mineralocorticoid receptors in intact vessels, as well as in cultured vascular smooth muscle cells, and has been shown to correlate with markers of vascular fibrosis.62", "Whether increased signalling through mineralocorticoid receptors plays a role in vascular fibrosis associated with aging has yet to be confirmed.", "ET-1 is a secreted peptide produced primarily in endothelial cells after conversion of preproendothelin to proendothelin and subsequently to mature endothelin, which has potent vasoconstrictor activity.", "The vascular actions of ET-1 are mediated by 2 distinct endothelin receptor subtypes: the ETA and ETB receptors located on both vascular smooth muscle and endothelial cells.", "In addition to well-established hypertrophic and mitogenic properties, ET-1 can modulate ECM remodelling by stimulating fibroblast-induced collagen synthesis.", "ET-1 stimulates synthesis of collagen through both ETA and ETB receptor subtypes.88,89", "Reduced cardiac and renal MMP activity and expression has been reported after administration of ETA receptor antagonists.90-92", "Similarly, treatment with an endothelin antagonist normalizes expression of the collagen I gene and leads to the regression of renal vascular fibrosis and improved survival.93", "Numerous findings have reported elevated ET-1 levels in healthy older adult humans.94,95", "In cultured aortic endothelial cells, ET-1 synthesis is greater in cells obtained from older donors vs young adult donors.96", "In Wistar-Kyoto (WKY) rats, aging is associated with a 3.6-fold elevation in kidney ET-1 protein expression in the kidney.", "In rodent models, dual ETA/ETB receptor antagonism had no effect on the age-associated increase in aortic MMP-2 activity in WKY rats but markedly reduced pro and active MMP-2 activity in aged hypertensive rats, demonstrating that ET-1 may represent an important mediator of vascular stiffness in aging in the presence of other vascular diseases.63", "With aging, the vasculature undergoes structural and functional changes characterized by arterial remodelling, vascular fibrosis, and stiffening, which are processes that are evident in aging and hypertension.", "Arterial stiffening is common, occurring in > 60% of individuals older than 70 years and is a major independent predictor for serious cardiovascular events.", "Accordingly, there is a need to understand the fundamental processes that cause vascular stiffness so that mechanism-based therapeutic strategies can be developed to ameliorate or prevent processes of “vascular aging” in hypertension and associated cardiovascular diseases.", "Arterial stiffening is caused primarily by excessive fibrosis from excessive accumulation of vascular collagen and degradation of elastin.", "It is a dynamic phenomenon, which initially is an adaptive repair response that is reversible.", "However, the fibrogenic process is progressive, leading to further worsening of arterial stiffness and fibrosis that gradually extends into the neighbouring interstitial space, causing tissue and organ damage.", "A number of noninvasive methods are currently available to evaluate large-artery stiffness in the clinical setting, including carotid-femoral PWV.", "Increased PWV in aging and hypertension reflects increased arterial stiffness and is emerging as a biomarker for cardiovascular risk stratification.", "Perhaps over the next decade, PWV assessment may become a routine investigation in the clinical tool kit to better predict hypertension and cardiovascular disease.", "This work was supported by grants from the British Heart Foundation (BHF) (RG/13/7/30099).", "R.M.T. is supported through a BHF Chair (CH/12/4/29762) and R.A.L. is supported by a PhD scholarship from FAPESP-Brazil (2012/12178-6)." ]
[]
Influence of service loading and the resulting micro-cracks on chloride resistance of concrete
Chloride-induced corrosion of steel in reinforced concrete structures is one of the main problems affecting their durability, but most previous research projects and case studies have focused on concretes without cracks or not subjected to any structural load. Although it has been recognised that structural cracks do influence the chloride transport and chloride induced corrosion in reinforced concrete structures, there is little published work on the influence of micro-cracks due to service loads on these properties. Therefore the effect of micro-cracks caused by loading on chloride transport into concrete was studied. Four different stress levels (0%, 25%, 50% and 75% of the stress at ultimate load - fu) were applied to 100 mm diameter concrete discs and chloride migration was measured using a bespoke test setup based on the NT BUILD 492 test. The effects of replacing Portland cement CEMI by ground granulated blast-furnace slag (GGBS), pulverised fuel ash (PFA) and silica fume (SF) on chloride transport in concrete under sustained loading were studied. The results have indicated that chloride migration coefficients changed little when the stress level was below 50% of the fu; however, it is desirable to keep concrete stress less than 25% fu if this is practical. The effect of removing the load on the change of chloride migration coefficient was also studied. A recovery of around 50% of the increased chloride migration coefficient was found in the case of concretes subjected to 75% of the fu when the load was removed.
[ "ENGI", "MATE" ]
[ "Chloride transport", "Load effect", "Micro-cracks", "Service life", "Stress level" ]
[ "2205", "2215", "2500" ]
[ "Chloride-induced corrosion of steel in reinforced concrete structures is one of the most serious problems affecting their service life and if unattended it can put the infrastructure at risk and endanger people’s lives.", "An understanding of the processes of chloride transport in concrete is very important for engineers attempting to predict the service life of reinforced concrete structures and numerous studies dealing with this topic can be found in the literature [1–9].", "However, most of them have focused on concretes which are neither cracked nor subjected to any structural loading.", "The work by Gowripalan et al. [10] showed that chloride diffusivity of concrete in the compression zone was much smaller than that in the tension zone when concrete beams were subjected to flexure.", "Win et al. [11] suggested that chloride can readily penetrate into the concrete through an open crack.", "Other studies on the influence of crack width on chloride transport [12–15] showed that the influence of micro-cracks on chloride ingress can be significant.", "Although it has been recognised that structural cracks do influence the chloride transport and chloride induced corrosion of steel in reinforced concrete structures, there is little published work on the influence of micro-cracks due to service loads on these properties.", "In some studies [12,16–21] chloride transport tests on concrete have been conducted after the load was removed, but the results of concrete under loading and after the load is removed can be different.", "Based on tests conducted under sustained compressive loading, Antoni et al. [22,23] reported a significant increase of chloride transport into plain concrete when the compressive stress level was above 50% of the ultimate stress – fu.", "In contrast, based on a numerical simulation a linear decrease of chloride diffusion in concrete under sustained compressive stress from an unloaded state up to 50% fu was reported by Wang et al. [24].", "It was found that some conflicting views exist in the literature on critical stress level and chloride transport properties of concretes under different loading conditions.", "Further, most of the work focused on concrete containing only Portland cement CEMI [24,25], and data on concrete with mineral additives are still limited.", "The existing service life prediction models, such as Life-365 [26,27], AGEDDCA model [28] and DuraCrete [29], do not consider any effect of loading or consequential cracking (macro-cracking or micro-cracking; micro-cracks are defined below as cracks <0.1 mm wide), hence the predicted service life may not be reliable for most structures in service.", "The effect of stresses and micro-cracks on chloride transport is not considered in the current Standards.", "The Eurocode 2 [30] restricts the crack width to 0.3 mm for reinforced concrete in chloride-laden environments but has no restrictions on concretes with small cracks (<0.3 mm) or micro-cracks (<0.1 mm).", "As a consequence, premature deterioration of structures in service is common.", "For example, on the basis of a field survey of 57 bridges in Kansas, USA, Lindquist et al. [31] have indicated that the chloride concentration at a depth of 76 mm from the location of a surface crack can exceed the corrosion threshold amount within the first year.", "Another survey of 219 marine structures along the Norwegian coastline has shown that the signs of corrosion could be seen as early as 5–10 years, which means that the time for corrosion initiation was even earlier [32].", "Almost all of these structures required major repair at the age of 20 years, which is significantly earlier than their design life of 100 years.", "Therefore, the influence of micro-cracks due to structural loads on the chloride transport in concrete was investigated and thereby recommendations for any change of service life designs of such structures are made.", "As concrete structures exposed to chloride environments are manufactured with cementitious materials containing mineral additives, the effect of such cementitious materials on micro-cracking under sustained loading and its consequential influence on chloride transport were part of the research reported in this paper.", "The coarse aggregate used was crushed 5–20 mm well graded basalt (size 5–10 mm 33% and size 10–20 mm 67%) and the fine aggregate was natural medium graded sand conforming to BS EN 12620:2002 [33].", "Both types of aggregates were oven-dried at 100 ± 5 °C for 24 h to remove the initial moisture content and then allowed to cool down to room temperature (∼18 °C) in air-tight containers before being used.", "Class 42.5 N Portland cement CEMI conforming to BS EN 197-1:2011 [34] was used.", "Ground granulated blast-furnace slag (GGBS) complying with BS EN 6699: 1992 [35], pulverised fuel ash (PFA) complying with BS EN 450 [36] and silica fume (SF) conforming to BS EN 13263 [37] were used to replace part of CEMI in the mixes studied.", "The proportions for each mix, along with their physical properties, are reported in Table 1.", "As can be seen from Table 1, some of the mixes were added with a polycarboxylate polymer-based superplasticiser conforming to BS EN 934-2 [38].", "The solid content of the superplasticiser was 29%.", "The water content of superplasticiser was considered to be part of the mixing water and hence this quantity was deducted from the water content reported in Table 1.", "Water from the mains at a temperature of 20 (±1) °C was used to manufacture all mixes.", "Four blocks (250 × 250 × 80 mm) and six 100 mm cubes were cast for each mix.", "The required quantities of the mix constituents were batched by mass and then mixed using a pan mixer in accordance with BS 1881-125: 1986 [39].", "The slump was measured immediately after batching the concrete.", "The concrete was placed in the moulds in three layers and each layer was compacted using a vibrating table until air bubbles appearing on the surface stopped.", "The cast surface of each concrete specimen was then finished with a trowel and covered by a thick black polythene sheet to prevent any loss of water.", "The samples were demoulded after 24 h, they were then placed in a water bath at 20 (±1) °C for 3 days, after which they were wrapped in wet hessian and thick black polythene sheet.", "The specimens were then placed in a constant temperature room at 20 (±1) °C.", "The cubes were used to carry out the compressive strength test at an age of 28 and 56 days for each mix, the results of which have already been reported in Table 1.", "Cores of size 100 mm diameter and 80 mm thickness were cut from the blocks at an age of 56 days and the outermost, approximately 15 mm thick layer, was cut off from the two end surfaces using a water-cooled diamond saw.", "This was done to eliminate the effects of the boundary layer on chloride transport and ensure a smooth test surface of the samples.", "The middle part of 50 (±1) mm was thus used as the test specimen.", "The thickness of each specimen was measured with a digital calliper to an accuracy of 0.1 mm.", "The chloride migration test was carried out using these disks.", "In order to test them under sustained loading, they were compressed by two diametrically opposite loads by using a special setup that was designed and manufactured, as shown in Figs. 1 and 2.", "This test setup is novel in the sense that it was not used before and was designed to stimulate micro-cracking of reinforced concrete under the sustained loading condition.", "Samples were saturated as per the preconditioning procedure outlined in NT BUILD 492 [40].", "The cores were placed in a vacuum container after they were surface-dried, by making sure both end surfaces were exposed.", "The pressure inside was reduced to 30 (±5) mbar and the vacuum was maintained for three hours.", "Then the container was filled with Ca(OH)2 solution to immerse all specimens, with the pump still running.", "The vacuum was maintained for a further hour before allowing air to enter.", "The cores were kept in the solution for another 18 h, after which they were taken out for placing in the test setup.", "The cores were then coated on the curved surface with a silicon coating material and clamped in position (Fig. 2).", "After the silicon had hardened, the specimens were loaded at a constant rate of 1.23 (±0.1) kN/s until failure.", "The average load of the three failure loads in each mix was taken as the maximum load and the related stress was taken as the ultimate stress – fu for each mix.", "The typical crack pattern at the failure of the core specimen is shown in Fig. 3.", "Three loading conditions (Control – no loading, under loading and after unloading) were utilised.", "Fig. 4 shows that cores under different loading conditions were treated separately before testing.", "The load was removed after 1 min of loading for the After Unloading condition and UPV tests were conducted before the load was applied and after the load was removed.", "The load was kept constant throughout the test period for the under loading condition, UPV tests were conducted before and after the load was applied.", "No load applying and UPV tests for the Control condition.", "Different stress levels (equivalent to 25%, 50%, and 75% of fu) were applied to the cores by fastening the nuts at the top of the brackets using a torque wrench.", "Strain gauges placed on bolts were used to determine the forces in the bolts, indicated in Fig. 2.", "An ultrasonic pulse velocity (UPV) test was conducted before and after the load was applied.", "Control: Specimens were ready for testing and no UPV test was conducted.", "Under loading: Different levels of load were applied and maintained for the tests conducted.", "An UPV test was conducted before and after the load was applied.", "After unloading: Different levels of load were applied and kept constant for 1 min, and then the load was released.", "UPV tests were conducted before applying and after releasing the load.", "Fig. 6 shows a case of typical changes in Dnssm due to the applied load.", "There is almost no change of Dnssm when the stress level increased to 25% fu; above this level and up to 50% fu, Dnssm increases a little; with the stress level increasing up to 75% fu, a significant increase of Dnssm is obtained.", "No visible cracks were observed in any of the specimens after testing.", "Mehta and Monteiro [42] have suggested that there is no change in behaviour of concrete up to 30% of the ultimate stress, slight damage to the structure between 30% and 50% of the ultimate stress, significantly increased damage between 50% and 75% of the ultimate stress, and the system becomes unstable beyond 75% of the ultimate stress.", "The changing behaviour of Dnssm of 0.65-CEMI concrete presented in Fig. 6 agrees well with the crack developing stages of CEMI concrete under compressive loading suggested by Mehta and Monteiro [42].", "Below 25% fu is within the elastic range of concrete, and there is no development of micro-cracks.", "Around 50% fu, there is a stable development of micro-cracks in the interfacial transition zone.", "Up to 75% fu, cracks begin to form and increase in the matrix and the crack system in the interfacial transition zone becomes unstable.", "Sustained loading above 75% fu should be avoided because the crack systems of concrete become extremely unstable and can easily fail [42].", "As shown in Fig. 6, a significant difference can be found between the values of Dnssm in concretes before and under loading.", "Concrete structures have to sustain service loading, such as live and dead loads; therefore, the stress level is an important factor to consider in the context of chloride ingress.", "The effect of loading on chloride ingress into concrete can be considered in the design stage of reinforced concrete structures if the range of the actual stresses on the structure is known.", "Typical relationships between characteristic values and design values of the concrete strength and the stress are shown in Fig. 7.", "The maximum stress level of concrete according to the design standard Eurocode 2 can be estimated as fd/fck.", "Then the maximum design stress level of in-service concrete can be calculated as in Eqs. (4) and (5).", "From the above calculation, the normal stress level is around 57% fu and the accidental stress level is around 71% fu, as shown in Fig. 8.", "Stress levels discussed here are related to design loads; in service, real loads are lower due to the use of partial safety factors in design.", "Further, real loads and stresses also depend on live to dead load ratios.", "It can be seen from Fig. 6 that Dnssm of concrete, within the range of stress levels 57% and 71% fu could increase by 100–200% of that without loading.", "Thus the service life predicted, based on concrete in unloaded state, might be too optimistic, which suggests that the actual service loading should be considered for the durability design stage.", "Fig. 9 shows the relationship between Dnssm and the stress level for each concrete mix.", "The Dnssm values might be lower than some of the published data, but the overall quality of the data is good and the difference between mixes is clear.", "As one would expect, the w/b has a significant influence on Dnssm for each type of concrete (i.e. binder type).", "The value of Dnssm of 0.45-CEMI and 0.45–10%SF is much lower than that of 0.65-CEMI and 0.65–10%SF respectively.", "This is because concrete with a higher w/b has a higher porosity as well as higher percolation.", "Fig. 10 shows the relationship between Dnssm and the actual value of the load applied for each concrete mix.", "The change in chloride migration coefficient for each mix due to the application of 43 kN (highest load that was resisted by the 0.65-CEMI concrete) is reported in Table 2.", "The load 43 kN was selected because Dnssm for all mixes are available under this load.", "Also reported are the changes in chloride migration coefficient due to approximately half of 43 kN (≅20 kN) applied to the samples.", "The highest increase of 208% is for the 0.65-CEMI concrete subjected to 43 kN.", "In comparison, the increase for the 0.45-CEMI concrete at 43 kN load was only 22%.", "This is because concrete with a lower w/b has a higher strength (and hence higher tensile resistance) and can withstand higher load before Dnssm begins to increase.", "As shown in Fig. 9, the curves of Dnssm under different stress levels for both PFA and GGBS concretes are quite similar, but they are different from that of the CEMI concretes.", "The addition of PFA and GGBS resulted in an almost no change in Dnssm.", "The SF concretes on the other hand show a trend similar to that of CEMI concretes, but the overall values of Dnssm are greatly decreased.", "These results suggest that the addition of PFA, GGBS and SF is beneficial to control the chloride ingress for concrete structures under sustained loading.", "From Table 2, it can be seen that at 43 kN sustained load, 0.65-CEMI concrete has a very high increase in Dnssm and both 0.65–35%PFA and 0.65–10%SF concretes have lower increases.", "However, under the same load, 0.65–35%GGBS and 0.65–65%GGBS concretes had a very low increase and even a decrease in Dnssm.", "The data in Table 2 were calculated based on curve fitting done to the measured data because actual data points were not available for both 43 kN and 20 kN for all mixes except 0.65-CEMI.", "As a result, no attempt is made to test for statistical significance of the effects reported in Table 2.", "Dnssm of 0.65-CEMI increased rapidly with increasing force applied due to relatively lower strength of this concrete.", "However, there was little change for 0.65–35%GGBS and 0.65–65%GGBS concretes with the same range of load applied.", "Thus, the binder type is an important factor to consider for the durability of concrete under service loads.", "Note that λL will always be ⩾1.", "λL < 1 should be taken as 1 for safety reasons.", "The data reported in this paper is not sufficient to establish the function λL in terms of the parameters in Eq. (7).", "Fig. 11 shows the effect of unloading from different stress levels on the change of Dnssm of each mix.", "The effects of loading and unloading on changes in chloride migration coefficient of each mix are reported in Table 3.", "Changes below 20% are not considered to be statistically significant and this suggests the load levels below 50% fu do not have a significant effect on chloride penetration resistance of concrete.", "It can be seen clearly from Fig. 11 that there is a significant recovery after the load was removed.", "The change of Dnssm at 25% fu (elastic range of concrete) was fully recovered when the load was released, but only a part of the increase in Dnssm due to sustained load was recovered when the load was ⩾50% fu, which might have been caused by the presence of residual cracks [42].", "The increase and recovery of Dnssm for each mix after unloading from 50% fu and 75% fu are compared and summarised in Table 4.", "Dnssm significantly decreased after the load 75% fu was removed and the recoverable part is about 50%.", "This suggests that the short-time loading might not cause a lasting detrimental effect on the chloride penetration resistance of concrete structures.", "The non-recoverable part might increase with the frequency of loading when concrete suffers cyclic loading [20,45], which is an important topic to consider for structures subjected to traffic, wind and earthquake loads.", "Life 365 v2.1 [27] was used to calculate the service life of the reinforced concrete element under assumed different stress levels.", "A square reinforced concrete column with a cover depth of 60 mm was assumed to be exposed in the tidal zone of the North Sea at Dornach in Scotland, where the data of the temperature and surface chloride content was collected [46].", "It was assumed that the load applied to the column resulted in the same level of stress in the concrete cover as in the interior.", "Similarly, it was assumed that the effect of a short term load (as studied) is the same as of a sustained load (as in the field).", "The time needed to build to maximum surface chloride concentration of 0.95% (weight of concrete) was assumed to be 5 years [46].", "The reported diffusivity reduction factor (m) in literatures for concrete with 30% PFA was 0.66–0.7 [3,47] and for concrete with 60–70% GGBS was 0.621–1.23 [48].", "The maximum value of m in Life 365 is 0.6 [27], thus, values of m for different types were assumed as: m = 0.5 for 35% PFA concretes, m = 0.6 for 65% GGBS concretes, m = 0.4 for 35% GGBS concretes and m = 0.2 for CEMI concretes.", "The default hydration time (25 years) and critical chloride content (0.05% of concrete by weight) were used.", "Only the corrosion initiation time was considered for the service life calculation.", "There are limitations on these assumptions.", "One limitation is to relate the result of discs to the columns on site because of their different stress distributions.", "Fig. 12 shows significant differences in service life predicted between concretes without loading and under loading.", "Service life of concretes under loading at above 75% fu could be less than half of that without loading.", "In all the 8 mixes, 0.65–65%GGBS concrete has the best performance with the longest service life at 25% fu.", "By adding GGBS, the service life of 0.65–65%GGBS at 75% fu is still much longer than that of any other mix under loading or without loading, which suggests that adding mineral additives might be a better way to control the detrimental effect caused by loading level in durability design of concrete structures.", "A simplified model of the service life of reinforced concrete under different loading conditions based on the data of this research is shown in Fig. 13.", "The chloride diffusion coefficient changes with time.", "It may decrease in the first one or two years due to continuous hydration [3,47,49], but then may increase with time because of micro-cracks caused by the service loading.", "The assumed service lives of reinforced concretes (0.65-CEMI) in different loading conditions are indicated in Fig. 13.", "As can be seen, it is not appropriate to predict the service life of reinforced concrete without considering the effect of loading, which is not the current practice.", "High levels of loading (>50% fu) may significantly reduce the service life of concrete structures.", "Fig. 14 shows the relationship between Dnssm of concrete under loading and no loading.", "All types of concrete show similar behaviour with a higher variation at 75% fu.", "Dnssm of concrete under 25% fu is almost the same as Dnssm of concrete with no loading, Dnssm of concrete under 50% fu is around 1.43 times of that with no loading and Dnssm of concrete under 75% fu is around 2.24 times of that with no loading.", "Dnssm of concrete under loading can be estimated from these relationships and adjustments to the service life based on no-load condition can be made.", "Figs. 15–17 show the relationship between damage degrees measured in 3 different directions (Fig. 5) and normalised Dnssm.", "Dnssm/Dnssm0, where Dnssm0 is Dnssm of concrete without loading, was taken as normalised Dnssm.", "In Fig. 15, the damage degrees obtained from the UPV measurement along the cracks (1–1 in Fig. 5) are plotted against the normalised Dnssm.", "As is evident from this figure, there was no relationship between them for the range of concretes tested for the two loading conditions.", "A similar finding was obtained in Fig. 16, which shows the damage degrees based on UPV measured parallel to the cracks on the test surface (2–2 in Fig. 5) and the normalised Dnssm.", "However, in Fig. 17, there is an exponential relationship between the damage degrees based on UPV measurement perpendicular to the crack on test surface (3–3 in Fig. 5) and normalised Dnssm.", "These differences can be interpreted by the directions of measurement compared with the direction of cracks.", "The damage degree can be determined from UPV measurements only if the direction of measurement is perpendicular to the direction of cracks.", "The negative values were mainly caused by testing errors, which include the accuracy of test locations and the contact between sensors and test surfaces.", "However, the negative values were not found at higher load levels, which suggests the sensitivity of UPV test increases with increasing width of cracks.", "The results of the three figures suggest that the test direction of UPV method should be carefully chosen in order to detect the cracks.", "From the above results and discussions, the following conclusions have been drawn:", "Compressive stresses in concrete can increase the ingress of chloride.", "Design stresses below 50% fu do not detrimentally reduce the concrete’s resistance to chloride ingress.", "However, overloading should be avoided because it can cause significant increases in the ingress of chloride ions into the concrete.", "Concrete in aggressive environments with service loading of over 50% fu can have a 2–4 times higher chloride migration coefficients compared to concrete without loading.", "Significant difference can be found in chloride resistance of concrete under loading and after unloading (load was removed).", "The chloride migration coefficient of concrete under loading at 75% fu can be reduced by half if the load is released.", "This indicates that there are two different situations of chloride ingress in concrete which are under live load as well as under cyclic load.", "Accidental loading is therefore unlikely to cause a lasting detrimental effect.", "Micro-cracks caused by service loads should be taken into account during durability design and service life prediction.", "The chloride migration coefficient of concrete under loading is different and can be significantly higher than that without loading.", "Thus a multiplier should be applied to take account of the effect of loading.", "The findings of this research have indicated that the afore mentioned factor is a function of water/binder (w/b), stress level, mineral additives (ma) and binder content (cc).", "Further work needs to be carried out to quantify the relative effect of each of these factors.", "In the context of all the data presented in this paper, the use of mineral additives such as GGBS would appear to be very effective in extending the service life of structures." ]
[ "Effect of microcracks due to sustained loading on chloride transport into concrete was obtained.", "Stress levels above 50% of the ultimate can cause a great increase in chloride ingress.", "Mineral additives perform well against chloride ingress in concrete subjected to sustained loading.", "At high stress levels, removal of the load results in a recovery of the chloride resistance." ]
Feasibility of using a smartphone app to assess early signs, basic symptoms and psychotic symptoms over six months: A preliminary report
Background: Psychosis relapses are common, have profound adverse consequences for patients, and are costly to health services. ‘Early signs’ have been used to predict relapse, in the hope of prevention or mitigation, with moderate sensitivity and specificity. We investigated the feasibility and validity of adding ‘basic symptoms’ to conventional early signs and monitoring these using a smartphone app. Methods: Individuals (n = 18) experiencing a relapse within the past year were asked to use a smartphone app (‘ExPRESS’) weekly for six months to report early signs, basic symptoms and psychotic symptoms. Above-threshold increases in app-reported psychotic symptoms prompted a telephone interview (PANSS positive items) to assess relapse. Results: Participants completed 65% app assessments and 58% telephone interviews. App items showed high concurrent validity with researcher-rated psychotic symptoms and basic symptoms over six months. There was excellent agreement between telephone call and face-to-face assessed psychotic symptoms. The primary relapse definition, based on telephone assessment and casenotes, compared well with a casenote-only definition but had better specificity. Mixed-effects models provided preliminary evidence of concurrent and predictive validity: early signs and basic symptoms were associated with most app-assessed psychotic symptom variables the same week and with a number of psychotic symptoms variables three weeks later; adding basic symptoms to early signs improved model fit in most of these cases. Conclusions: This is the first study to test a smartphone app for monitoring early signs and basic symptoms as putative relapse predictors. It demonstrates that weekly app-based monitoring is feasible, valid and acceptable over six months.
[ "MEDI", "NEUR" ]
[ "Psychosis", "Relapse", "Schizophrenia", "Smartphone", "Telemedicine", "mHealth" ]
[ "2738", "2803" ]
[ "Psychosis relapses are associated with worse outcomes by almost every measure (Almond et al., 2004; Andrew et al., 2012; Appleby, 1992; Birchwood et al., 2000; Gumley and Schwannauer, 2006; Iqbal et al., 2000; Maclean, 2008; Wiersma et al., 1998; Wu et al., 2005).", "Relapse signatures, idiosyncratic combinations of warning signs, have been used to predict relapse in the hope of prevention or mitigation but have only moderate sensitivity and specificity (Eisner et al., 2013).", "To improve predictive power, we investigated adding basic symptoms (Schultze-Lutter et al., 2007a) to pre-existing putative predictors (conventional early signs) (Birchwood et al., 1989) and using a smartphone application (‘app’) to facilitate prompt identification of these.", "Incorporating both basic symptoms and conventional early signs of relapse into personalized relapse signatures will likely achieve better relapse prediction than has previously been demonstrated (Eisner et al., 2013).", "Basic symptoms are subtle, subjective changes in individuals' experiences of themselves (e.g. mild cognitive problems) and the world around them (e.g. more vivid colors) which predict first episodes of psychosis (Fusar-Poli et al., 2012; Schultze-Lutter et al., 2007b).", "There is preliminary evidence that basic symptoms also predict relapses of psychosis (Bechdolf et al., 2002; Eisner et al., 2018; Gaebel and Riesbeck, 2014) but there are no comprehensive, prospective studies examining this.", "A well-powered, methodologically sound, prospective study to establish whether basic symptoms predict relapses of psychosis is needed.", "We tested the feasibility of carrying out such a study using a smartphone app, ExPRESS (Experiences of Psychosis Relapse: Early Subjective Signs) (Eisner et al., 2019), which collects weekly assessments of early signs, basic symptoms and psychotic symptoms.", "Existing early signs studies have typically used pen and paper questionnaires (Birchwood et al., 1989; Gaebel et al., 1993; Gaebel and Riesbeck, 2007, 2014; Gleeson et al., 2005; Gumley et al., 2015; Hirsch and Jolley, 1989; Jørgensen, 1998; Malla and Norman, 1994; Marder et al., 1991; Marder et al., 1994; Subotnik and Neuchterlein, 1988; Tait et al., 2002; Tarrier et al., 1991) or text message systems (Spaniel et al., 2018; Spaniel et al., 2007; Spaniel et al., 2008) to examine the predictive value of conventional early signs of relapse.", "Compared to these methods, smartphone apps have a number of advantages: apps can be accessed at the individual's convenience (Ben-Zeev et al., 2013), decreasing participant burden and increasing ecological validity (Bucci et al., 2018); apps can automatically supply surveys and securely upload responses; finally, apps are more acceptable to individuals with psychosis than text message systems (Ainsworth et al., 2013).", "A number of symptom monitoring apps prospectively assessing symptom course have been tested (Ainsworth et al., 2013; Barnett et al., 2018; Ben-Zeev et al., 2014; Ben-Zeev et al., 2017; Ben-Zeev et al., 2018; Bucci et al., 2018; Kumar et al., 2018; Meyer et al., 2018; Niendam et al., 2018; Palmier-Claus et al., 2012).", "Most monitor individuals' current mental state rather than aiming to elicit symptoms predictive of relapse.", "Two assessed symptoms overlapping somewhat with conventional early signs (e.g. anxiety, confusion), but did not measure relapse (Kumar et al., 2018; Niendam et al., 2018).", "A small pilot study (Barnett et al., 2018) collected both self-reported early signs via an app and relapse as an outcome, but the focus of the published paper was mainly on the predictive value of passively collected data, with limited details of early signs data reported.", "Although a number of studies have reported good correspondence between passively collected data and outcomes (Barnett et al., 2018; Meyer et al., 2018; Wang et al., 2016; Wang et al., 2017), this was not the focus of the current study.", "Instead, we aimed to further refine the predictive value of app-based monitoring by adding basic symptoms to conventional early signs as putative relapse predictors.", "The current study tested the methodology for a planned large-scale study examining whether basic symptoms are valuable relapse predictors.", "There were four specific aims:", "to explore the feasibility of using a smartphone app (ExPRESS) for weekly monitoring of early signs, basic symptoms, psychotic symptoms and relapse over six months;", "to assess the concurrent and preliminary predictive validity of using personalized relapse signatures integrating basic symptoms and conventional early signs;", "to examine the validity of an operational definition of relapse using a combination of smartphone app assessment, verbal telephone assessment and casenote examination;", "to examine the acceptability of the study procedures.", "This study consisted of three phases.", "First, cross-sectional assessments characterized the sample and checked eligibility for the next phase.", "Second, eligible participants used ExPRESS for six months and received telephone calls from the researcher (prospective, longitudinal phase).", "Finally, after six months the acceptability of the study procedures was explored using qualitative interviews.", "The study was carried out in accordance with the Declaration of Helsinki (World Medical Association, 2013), ethical approval was obtained from Greater Manchester West Research Ethics Committee (14/NW/1471) and the study was registered (ClinicalTrials.gov: NCT03558529).", "Participants were recruited from three Mental Health Trusts in North-West England between June 2015 and June 2016.", "Inclusion criteria were: schizophrenia spectrum diagnosis (Diagnostic and Statistical Manual of Mental Disorders, 4th Edition; DSM-IV) (Sheehan et al., 1998); ≥1 acute psychotic episode in the past year (admission to crisis team or hospital; or exacerbation of psychotic symptoms lasting ≥2 weeks and leading to a change in management), or ≥2 episodes in the past 2 years, including index episode; currently prescribed antipsychotic medication; age over 18 years; fixed abode; fluent in English; sufficiently stable to take part (able to complete screening assessment); no current alcohol or drug dependence (Structured Clinical Interview for DSM-IV) (First et al., 1996); informed consent.", "To progress beyond baseline assessment, individuals must have reported basic symptoms which began or increased prior to a recent episode of psychosis.", "App design is detailed elsewhere (Eisner et al., 2019), with screenshots provided in Supplementary Fig. 1.", "Briefly, ExPRESS is an android smartphone app which prompts participants once a week to answer a personalized set of questions regarding psychotic symptoms (PANSS positive items) (Ainsworth et al., 2013; Kay et al., 1987; Palmier-Claus et al., 2012), mood symptoms (Calgary Depression Scale) (Addington et al., 1993), basic symptoms (Basic Symptoms Checklist, BSC) (Eisner et al., 2019) and early signs of relapse (Early Signs Scale, ESS) (Birchwood et al., 1989) within the past week.", "Participants have a 24-h window each week to respond to the question set.", "Responses are uploaded automatically to a secure server, accessible to the research team via a password protected web interface.", "Weekly self-reports were deemed sufficiently frequent to allow early signs to be meaningfully detected but not so frequent as to overburden participants, since beta-test participants considered weekly app use acceptable but would not have wanted it to be more frequent (Eisner et al., 2019).", "An overview of all baseline and follow-up assessments is provided in Fig. 1.", "At baseline, the Schizophrenia Proneness Instrument Adult Version interview (SPI-A) (Schultze-Lutter et al., 2007a) identified whether participants had experienced basic symptoms beginning or increasing prior to their most recent psychotic episode.", "Early signs (ESS) (Birchwood et al., 1989) were assessed for the same period.", "Psychotic symptoms and mood symptoms during the previous week were assessed using the PANSS (Kay et al., 1987), PSYRATS (Psychotic Symptom Rating Scales) (Haddock et al., 1999) and Hospital Anxiety and Depression Scale (Zigmond and Snaith, 1983).", "Substance use was assessed using a 4-point scale (Tarrier et al., 2006), medication adherence using a 7-point scale (Kemp et al., 1996) and cognitions related to imminent psychosis relapse using the Fear of Recurrence Scale (FoRSE) (Gumley et al., 2015).", "Demographic information was gathered using a standard questionnaire.", "Assessors were trained and supervised by senior colleagues.", "Mean intra-class correlations (ICC) compared to gold standard PANSS ratings were excellent (positive = 0.92; negative = 0.83; general = 0.89; total = 0.92).", "Using SPI-A and ESS assessments, the researcher and participant defined a ‘relapse signature’ combining early signs and basic symptoms.", "Items from the participant's relapse signature were entered into the ExPRESS app so that individuals could monitor a personalized set of early signs.", "Participants were trained on ExPRESS and asked to use it weekly for 6 months or until relapse, whichever was sooner.", "Those owning an android smartphone used their own phones and the remaining participants used a study phone.", "The latter received weekly text messages to their own phone reminding them to use ExPRESS.", "Participants were telephoned by the researcher (weekly for four weeks; monthly thereafter) to encourage participation and troubleshoot any difficulties with app use.", "During the 3-month telephone call, the researcher assessed the PSYRATS, five PANSS positive items (delusions, hallucinations, suspiciousness, grandiosity, conceptual disorganization), and a subset of SPI-A items (Cognitive Disturbances (COGDIS) and Cognitive-Perceptive (COPER)) (Schultze-Lutter et al., 2007a).", "This was to screen for additional delusions or overlooked relapses and to check app item validity.", "The primary relapse definition (Wunderink et al., 2007) (Supplementary Fig. 2) required a symptom increase for ≥1 week resulting in a management change (casenote-reported medication change or increased observation by the clinical team, including admission).", "Symptom increase criteria, assessed via PANSS telephone interview (five items: delusions, hallucinations, suspiciousness, grandiosity, conceptual disorganization), were: for remitted individuals, an increase to ≥4 or an increase of ≥2 points (whichever was higher) on any item; for non-remitted individuals with all baseline PANSS positive items <5, at least one item ≥5; for non-remitted individuals with ≥1 baseline item ≥5, an increase to ≥4 or an increase of ≥1 point (whichever was higher) on any item.", "Individuals' remission status was initially determined from baseline PANSS using standard remission severity criteria (Andreasen et al., 2005).", "To identify participants remitting during the app-use phase, we modified these criteria to accommodate app content: participants scoring ≤3 for two consecutive weeks on all app-assessed psychotic symptom items (delusions, hallucinations, grandiosity, suspiciousness; scaled the same as corresponding PANSS items) were classified as remitted.", "PANSS telephone interviews were triggered when app-reported psychotic symptoms exceeded a pre-specified threshold (initially equivalent to the symptom increase criteria).", "If symptom increase criteria were met, the telephone assessment was repeated one week later to check duration.", "If app-reported symptoms exceeded the threshold two consecutive weeks without any increase in symptoms detected during the resultant telephone call, the threshold for receiving a call was recalibrated (for each app-assessed symptom, the new threshold was one point above the average of the previous four app responses).", "Two aspects of the above relapse assessment procedure were added seven months into the study.", "Firstly, the original definition did not account for remission status, with identical symptom increase criteria applied to non-remitted and remitted individuals.", "Secondly, the telephone call threshold recalibration was formerly absent, so participants with high residual symptoms were telephoned every week.", "Following these changes, four were deemed remitted at baseline, three as remitted during the app-use phase, four had their telephone call threshold recalibrated and five were unaffected.", "The secondary relapse definition, assessed using casenotes alone, required a symptom increase for ≥1 week resulting in a management change.", "For each participant, the relapse start date was recorded, with verbatim extracts from casenotes describing changes in symptoms and management.", "The researcher conducting casenote screening was trained to protocol (Barrowclough et al., 2010); ratings on reliability cases showed perfect correspondence with gold standard assessors (relapse presence/absence kappa = 1.00; relapse start date ICC = 1.00).", "On longitudinal phase completion or dropout, face-to-face assessments (SPI-A; PANSS positive items) and qualitative interviews were conducted.", "The brief (average 6 min), audio-recorded qualitative interview topic guide included: reasons for participation; researcher support; telephone call acceptability and financial reimbursement; study highlights and lowlights.", "PANSS positive items were assessed via telephone within three days prior to face-to-face assessment for validity checks.", "Participants received: £10 per completed study phase; £10 monthly to cover phone credit (longitudinal participants); £5 per telephone interview (last 6 months of the study only; to test whether engagement increased).", "Analyses were conducted in Stata (StataCorp, 2015), with bootstrapping where appropriate, and considered statistically significant at p < 0.05.", "App-assessed items scored on a 4-point scale were linearly transformed to a 7-point scale to aid comparison with other measures.", "Feasibility was assessed using descriptive statistics summarizing: recruitment to baseline assessments, recruitment to longitudinal phase (proportion of baseline participants eligible and consenting), app engagement (percentage of assessments completed), timing of app responses across the 24-hour response window, proportion of standard study telephone calls and telephone call PANSS assessments completed, and proportion of study phones returned.", "The 6-month relapse rate was noted, to inform future power calculations.", "The pattern of app completion during the app-use phase was examined in a mixed-effects model with a random effect of participant and a fixed-effect of time.", "Effects of baseline variables on percentage app completion were examined using Spearman's correlation (continuous variables), Mann-Whitney or Kruskal-Wallis test (categorical variables).", "To measure concurrent validity, the number of app-reported basic symptoms during the first three and full six months of app use were compared to retrospective telephone (3 months) and face-to-face (6 months) researcher-rated SPI-A assessments using ICCs (two way mixed, absolute agreement, single measures).", "Preliminary predictive validity was assessed in two ways.", "Firstly, mixed-effects models were estimated, with app-assessed psychotic symptoms as the dependent variable, a fixed-effect of early signs and/or basic symptoms and a random effect of participant.", "Likelihood ratio tests explored whether adding basic symptoms to early signs improved model fit.", "Secondly, patterns of basic symptoms and early signs were examined graphically in the participants meeting full or partial relapse definitions.", "Validity was examined by comparing: 6-month relapse rates using primary and secondary relapse definitions (kappa); telephone interviews of PANSS items and the same items assessed face-to-face (two way mixed, absolute agreement, single measures ICC); researcher-rated symptoms and app-reported symptoms.", "For the latter comparison, the researcher-rated symptom variables came from a face-to-face interview, where available, or telephone interview.", "Spearman's correlation was calculated to aid comparison with previous studies (Palmier-Claus et al., 2012) but this does not account for the nested data structure.", "Therefore, mixed-effects models were constructed, with app-reported symptoms as the dependent variable, a fixed-effect of researcher-rated symptoms and a random effect of participant.", "The fixed-effect coefficient can be interpreted as the average change in app-reported symptoms for a 1-point change in researcher-rated symptoms.", "We used the framework method (Gale et al., 2013) to analyze verbatim transcriptions of qualitative interviews.", "The research team developed the initial analytical framework using the topic guide and independently coded two of the first three transcripts each, updating the framework as necessary.", "The first author systematically coded the remaining transcripts and charted the data into a framework matrix to aid discussion and interpretation by the whole team.", "Demographic and clinical characteristics of longitudinal participants are shown in Table 1.", "Fig. 2 shows recruitment and retention.", "Only 11% (22/193) of individuals that clinical staff considered potentially suitable participated in baseline interviews.", "The main reasons for exclusion were that individuals were ineligible, declined to participate, or were not given study information by staff.", "Most baseline participants (18/22) were eligible for and consented to the longitudinal phase and engagement was high, with 78% completing ≥33% app assessments and 72% completing ≥50% app assessments.", "Most longitudinal participants (89%) completed a qualitative interview.", "Thirteen participants used ExPRESS on a study phone; all returned the phone in good condition except one who returned it to a clinician, who lost it.", "Participants completed 65% app assessments and 65% supportive telephone calls.", "App-reported symptoms increased above the telephone interview threshold 31 times, of which 18 (58%) resulted in completed PANSS positive telephone interviews; participants were unavailable for the remaining 13 telephone interviews.", "The distribution of app completion across the sample is shown in Fig. 3.", "Three clusters are apparent: half the sample completed ≥90% of app assessments, four completed 60–70% and four completed <13% of assessments.", "The exception was unexpectedly abroad for several months, precluding app completion; while in the UK, their app completion was 60%.", "Supplementary Fig. 3 shows app response timing across the 24-hour response window; most responses occurred in the first 12 h, with a substantial peak immediately after the initial alert (1.30 pm).", "Participants responded to fewer prompts as the study progressed (OR = 0.89 per week follow-up; p < 0.001; Supplementary Fig. 4).", "Percentage app completion was significantly and inversely correlated with baseline depression and fear of relapse, with baseline anxiety approaching significance and all other baseline variables non-significant (Table 1).", "The mean number of basic symptoms reported at baseline as having begun or increased prior to a recent psychotic episode was 6.4 (sd = 4.3, range = 1–17); mean ESS score for this period was 84.4 (sd = 20.6, range = 41–121).", "ICCs comparing the number of app-reported and researcher-rated basic symptoms suggested poor agreement at 3 months (ICC = 0.37, p = 0.307, n = 8; telephone call COPER/COGDIS items) but excellent agreement at 6 months (ICC = 0.76, p < 0.001, n = 12; face-to-face full SPI-A).", "In mixed-effects models (Table 3) both early signs and basic symptoms were significantly associated with all psychotic symptom measures, except grandiosity, at the same time point.", "Adding basic symptoms to early signs improved model fit in three cases.", "One week later: early signs significantly predicted suspiciousness; neither early signs nor basic symptoms predicted any other psychotic symptom variables.", "Two weeks later: basic symptoms approached significance in predicting suspiciousness; no other predictions two weeks later were significant.", "Three weeks later: early signs significantly predicted psychotic symptoms and hallucinations; basic symptoms significantly predicted psychotic symptoms and delusions, with hallucinations and suspiciousness approaching significance; adding basic symptoms to early signs improved model fit for psychotic symptoms and delusions, with suspiciousness approaching significance.", "Graphs of app-assessed items for participants meeting full or partial relapse definitions (Supplementary Fig. 5) indicate greater variability over time in severity of app-assessed early signs and basic symptoms than app- or researcher-assessed psychotic symptoms.", "Since they respond more than psychotic symptoms to relapse triggers and treatment, this probably signifies greater sensitivity to underlying changes rather than more random noise.", "Two participants met both primary and secondary relapse definitions, and one met the secondary definition alone, a substantial agreement (kappa = 0.76, p = 0.003).", "The relapse which met the secondary but not primary definition appeared to be a false positive (see Supplementary Textbox 1).", "Agreement between five PANSS positive items assessed during a telephone call and the same items assessed face-to-face was extremely high (Table 2), with ICCs ranging from 0.94 to 0.96 (p < 0.001).", "High agreement was also demonstrated for PSYRATS delusions (ICC = 0.97, p < 0.001) and hallucinations (ICC = 0.89, p < 0.001) subscales.", "The correlation between researcher-rated symptoms and app-reported symptoms was high, with Spearman's rho ranging from 0.80 to 0.87 (p < 0.001; Table 2).", "Fixed-effects coefficients from mixed-effects models ranged from 0.66 to 1.08 (p < 0.05), with all confidence intervals crossing 1.00.", "We report the acceptability of study participation (Supplementary Table 1).", "A detailed analysis of ExPRESS's acceptability is reported elsewhere (Eisner et al., 2019).", "Participants gave various reasons for taking part, including altruism, wanting to increase knowledge about psychosis, curiosity about the research, feeling it might help them personally and previous positive research experiences.", "One individual, initially attracted by the financial reimbursement, became genuinely interested in the study.", "Several participants reported liking answering the questions, enjoying their novelty and finding them easy to answer even during difficult weeks.", "Others enjoyed the normalizing effect of ExPRESS, satisfaction of helping with research and increased understanding of their illness.", "All participants found study telephone interviews acceptable and felt they received enough researcher support, with several mentioning that the reminder texts were helpful.", "Participants commented that the telephone calls were not intrusive and their frequency was appropriate; some found them encouraging and reassuring.", "Views about whether financial reimbursement for telephone interviews was needed were mixed.", "Although not specifically asked about the baseline interview, two participants mentioned finding it long and a little stressful initially; another commented that he found it acceptable.", "This study demonstrates the feasibility and acceptability of using an app for weekly monitoring of early signs, basic symptoms, psychotic symptoms and relapse over a six-month period, alongside telephone calls from a researcher.", "We show preliminary evidence of concurrent and predictive validity: early signs and basic symptoms were separately associated with most app-assessed psychotic symptom variables the same week and with a number of psychotic symptoms variables three weeks later, and adding basic symptoms to early signs improved model fit in most of these cases.", "App items showed high concurrent validity with researcher-rated psychotic symptoms and basic symptoms over six months.", "There was also excellent agreement between telephone call and face-to-face assessed psychotic symptoms.", "The primary definition of relapse, based on telephone interviews and casenotes, compared well with a casenote-only definition but had better specificity.", "Since this study has the longest app-use phase of any to date reporting a symptom monitoring app in a sample with established psychosis, participants' app engagement is of key interest.", "Participants completed 65% app assessments, with 78% completing ≥33% assessments.", "Although lower than studies with a follow-up ≤2 months (Ainsworth et al., 2013; Ben-Zeev et al., 2014; Meyer et al., 2018; Palmier-Claus et al., 2012), this compares favorably to those with 3-month (Bucci et al., 2018) or 5-month (Kumar et al., 2018) follow-up periods.", "Unlike previous studies (Ainsworth et al., 2013; Ben-Zeev et al., 2014; Bucci et al., 2018; Kumar et al., 2018; Palmier-Claus et al., 2012), we used weekly (rather than daily) app assessments which appear to be better tolerated over longer follow-up periods.", "However, the only study with a longer follow-up (14 months) (Niendam et al., 2018) included daily app assessments and averaged 69% app completion in a clinical high risk and recent onset psychosis sample.", "Participants were paid per completed assessment which likely increased engagement.", "Their young age may also have engendered higher app completion since they are likely to be more familiar with smartphone apps than our older sample with established psychosis (Bonet et al., 2018).", "Although neither we, nor others (Meyer et al., 2018; Palmier-Claus et al., 2012), found an effect of age on app completion, our sample was possibly too small or had an insufficient age range to detect this.", "Other predictors of app engagement have been examined but a consistent picture is yet to emerge (Killikelly et al., 2017).", "Studies have reported that higher positive (Meyer et al., 2018; Palmier-Claus et al., 2012), negative (Kumar et al., 2018; Meyer et al., 2018) or agitation/mania symptoms (Kumar et al., 2018) predicted lower app engagement, whereas the current study found a significant effect of depression and fear of relapse.", "Arguably, those who are more fearful of relapse and more depressed are at greater risk of relapse (Conley, 2009; Gumley et al., 2015), more likely to avoid help seeking (Gumley and Park, 2010) and thus more difficult for services to engage in treatment.", "If symptom monitoring apps become commonplace, clinicians must recognize that these may not suit everyone and take additional steps to engage these individuals.", "Interestingly, neither we, nor others (Kumar et al., 2018), found that using a study phone significantly reduced app engagement, despite qualitative feedback suggesting that participants prefer their own phones (Ainsworth et al., 2013; Eisner et al., 2019; Palmier-Claus et al., 2012).", "Return rate of study phones in the current study was excellent (92%), and comparable to previous studies (Biagianti et al., 2017; Granholm et al., 2012), contrasting with clinicians' fears that patients might lose/sell study phones (Berry et al., 2017).", "This study provides a novel approach to operationally defining relapse by using a combination of telephone interviews and casenotes.", "While numerous relapse definitions exist, these rely on face-to-face assessments or solely on casenotes (Eisner et al., 2013; Falloon et al., 1983; Gleeson et al., 2010; Olivares et al., 2013).", "We suggest that remote relapse assessment is more easily integrated into participants' lives, decreasing both participant and researcher burden; this is likely to increase engagement and allow more frequent, long-term monitoring.", "Our operational relapse definition compared well with a casenote-only definition but had better specificity, since the latter generated a false positive.", "However, conversely, our operational definition may have generated false negatives; since the proportion of unanswered telephone calls was high (42%), some symptom increases may have been missed.", "As the first study comparing telephone and face-to-face interview PANSS items, our finding of extremely high agreement between these is encouraging but needs replicating in a larger sample.", "Nevertheless, this finding suggests that our definition may be comparable to other relapse definitions that use PANSS positive items.", "We found a strong association between app-reported and researcher-rated psychotic symptoms, replicating previous findings (Niendam et al., 2018; Palmier-Claus et al., 2012) and suggesting that symptom monitoring apps are a valid means of assessing symptom course.", "Agreement between app- and researcher-assessed basic symptoms was also excellent over 6 months, improving upon previous self-report measures of basic symptoms which show poor concurrent validity (Mass et al., 1997; Michel et al., 2016).", "Although we found low agreement at 3-month assessment, key differences between the 3- and 6-month SPI-A assessments may explain this disparity: the 3-month researcher-rated assessment was conducted via telephone rather than face-to-face, with a smaller sample and only included a sub-set of SPI-A items.", "There are a number of important limitations.", "Firstly, the sample was small, precluding a definitive examination of predictive validity: sensitivity and specificity could not be calculated, making it difficult to compare with other early signs studies.", "Nonetheless, mixed-effects models (using changes in psychotic symptoms as a proxy for relapse) and graphs of relapsers provided sufficient evidence of predictive validity to warrant further investigation.", "Secondly, the sample is unlikely to be representative, as the decline rate was high (42%), albeit comparable with other symptom monitoring app studies (Ainsworth et al., 2013: ~30% decline; Bucci et al., 2018: 30%; Ben-Zeev et al., 2014: 39%; Kumar et al., 2018: 44%; Niendam et al., 2018: 47%; Palmier-Claus et al., 2012: ~50%).", "Thirdly, researchers were not formally blind to participants' early signs or basic symptoms prior to conducting PANSS telephone interviews; in practice neither early signs nor basic symptoms were known but future studies should use an independent, formally blinded assessor.", "Fourthly, regarding the mixed-effects models, the dependent variable (psychotic symptoms) and predictors (early signs and basic symptoms) are related experiences which were self-reported by participants using the same method (an app), increasing the risk of spurious correlations arising from common method variance and expectancy effects.", "Fifthly, while baseline assessments were conducted by a single assessor, a second assessor carried out half the 6-month SPI-A assessments but inter-rater reliability was not evaluated.", "Finally, since this was a feasibility study, protocol changes were made during data collection.", "However, these changes refined the relapse definition and reduced the number of telephone interviews.", "In conclusion, we found that weekly app-based assessment of symptoms was feasible, acceptable and valid over a six month period, offering support for a large-scale study using this methodology.", "More generally, these findings provide further evidence that symptom monitoring technology could be a valuable addition to routine mental health service delivery, since they closely correspond to researcher-rated assessments and are well-tolerated over an extended period of time.", "Sandra Bucci is a director of Affigo CIC, a not-for-profit social enterprise company spun out of the University of Manchester in December 2015 to enable access to social enterprise funding and to promote ClinTouch, a symptom-monitoring app, to the NHS and public sector.", "All authors were involved in the design and ongoing management of the study, and contributed to drafts of this report.", "EE, the principal investigator, prepared the protocol, was responsible for the day-to-day running of the study, collected, analysed and interpreted the data and took a lead on writing the manuscript.", "SB and RD provided clinical and research supervision.", "RE advised on statistical aspects of the study design and analysis.", "NB provided maternity cover.", "The Medical Research Council did not play a role in the study design, data collection, data analysis, data interpretation, writing of the report or in the decision to submit the article for publication." ]
[]
Cellular electron cryo tomography and in situ sub-volume averaging reveal the context of microtubule-based processes
Electron cryo-tomography (cryoET) is currently the only technique that allows the direct observation of proteins in their native cellular environment. Sub-volume averaging of electron tomograms offers a route to increase the signal-to-noise of repetitive biological structures, such improving the information content and interpretability of tomograms. We discuss the potential for sub-volume averaging in highlighting and investigating specific processes in situ, focusing on microtubule structure and viral infection. We show that (i) in situ sub-volume averaging from single tomograms can guide and complement segmentation of biological features, (ii) the in situ determination of the structure of individual viruses is possible as they infect a cell, and (iii) novel, transient processes can be imaged with high levels of detail.
[ "BIOC" ]
[ "Adenovirus", "Cellular architecture", "Cytoskeleton", "Dynein", "Electron cryo-microscopy", "Electron cryo-tomography", "Endocytosis", "Microtubules", "Retrograde transport", "Sub-volume averaging", "Viral entry", "Virus trafficking", "in situ structure determination" ]
[ "1315" ]
[ "Electron cryo tomography (cryoET) is a technique used to reconstruct 3-dimensional structures of biological samples in their native, frozen hydrated state.", "By taking a tilt series of images of vitrified cells (or other relevant specimens) in an electron microscope it becomes possible to visualize unique pleomorphic events, structures with short-lived and transient states, and large macromolecular organisations (e.g. lattices) that would be otherwise very difficult to reconstitute in vitro (Asano et al., 2015; Brandt et al., 2010; Hagen et al., 2015; Schur et al., 2015; Zeev-Ben-Mordehai et al., 2016).", "Furthermore, repetitive or frequently occurring molecular structures within these pleomorphic objects can be averaged together to create higher resolution maps, a technique generally referred to as sub-tomogram averaging.", "Recent technical developments in electron cryo microscopy (cryoEM) have resulted in the ability to image and collect data of vitrified specimens with unprecedented signal-to-noise ratios, allowing the analysis of biological structures and features at a much higher level of detail than previously attainable.", "These developments include better zero-loss energy filters and direct electron detectors, both of which have vastly improved the quality of tomographic data acquisition (Koster et al., 1997; Li et al., 2013; McMullan et al., 2014), and led to the high-resolution determination of novel macromolecular complexes and viruses, as well as the structural determination of pleomorphic complexes and the in situ architecture of cells (Asano et al., 2015; Bartesaghi et al., 2015; Fukuda et al., 2015; Mahamid et al., 2016; Matthies et al., 2016; Schur et al., 2015; Sirohi et al., 2016; Zeev-Ben-Mordehai et al., 2016).", "This move to analyze native cellular structures in situ has come concomitantly with a push towards the development of technologies such as Zernike phase plates, and more recently Volta phase plates (VPP) (Danev et al., 2014; Murata et al., 2010), that improve the image contrast.", "These improvements further include specimen thinning methods mitigating some of the detrimental impact of thick specimens – cells are typically too thick to image through standard methods, as the penetration limit of electrons through a frozen-hydrated specimen at 300 keV is around 1 μm (Baumeister, 2002) – and allow us to use cryoET to image processes anywhere in the interior of cells (Al-Amoudi et al., 2004; Rigort et al., 2012).", "However, such techniques are still limited and challenging to implement technically.", "Alternatively, it is also possible to avoid this thickness limit by focusing attention to the cell periphery.", "These areas of the cell are generally thin enough to be successfully imaged, and are accessible directly after plunge freezing such offering a much more widely and routinely used route of specimen preparation for many structural and cell biologists.", "Understanding the cell periphery is crucial to comprehending many biological mechanisms.", "These include, but are not limited to, cell signaling, translation, exocytosis, endocytosis, pathogen entry and egress and cytoskeletal organization and trafficking (Brandt et al., 2010; Cyrklaff et al., 2007; Dodonova et al., 2015; Ibiricu et al., 2011).", "In this work, we assess what can be achieved by targeting the cellular periphery of whole intact non-thinned cells in individual tomograms, particularly through sub-volume averaging and the localization of rare events (Fig. 1A).", "We discuss the opportunities that novel developments offer to structural cell biologists when using direct electron detectors, especially when combined with an energy filter.", "Our assessment shows that the majority of information to determine usable and contextual structures is contained within the tomogram.", "As examples we reveal a range of regular structures that are discernible throughout the tomograms and assess their roles and interaction in the course of viral entry.", "U87MG cells and U2OS cells were seeded and grown on poly-l-lysine-coated gold mesh carbon coated holey carbon grids (Electron microscopy sciences) in Dulbecco’s modified eagle medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum.", "The cells were grown to approximately 60–80% confluency, or until 1–2 cells per grid square.", "The grids were supported in 2×9-well μ-slide co-culture dishes (Ibidi).", "Adenovirus particles were cultured and isolated as has been described previously (Wodrich et al., 2010).", "For infection of U2OS cells with adenovirus, 4 μl of adenovirus was mixed in 200 μl of DMEM.", "50 μl was applied to each grid and incubated with the cells for 10–20mins before plunge freezing.", "Grids were plunge frozen in an ethane/propane mix, cooled to liquid nitrogen temperature using a manual blotting apparatus, blotted from the backside of the grid for between 2 and 4 s.", "Tomograms were acquired using a Tecnai TF30 “Polara” Microscope (FEI, Eindhoven) operating at 300 keV.", "The microscope was equipped with a Gatan Imaging Filter “Quantum” energy filter operated in zero-loss mode with a 20 eV wide slit, and a post energy filter mounted K2 Summit direct electron detector (Gatan, Inc.) Data were acquired in counting mode, with aligned frames produced ‘on the fly’, using the combined filter for image alignment in Digital Micrograph (Gatan, Inc.).", "Dose, pixel size, magnification and tilt angle information are provided in Table S1.", "Data were acquired using SerialEM (Boulder, Colorado) (Mastronarde, 2005).", "Tilt series outputted from the microscope were aligned and reconstructed using the IMOD software package (Kremer et al., 1996).", "CTF correction, where applicable, was also performed using IMOD.", "A global defocus was determined for the whole tomogram, which was then used to correct individual tilts in the tilt series, taking into account the change in eucentricity across the projection images at high tilts.", "Phase flipping was then performed on each tilt image and the tomogram reconstructed using this phase flipped image stack.", "Sub-volume averaging of microtubules was performed using PEET (also part of IMOD) (Nicastro et al., 2006).", "Particles were picked from tomograms binned by a factor of 4, using scripts based on TEMPy (Farabella et al., 2015) that evenly sampled at 4 nm intervals (based on the size of one tubulin monomer) through a straight line defined by the user, with each particle oriented parallel to the line.", "These particles were then used for iterative sub-volume refinement; with further refinement applied on factor 2 binned and unbinned tomograms.", "Custom scripts were used to relate reconstructions between the binning levels of the tomograms to utilize the respective Euler angles and translational information.", "Sub-volume averages were acquired at binning factors of 4,2 and 1 for the U87MG tomogram (final pixel size = 4.22 Å unbinned), and binning factors of 4 and 2 for the U2OS tomogram (final pixel size = 4.6 Å at binning factor 2).", "Symmetrisation of the microtubules was performed using custom scripts, and utilized the prior knowledge from the unsymmetrised structure that the microtubules contained 13 protofilaments, with 4 nm tubulin dimer spacing (on average).", "The picked microtubule particles were split in half at random prior to sub-volume averaging and each half dataset independently averaged during refinement, including symmetrisation.", "Each run of sub-volume averaging at every binning level for the reconstruction was performed three times: one on the first random half dataset, the second on the second half and a third on the combined data.", "In the case of the U2OS tomogram, at the end of refinement, particles with a cross correlation of less than 0.2 were excluded.", "In the case of both tomograms, if any particles had centres within 4 nm of each other, the particle with the lower cross correlation was excluded.", "Resolution determination was assessed through FSC at 0.5 and 0.143 between the two half set reconstructions (the ‘gold standard’ criterion).", "Structures shown in the paper are from the combined dataset, filtered to the resolution cut-off determined by FSC0.143 between the two half sets.", "Curves were determined using a number of different masks, specifically: no mask, the final microtubule structure binarised at a noiseless threshold and extended with a soft edge (threshold mask), or a hard edge cylinder or soft edge cylinder of approximate microtubule diameter (Fig. 3F).", "FSC curves and masks for “gold-standard” FSC determination were generated using EMAN2 (Tang et al., 2007).", "Refinement of the adenovirus structure was performed using PEET, utilising an option to not produce a new map after each iteration of refinement.", "The adenovirus reference structure (EMDB-1574) was filtered to 60 Å prior to alignment.", "The determined position and orientation of the picked particle was then used to symmetrize it and produce a reconstruction.", "We also produced a “back-plotted” tomogram, where the virus particles in the tomographic volume were substituted with the reconstruction using a custom script that utilized the translation offsets and Euler angles for each given particle position.", "Segmentation and representation of tomograms (including back-plotted tomograms) was performed and viewed using the visualization software Amira (FEI, Eindhoven).", "Structures resulting from sub-volume averaging and backplotted coordinate models were visualized using Chimera (UCSF) (Pettersen et al., 2004).", "Using a 300 keV FEG electron microscope with post-energy filter mounted direct electron detector (Gatan K2 Summit), we first aimed to assess visually what information could be gained by following relatively regular and populous features within single tomograms of adherent human cells.", "We comparatively performed this analysis on two tomograms from two different cells lines: 1) U87MG cells and 2) U2OS cells in areas of ∼80–200 nm thickness (Figs. S1 and S2).", "Visual inspection of tomograms is a useful first approach in analyzing and characterizing the major components (cf. its use in some notable recent studies (Fukuda et al., 2015; Mahamid et al., 2016)).", "We clearly observed and easily discriminated the different components of the cytoskeleton, such as intermediate filaments, F-actin and microtubules (Fig. 1B, F) due to their characteristic structural signatures.", "The overall level of detail in these tomograms was very high, allowing us to clearly discern, e.g. for the cytoskeletal structures, considerable sub-structural detail.", "For F-actin, we could observe the zig-zag nature of F-actin (Fig. 1H, green line), a ∼2.8 nm repeat feature, as well as the globular features of individual actin monomers within the filament (Fujii et al., 2010).", "Similarly, the intermediate filaments showed a rather smooth parallel tubular architecture slightly wider in diameter (∼10 nm) than actin filaments (∼7 nm), consistent with their tightly packed, cylindrical structure (Herrmann et al., 2007).", "However, the greatest level of detail visible in the tomogram was found when considering the microtubules.", "Protofilaments were clearly visible in the top and bottom views of the microtubules.", "Furthermore, the tubulin lattice in the microtubule wall was evident with the 4 nm spacing of tubulin monomers being clearly distinguishable through top, bottom and middle slices of the microtubules (Fig. 1E, G), suggesting a high level of detail being contained within the tomogram.", "The high signal-to-noise ratio further allowed us to visualize more transient states in these filaments, such as a microtubule plus end in the process of disassembly (Fig. 1D) (Koning et al., 2008).", "As well as elements of the cytoskeleton, the tomogram also showed less regular, more globular macromolecules.", "Ribosomes were often arranged into polysomes (Fig. 1C), as has been characterized previously (Brandt et al., 2010).", "The level of detail allowed us to differentiate the 50S and 40S subunits.", "A ribonucleoprotein “Vault” (Woodward et al., 2015) could be observed, in close proximity to a microtubule and potentially engaged with a dynein motor (Figs. 1I, S2).", "Putative AAA+ domains of a dynein motor complex as well as the respective stalk regions projecting onto a nearby microtubule are visible (Figs. 1I, S2) (Roberts et al., 2009).", "Vaults are ribonucleoprotein organelles that are thought to recruit and store mRNAs.", "This function has led to the suggestion that they act as a transport cages within the cytosol (Chugani et al., 1993).", "Our observation adds credit to the idea that Vaults are important in the transportation of mRNAs and other cargoes inside cells.", "Further to macromolecules, various organelles could also be identified within the tomogram, such as the lysosome and the endoplasmic reticulum (Fig. 1B, arrows), with their 6–8-nm-thick bilayer distinctly visible.", "In order to assess the potential resolution achievable from this tomogram of a peripheral U87MG cell area, we performed sub-volume averaging of microtubule segments (Fig. 2).", "Due to the fact that the microtubules were oriented roughly perpendicular to the tilt-axis for the tomographic series, we expected that we would be able to compensate to some extent the information lost due to the CTF, as different apparent defoci of extracted volumes caused by tilting of the sample would compensate for some of the lost information.", "The resultant microtubule structure has 13 protofilaments, with a tubulin monomer spacing of ∼4 nm, consistent with the current understanding of the microtubule structure in vivo (Evans et al., 1985; Kollman et al., 2010).", "As the sampling of the tomogram precluded the possibility of discerning the microtubule seam in these structures, we applied a 13-fold helical symmetry to the final model to produce the final structure.", "The gold standard Fourier Shell Correlation curve of the resultant microtubule structure (Fig. 2B) indicated a resolution of 18.5 Å at the 0.143 threshold (Fig. 2D).", "A prominent local peak occurred at a spatial frequency equivalent to ∼22 Å resolution (Figs. 2D and 4A).", "Analysis of the Fourier spectrum of the microtubule structure indicated a strong layer line at this resolution, as has been seen for other microtubule structures (Alushin et al., 2014).", "The calculated defocus and the nominally acquired microscope defocus differed by up to 1 μm, and accounting for inaccuracies of the defocus due to spherical aberration in the microscope, as well as the range of defoci across the tomogram, it is difficult to determine an overall single defocus.", "We therefore proceeded with the IMOD determined defocus (Xiong et al., 2009) as basis for comparisons.", "The resulting microtubule structure is of correct size and shape to fit a crystal structure of the tubulin αβ-dimer (PDB: 1TUB) (Fig. 2B, inset).", "Due to the nature of tomographic data acquisition, where a range of defoci likely exist within the same dataset, the zeroes of the CTF will not be clearly visible in a sub-tomogram average if a structure that prevails throughout the field of view in a single tomogram is used for sub-volume averaging.", "Our analysis suggests that the sub-volume averaging performed from this single tomogram is however very likely limited in resolution by the number of particles contained within the tomogram; here 4056 overlapping sub-volumes of 25 nm × 25 nm contributed to the final average.", "In the case of single particle reconstruction methods, resolution has been shown to be very sensitive to the applied electron dose (Bartesaghi et al., 2014).", "An element where the dose may be important in sub-tomographic averaging is when incorporating high tilts into the final sub volume average.", "Studies have shown that lower tilt images are the main contribution to good resolution sub-volume averages (Bartesaghi et al., 2012) and recent strategies have been developed to down weight the contribution of higher tilts (Bharat et al., 2015).", "However, high tilts can be very important in the reduction of back-projection artifacts from gold fiducials in tomograms, which may otherwise interfere with the interpretation of rare individual events in cellular tomograms that do not benefit from sub-volume averaging.", "Sub-volume averaging of microtubules in the U2OS cellular tomogram (Fig. 3) resulted in a microtubule structure of 25 Å.", "This resolution was determined without a mask as use of a mask appeared to produce an FSC at 0.143 in noisy regions of the FSC curve, as shown in Fig. 4C. Due to the higher magnification, and resultant smaller field of view, only a quarter of the number of particles could be picked (Table S1).", "Only particles with a cross-correlation coefficient (CCC) score above 0.2 after alignment were included in the final average.", "Removal of particles below this threshold removed the majority of the microtubule particles that were in the thicker regions of the tomograms (Fig. S4).", "A thickness gradient from ∼90 to ∼180 nm existed across this tomogram (Fig. S3).", "It therefore seems likely that the main contribution to the low CCC in this model is due to the increased thickness.", "However, it has been shown before that filamentous objects perpendicular to the tilt axis suffer from distortions that may affect their potential to align them accurately (Mastronarde, 1997).", "Interestingly, these microtubules also aligned parallel to the tilt axis, suggesting the alignment may possibly be dependent on the tilt axis.", "The reduced signal to noise and low cross correlation, and subsequent removal of bad particles most likely contributed to the difficulty in resolution determination during masking.", "In the case of this second microtubule sub-volume average structure, the fall-off in cross correlation between the two independent halves of the dataset did not fully reach zero (Fig. 3F), suggesting that there is inherent noise in this structure, possibly introduced by particles from the thicker regions of the sample.", "This is further validated by comparison with the non-CTF corrected FSC curve, which was generated by replacing the CTF-corrected tomogram with its non-CTF corrected equivalent, without further alignment of the particles.", "Since this changes the noise floor, the noise correlation disappears, as evidenced by the near zero values of the non-CTF corrected FSC curve at higher resolutions (Fig. S5B).", "The ability to gain biological insights from cryoET data depends intrinsically on our ability to mine and detect the in situ context of our biological feature of interest.", "To allow better probing the relative localization of features in the cell, we used the refined positions and orientations of the individual particles contributing to the average.", "The microtubule average was back plotted (see Section 2) into the raw tomogram, which are of comparably low signal-to-noise and thus make it difficult to directly visualize a molecular interaction.", "In this instance, we applied two methods: a) direct back plotting of the sub-tomogram average structure using the positions and orientations determined during alignment, and b) refining the orientation of a known structural feature into the respective position in the tomogram, in this case performed for an adenovirus virus capsid.", "Sub-volume averaging of the microtubule was performed as described above.", "For the adenovirus re-orientation, we used PEET to fit a previously determined viral structure (EMDB ID 1574, (Fabry et al., 2008)) into our tomographic viral feature.", "The orientation of this structure was then subsequently refined and placed into the tomogram according to the determined orientation.", "The fitting of the structure was then compared to the original tomogram, to verify and compare whether the adenovirus densities overlapped with features in respective slices through the back-plotted volume.", "To further validate the accuracy of the alignment of the EMDB-structure to the adenovirus contained within the tomogram, we performed icosahedral averaging of the single adenovirus within the tomogram, according to the Euler angles determined during the alignment of the EMDB-map.", "The resultant symmetrically averaged structure yielded a structure of 77 Å, when compared to a low-pass filtered map of EMDB-map at 20 Å (Fig. 3F).", "The icosahedral average of the single virus before and after orientation refinement is presented in Fig. S6.", "The symmetrized adenovirus structure allowed for direct visualization of the trimeric hexon proteins on the virus surface, as well as of the 12 penton bases.", "Icosahedral symmetrisation of the virus in the absence of any refinement yielded no discernible structure (Fig. S6) indicative of a lack of symmetry axis orientation and possible position inaccuracy.", "This analysis shows that the information contained in our tomogram was able to produce correlative signal to 77 Å (Fig. 3E, F) from only 60 copies of an asymmetric unit, with the noisy FSC curve being indicative of particle number based limitation and even further information content.", "The demonstrated potential for the differential structural determination of sub-populations of virus and protein structures in situ provides promising and exciting possibilities for dissecting dynamic processes and subtle structural changes in situ.", "The resultant “segmented” tomogram (Fig. 3C, D), allowed for directly viewing the 3-dimensional spatial organization of the microtubule network of an adenovirus undergoing viral entry.", "We were able to observe the topology and specific orientation of an adenovirus as it is enveloped into the periphery of a cell.", "Given the contextual situation of the virus, we were able to define that the virus is undergoing the process of endosomal uptake into the cell.", "The ability to determine the orientation of the virus with respect to the cell membrane allowed the visualization of the position of the adenovirus penton fibre as it interacts with the plasma membrane, being in this instance directly found centrally in the invaginated plasma membrane patch (Fig. 3C, D).", "The bilayer of the plasma membrane could clearly be followed around the virus in x, y and z, the curvature being indicative of the process of entry being fairly advanced.", "The immediate proximity of microtubules to the virus undergoing internalization by endocytosis suggests that the subsequent process of viral retrograde transport towards the nucleus is closely coupled to microtubules, consistent with observed speeds for incoming adenoviruses in the range of ∼1 μm s−1 (Bremner et al., 2009), which allows the virus to quickly gain entry to the inner regions of the cell.", "Sub-volume averaging of structures from single tomograms is routine until the resolution approaches the first zero of the CTF function for the given defocus at which the tomogram was acquired.", "In order to achieve accurate information past the first zero, it is important that one efficiently corrects the CTF.", "In our instance and conditions phase flipping in IMOD was sufficient in a single tomogram to recover significant information contained across the field of view.", "This is the case despite a large nominal defocus range determined during the acquisition of the tomogram (Table S1).", "This inaccuracy in the defocus during tomogram acquisition is not uncommon, and the lack of eucentricity across the sample and non-uniformity of the defocus will make the CTF model of individual particles within the tomogram more difficult to determine.", "The comparison of microtubule structures reconstructed using CTF and non-CTF corrected tomograms (Fig. 4A and B) for sub-volume averaging from each tomogram clearly shows that even in the case of one tomogram, the resolution to which we can interpret a structure is significant (Fig. 4A).", "The CTF corrected structure of the microtubule from the U87MG tomogram has an FSC(0.143) of 18.5 Å, and interpretable information persists in the FSC to around 17 Å.", "Without CTF correction, the amount of information in the structure falls off at around 25 Å (Fig. 4A).", "In the case of the U2OS tomogram, the non-CTF corrected structure is interpretable to 32 Å due to the CTF induced phase change, but is increased to a resolution of 25 Å after CTF correction (Fig. 4B).", "Interestingly, the calculation of the defocus in IMOD of each tomogram was different to the nominal defocus of the microscope by as much as 0.5 μm (Table S1).", "Even accounting for inaccuracies in defocus determination of the microscope (Danev and Baumeister, 2016), this is still a large discrepancy.", "The calculated CTF of the calculated defoci is shown in Fig. 4C for each tomogram.", "To better determine the accuracy of the CTF correction, an ‘ideal’ microtubule structure was built by fitting atomic models of symmetrized tubulin monomers from a microtubule structure (PDB ID: 3J2U) into the sub-tomogram averages determined here.", "These atomic models were then Gaussian blurred in Chimera to Nyquist frequency and matched in sampling and box size to the respective sub-tomogram average.", "The FSCs between this ‘ideal’ structure and the CTF and non-CTF corrected averages are shown in Fig. 5.", "For both structures, CTF correction resulted in a more accurate structure, with the U87MG cell microtubule average (Fig. 4A) displaying a particularly obvious phase flip correction.", "However, a small dip below zero at around 25 Å in the U2OS cell microtubule average CTF-corrected FSC (Fig. 4B, purple line) may well indicate an inaccuracy in defocus determination.", "We would suggest that an “all-tomogram” approach to CTF correction, especially for datasets with low particle numbers, likely results in corrections that are less accurate or not weighted correctly.", "Approaches that have been implemented recently (Bharat et al., 2015; Galaz-Montoya et al., 2016) whereby individual sub-volumes are tilt-dependently CTF corrected should prove to be much more accurate in determining a per-particle CTF correction.", "These approaches would yield a much more accurate determination of particle eucentricity across the tilt series, as a custom per-particle defocus and z-position within the tomogram can be determined.", "This should result in sub-volume averages that yield better results, particularly for single tomograms, or datasets with only a low number of particles.", "The current structural understanding of virus-host interactions stem mainly from studies using co-crystalized complexes, small angle scattering and sub-volume averaging of in vitro constituted systems, such as virus-like particles.", "Direct molecular imaging of the viral interactions with the host has hitherto not been a mainstay of investigation.", "The increased signal-to-noise ratio in the images gained from the use of direct detection allowed us to see much greater details inside the cell, as well as to average structures to an intermediate resolution capable of bridging the gap between structural context and molecular resolution (Figs. 2 and 3).", "This will surely allow us to provide novel insights into the cellular biology of viral infection.", "The structural context of virus-cell processes in a model cell line is currently the closest one may currently come to reconstituting a disease-like state.", "The ability to localize structures in their cellular context (Fig. 3), and to sub-volume average different conformers of the virus as it progresses through various stages of its life cycle (Fig. 3C), will allow determining which viral proteins are lost, which cellular proteins it interacts with, and what other biological macromolecules and pathways the virus recruits or hijacks in this dynamic process.", "Being often easily recognizable due to their unique structural signatures viruses also constitute ideal tools to probe cellular processes.", "Furthermore, the structural determination of cellular factors co-localising with the virus during infection gives clues to potential novel forms of interventions to be explored.", "Many biological phenomena are intrinsically transient, and rare.", "Structural biology currently relies on approaches that can enrich for a particular biological event, state, or process of biological relevance, e.g. by overexpression of a particular protein, though this often comes with the sacrifice that the system is no longer near native.", "These are typified by the approaches based on purified material and can be combined with X-ray crystallography, single particle cryoEM or sub-tomographic averaging.", "However, these necessary and, in many cases sensible approaches, cannot always be applied due to the difficulty of producing substantial amounts of protein, DNA/RNA or other macromolecule in a structurally determinable state.", "Furthermore, the lack of in situ context for these structures means that in many cases crucial pieces of information from the overall model of how a biological process occurs are missing, such severely limiting a mechanistic understanding.", "In this paper, we have discussed methods and data that allow for assessing the wealth of detail that can be mined from a single tomogram.", "Resolutions of 77 Å from a single symmetrized icosahedral virus and 18.5–25 Å from relatively few numbers of microtubule segments gives us a good indication that in many cases, the amount of information present even in a single tomograms is substantial, and something that, given the right biological problem and system, may lead to novel insights to complement the structural models many laboratories are building using canonical techniques.", "The direct observation of biology just at the cryoET accessible periphery of a cell provides a severe limitation; as the feature of interest needs to reside in an area thin enough to image with sufficient counts.", "This limits the range of biological processes one can image and the number and quality of observed features.", "Accordingly, several approaches have been implemented addressing this shortfall by not only making localization of a biological feature of interest easier to find, but also adding to the overall interpretation of the cryoET data (Schellenberger et al., 2014; Schorb and Briggs, 2014).", "The recent success provided by the use of thinning methods (see below) is another promising direction.", "Recent developments in phase plate technologies and in cryoFIBSEM (Asano et al., 2015; Danev et al., 2014; Mahamid et al., 2016; Rigort et al., 2012) have given a flavor of the insights that may be gained in the future, specifically with the ability to investigate by cryoET processes and structures deeper inside a cell, and e.g. probe nuclear biology in its native context.", "However, these applications are currently still not routine or for higher throughput uses, and require specialist knowledge for their operation and application.", "These current drawbacks are significant, as they restrict the range of biological applications that may benefit from the improved insight that could be determined.", "Future developments need to address these, and make the technologies much easier to use and robust for routine everyday use.", "A potentially powerful technique to provide further context for in situ-determined viral structure is the coupled use of correlative light electron microscopy (CLEM).", "Techniques such as light sheet fluorescence microscopy (LSFM), 3-dimensional structured illumination microscopy (3D-SIM) and localization microscopy (Chen et al., 2014; Schermelleh et al., 2008), especially when utilized in combination (Hoyer et al., 2016) offer potential for coupling the many complimentary techniques for cryoET, such as CLEM and cryoFIB/SEM, to offer a multi-scale resolution view of a molecular process in situ (Mahamid et al., 2015).", "Performing super-resolution fluorescence microscopy modalities routinely under cryo conditions and ultimately in a CLEM fashion (cryoCLEM) is the next challenge, but the few pilot studies performed demonstrate the potential and feasibility of such an approach (Wolff et al., 2016).", "The examples highlighted in our study suggest that it is possible to gain valuable structural insight from only few tomograms, and a 25 Å 3D structure should be possible from fewer than 300 particles.", "Subsequent classification, back plotting into the tomogram, and segmentation can provide a diverse range of biological information, in the most native of contexts, over a wide range of resolutions.", "The work presented in this article provide a significant and exciting frontier in cellular biology that may one day lead to the structural determination of novel structural complexes in situ.", "The multitude of classical electron microscopy applications many of which could be imaged under the much more native environment provided by cryoET, promise a rich and plentiful vein of discovery that can be judiciously mined through appropriate fluorescently tagged biological systems.", "This link between the high resolution imaging of cellular structure and structure determination is so far poorly explored and advancements would lead to a wealth of novel biological insight.", "CryoET of cells offers a genuine route to the structural determination of subcellular complexes and organelles in their native state in the course of their function, and at a resolution gradually closer approaching that of single particle analysis.", "Improvements in microscopic hardware, software workflows, sample preparation and transfer, and computational processing will require future investment if cryoET is to realize its full potential.", "The microtubule sub-volume average reconstructions have been deposited in the Electron Microscopy Data Bank (EMDB) at PDBe (http://www.ebi.ac.uk/pdbe/emdb/) as EMD-4043 and EMD-4045." ]
[]
Dietary nucleotide supplementation enhances immune responses and survival to Streptococcus iniae in hybrid tilapia fed diet containing low fish meal
A feeding trial was conducted to evaluate the effects of nucleotide (NT) supplementation in diet on immune responses and disease resistance of juvenile hybrid tilapia, Oreochromis niloticus × Oreochromis aureus. Nucleotide was added at 0, 120, 240, 360, 480 and 600 mg NT/kg to low fish meal (6%) and high soybean meal (56%) basal diet for a total of 6 experimental diets. Each diet was fed to triplicate groups of tilapia (initial body weight 0.15 ± 0.005 g) in a recirculated freshwater rearing system for 10 weeks. Head kidney leukocyte superoxide anion production ratio was higher (P < 0.05) in fish fed diets supplemented with ≥240 mg NT/kg than that in fish fed the NT unsupplemented control diet. Fish fed the diet supplemented with 240 mg NT/kg had higher plasma lysozyme activity than fish fed diets supplemented with ≤120 mg NT/kg. The stimulation index (SI) of head kidney leukocyte stimulated with ConA was higher in fish fed diets supplemented with ≥120 mg NT/kg than that in fish fed the control diet. The SI of leukocyte stimulated with PHA-P was higher in fish fed diets supplemented with ≥240 mg NT/kg than in fish fed diets supplemented with ≤120 mg NT/kg. After the feeding trial, 10 fish were randomly selected from each aquarium and were challenged with Streptococcus iniae for 7 days, higher survival (>80%) were observed in fish fed diets supplemented with NT than fish fed the NT unsupplemented control diet (56.7%). These results suggest that nucleotides supplemented at 120-240 mg NT/kg in diet enhances immune responses and survival of tilapia fed low fish meal and high soybean meal diet.
[ "AGRI" ]
[ "Immune responses", "Low fish meal diet", "Nucleotides", "Tilapia" ]
[ "1103", "1104" ]
[ "Nucleotides (NT) have essential physiological and biochemical functions including encoding and deciphering genetic information, mediating energy metabolism and cell signaling as well as serving as components of coenzymes, allosteric effectors and cellular agonists in terrestrial animals (Carver and Walker, 1995).", "Because of active de novo synthesis of NT mainly in liver, most animal appear to be almost independent on exogenous NT (Jyonouchi, 1994).", "However, the requirements for exogenous NT may increase under certain conditions, e.g. tissue injury, dysfunction of liver, under disease or stress, or in fast-growth life stage.", "For aquatic animal, dietary NT supplementation has been demonstrated to enhance growth (Li et al., 2007a; Lin et al., 2009; Abtahi et al., 2013), disease resistance and immune responses (Ramadan et al., 1994; Burrells et al., 2001a,b; Li et al., 2004, 2007; Lin et al., 2009).", "The immune system in fish is customary divided into innate (non-specific) and acquired (specific) system (Magnadóttir, 2006).", "Superoxide anion production and lysozyme activity are widely used as non-specific immune parameters in fish.", "Several reactive oxygen species (ROS) are produced by fish phagocytes during the respiratory burst.", "Once bacteria or fungi are engulfed by leucocytes, the host’s NADPH-oxidase is activated, which in turn increases oxygen consumption and subsequently produces ROS such as superoxide anion (O2−) (Roch, 1999).", "The release of superoxide anion is known as the respiratory burst, and together its derivates are bactericidal (Secombes and Fletcher, 1992).", "Lysozyme found in cutaneous mucus, peripheral blood and certain tissues rich in leucocytes, is an enzyme which catalyses the hydrolysis of N-acetyl muramic acid and N-acetyl glucosamine of peptidoglycan in bacterial cell walls (Jolles and Jolles, 1984) which plays a crucial role in the defense system.", "Head–kidney lymphocyte proliferation is often used as specific immune parameter in fish.", "The proliferation response can be observed when lymphocytes are stimulated by mitogens.", "Tilapia is widely cultured in tropical and subtropical regions of the world.", "The production of tilapia has increased from 332,186 MT in 1990 to 4,080,898 MT in 2012 (FAO, 2014) and has been recognized by FAO as the most potent culture fish species in supplying human protein source of the Century.", "Feed is the most expensive cost item in aquaculture industry, often ranging from 50 to 60% of the total variable expenses.", "Fish meal is the major dietary protein source, comprising between 20 and 60% of fish diet in general.", "Due to increasing demand, limited supply, and the dramatic increase in fish meal price, efforts to replace fish meal by other plant protein source such as soybean meal have been increasing in aquafeed (Lim et al., 2011).", "It should be noticed that, however, NT concentration of soybean meal (0.038 mg/g) is about only half of fish meal (0.075 mg/g) (Mateo et al., 2004).", "While the soybean meal are used to replace fish meal in diet, it is unclear that the reduced NT levels whether influence the performances, e.g., growth or immune responses of fish.", "Thus, the purpose of this study was to evaluate the effects of dietary nucleotides supplementation on growth, immune responses and resistance to Streptococcus iniae in hybrid tilapia Oreochromis niloticus × Oreochromis aureus fed diet containing low fish meal (6%) and high soybean meal (56%).", "Nucleotide (Rovimax NX, 80% nucleotides, DSM nutritional Products, Basel, Switzerland) was supplemented at 0, 150, 300, 450, 600 and 750 mg/kg diet in a basal diet with 6% fish meal and 56% soybean meal (Table 1), providing 0, 120, 240, 360, 480 and 600 mg NT/kg diet.", "Fish meal (blue whiting, imported from Norway) and soybean meal (TTET Union, Taiwan), fish oil (Semi-refined fish oil, Oleaginosa Victoria S.A., Peru) and corn oil (Tai-Tang Industrial, Taiwan), corn starch (Sigma Chemical Co., St. Louis, MO) were used as dietary protein, lipid, and carbohydrate sources, respectively.", "The vitamin and mineral mixtures were similar to those described by Hsien and Shiau (2000).", "The diets were prepared by thoroughly mixing the dry ingredients with oil and then adding cold water until a stiff dough resulted.", "This was then passed through a mincer with die and the resulting strings were dried using an electrical fan at 24 °C.", "After drying, the diets were broken up, sieved into pellets and stored at −20 °C.", "This study, involving animal experiment, conformed to the principles for the use and care of laboratory animals, in agreement with the Institutional Animal Care and Use Committee (IACUC) in National Pingtung University of Science and Technology (NPUST).", "Male hybrid tilapia (O. niloticus × O. aureus) obtained from the Far East Hatchery (Cha-Yi, Taiwan) were used in the study.", "Upon arrival, they were acclimated to laboratory conditions for 4 weeks in a 1000 L plastic tank and fed a commercial diet (Uni-President Enterprise Corp., Tainan, Taiwan; moisture, 11.7%; crude protein, 43.3%; lipid, 8.8%; ash, 9.3%) for 1 week to make sure the health status of tilapia and thereafter fed basal diet for 3 weeks.", "At the beginning of the experiment, 25 fish (mean weight: 0.15 ± 0.005 g) were stocked in each experimental aquarium (30.5 × 61.0 × 55.5 cm).", "Each experimental diet was fed to three groups of fish.", "The fish were chosen for the experiment and the diets were assigned to groups of fish randomly.", "Each aquarium was part of a closed-recirculating system with a common water reservoir maintained at 27 ± 1 °C.", "The water was circulated at 2 L/min through two separate biofilters to remove impurities and reduce ammonia concentrations.", "A photoperiod of 12 h light (0800–2000 h), 12 h dark was used.", "The fish were fed 5% of their body weight per day.", "This amount was close to the maximal daily ration for tilapia according to feed consumption during the acclimation period of the study.", "The daily ration was divided into two equal meals fed at 0830 and 1630 h. Fish were weighed once every two weeks and the daily ration adjusted accordingly.", "The remaining feed and feces were removed by a siphon immediately after feeding.", "Any dead fish were removed and not replaced during the experiment.", "The fish were fed the test diets for a 10-week period.", "At the end of the feeding trial, percentage of body weight gain (WG) in each aquarium [100×(final body weight – initial body weight)/initial body weight], feed efficiency (FE) [(final body weight – initial body weight)/feed intake], and survival [100×(final fish number/initial fish number] were calculated.", "After the final weighing, blood was collected from the caudal vein by syringe with heparin as the anticoagulant from 8 fish selected randomly per aquarium and pooled.", "The blood was centrifuged at 3000 × g for 15 min.", "Plasma was removed for lysozyme activity determination according to the methods of Obach et al. (1993).", "After blood collecting, head kidney were removed from the same fish and pooled.", "Head kidney leukocytes were isolated by 35% and 51% Percoll solution (Pharmacia Biotechnology Inc., Uppsala, Sweden) described by Secombes (1990).", "Leukocyte superoxide anion (O2−) production ratio and stimulation index (proliferation) were analyzed by the method of Secombes (1990) and Daly et al. (1995), respectively.", "The O2− production ratio of leukocytes was quantified using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of superoxide anion production.", "The assay of leucocyte proliferation used the colorimetric 3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyl tetrazolium bromide (MTT) method with some modifications.", "A 90 μL aliquot of proliferation medium containing 5 × 105 leucocytes and 10 μL mitogen solution was added to wells of a 96 well flat-bottomed microtitre plate and incubated for 48 h at 27 °C in a humidified atmosphere with 5% CO2.", "The mitogen solution contained one of the following mitogens: 200 μg/mL Escherichia coli O26:B6 lipopolysaccharide (LPS), 10 μg/mL phytohaemagglutinin (PHA-P), or 25 μg/mL concanavalin (Con A) (all from Sigma Chemical).", "A volume of 20 μL MTT (5 g/mL, Sigma Chemical) was added to each well and incubated for 4 h at 27 °C.", "The microtitre plate was then centrifuged at 500 × g for 10 min, the supernatant discarded and the formazan crystals in each well were dissolved by adding 200 μL dimethyl sulphoxide (Sigma) and 25 μl of 0.1 M glycine buffer.", "The contents of the wells were then thoroughly mixed with a multichannel pipette.", "The O.D. at 600 nm of the resulting suspension was measured in the microplate reader 10 min later.", "The proliferation of the leucocytes was expressed as a stimulation index (SI).", "SI was calculated using the formula: Stimulation indexn= (mean OD600 nm of leukocyte wells with test mitogen/mean OD600 nm of leukocyte wells without mitogen) – 1.", "All analyses were conducted in triplicates.", "After feeding trial, 10 fish from each aquarium were challenged with S. iniae for 1 week, survival of the fish were recorded.", "The S. iniae used in this study was strain BCRC 14.744, obtained from Bioresources Collection and Research Center (BCRC) of the Food Industry Research & Development Institute in Taiwan.", "Ten fish were randomly selected from each aquarium and injected by intraperitoneal (i.p.) injection with 1.0 × 107 CFU/g fish body weight of S. iniae (Ndong et al., 2007).", "Another 10 fish were i.p. injected with saline as a blank.", "All the challenged fish were returned to their respective rearing aquarium.", "Survival was recorded daily for 7 days and dead fish were removed.", "Each experimental diet was fed to three groups of fish according to a completely randomized design.", "Results were analyzed by one-way analysis of variance (ANOVA) with dietary NT levels as main effects using the SAS/PC statistical software (SAS Inst.", "Inc., Cary, NC, USA), and significance was set at P < 0.05.", "Multiple comparisons among means were made with Duncan’s new multiple range test.", "Tilapia fed different diets for 10 weeks, WG, FE, PER and survival of the fish were not significantly (P > 0.05) different among the dietary groups (Table 2).", "Head kidney leukocyte O2− production ratio was higher in fish fed diets supplemented with ≥240 mg NT/kg than in fish fed the NT-unsupplemented control diet (Table 3).", "Fish fed the diet supplemented with 240 mg NT/kg had higher plasma lysozyme activity than fish fed diets supplemented with ≤ 120 mg NT/kg.", "The stimulation index (SI) of head kidney leukocyte stimulated with ConA was higher in fish fed diets supplemented with ≥120 mg NT/kg than that in fish fed the control diet (Table 4).", "The SI of leukocyte stimulated with PHA-P was higher in fish fed diets supplemented with ≥240 mg NT/kg than in fish fed diets supplemented with ≤120 mg NT/kg.", "The SI of leukocyte stimulated with LPS was higher in fish fed diets supplemented with 240–480 mg NT/kg than in fish fed the diet supplemented with 120 mg NT/kg.", "After challenged with S. iniae for 1 wk, significantly higher survival (80–86.7%) were observed in fish fed diets supplemented with NT than fish fed the NT-unsupplemented control diet (56.7%).", "The immune system of fish can be influenced by a wide range of factors including diseases, pollutants, hormones and feed (nutrition).", "Feed formulation of tilapia culture is largely depending on plant ingredients.", "Fish meal replaced by plant ingredients caused depression of immune responses in fish (Sitjà-Bobadilla et al., 2005; Kokou et al., 2012).", "Nucleotide concentration of soybean meal (0.038 mg/g) is about half of fish meal (0.075 mg/g) (Mateo et al., 2004).", "The NT concentration of the basal diet used in the present study was calculated as 25.78 mg/kg diet.", "Superoxide anion production and lysozyme activity are widely used as non-specific immune parameters in fish.", "In the present study, both head kidney O2− production ratio and plasma lysozyme activity were enhanced in tilapia fed diet supplemented with 240 mg/kg diet of NT (Table 3).", "Nucleotides (NT) functions in encoding and deciphering genetic information, mediating energy metabolism, cell signaling and serving as components of coenzymes, allosteric effectors and cellular agonists in terrestrial animals (Carver and Walker 1995).", "This may explain why the supplementation of dietary NT can enhance some non-specific immune responses.", "Our results were similar with previous works regarding of the enhancement of immune responses by the administration of dietary NT for fish.", "Similar results were found in Nile tilapia (Ramadan et al., 1994), Atlantic salmon (Burrells et al., 2001a,b) hybrid striped bass (Li et al., 2004), red drum (Li et al., 2007) and grouper (Lin et al., 2009).", "Head-kidney lymphocyte proliferation is often used as specific immune parameter in fish.", "Fish have two kinds of lymphocytes, B cells and T cells, respectively.", "The PHA-P and Con A responsive cells belong to the T-lymphocytes (DeLuca et al., 1983; Caspi and Avtalion, 1984; Jokinen et al., 2001), and are distinct from the B-lymphocytes that is LPS-responsive (Sizemore et al., 1984; Jokinen et al., 2001).", "In the present study, lymphocyte proliferation were clearly stimulated with all these three mitogens, i.e., Con A, PHA-P and LPS, in tilapia fed diets supplemented with 120–240 mg/kg diet (Table 4).", "This phenomenon were in line with the major function of NT involved in translation and/or transcription for animals.", "Administration of dietary NT has been reported to enhance growth of Atlantic salmon (Burrells et al., 2001a,b), Caspain brown trout (Abedian Kenari et al., 2013), red drum (Li et al., 2007), grouper (Lin et al., 2009) and Beluga sturgeon (Abtahi et al., 2013).", "However, the growth-promoting effect of NT supplementation was not observed in hybrid striped bass (Li et al., 2004), barramundi (Glencross and Rutherford, 2010), channel catfish (Welker et al., 2011) and pikeperch (Jarmolowicz et al., 2012).", "Generally, animal during periods of rapid growth require high levels of NT (Calver, 1994).", "In the present study, dietary NT supplementation had no growth promoting effect for the juvenile tilapia with body weight of 0.15–5.1 g.", "Streptococcal disease is the cause of significant economic losses to the world aquaculture industry (Shoemaker and Klesius, 1997).", "The impact of Gram-positive bacterium S. iniae on tilapia aquaculture has been known for more than a decade (Klesius et al., 2008).", "In the present study, tilapia fed diets supplemented with NT (≥120 mg NT/kg diet) had significantly higher survival after being challenged with S. iniae than that of fish fed the NT unsupplemented diet (Table 5).", "The results suggest that nucleotides supplemented at 120–240 mg NT/kg in diet enhances immune responses and survival to S. iniae for tilapia fed low fish meal and high soybean meal diet." ]
[ "We have evaluated the effects of dietary nucleotide (NT) on immune responses and disease resistance of tilapia.", "Dietary NT at 120–240 mg NT/kg enhances immunity and survival for tilapia.", "Additional NT is required for tilapia fed with low fish meal diet." ]
CRUD deposition in accelerated high-temperature water: Investigation on the effect of substrate material and water chemistry
Deposition of corrosion products (CRUD) on micro-orifice specimens under accelerated flow conditions was investigated in high-temperature water on several substrate materials (SS 304L, Titanium, Alloy 690, Zirlo and magnesia stabilised Zirconia) and water chemistries (0–2 ppm of Li, 0–2.5 ppm of H2). Regardless of the substrate material, no significant differences in radial CRUD build-up were observed in high purity water (0 ppm of Li and either 0 or 2.5 ppm of H2). Conversely, in lithiated water (2 ppm of lithium and 2.5 ppm of H2), the CRUD surface build-up was dependent on the oxide electrical conductivity, which in turn depends on the substrate material. In fact, no build-up occurred on Zirconia and only a minor deposition on Zirlo. The deposition mechanism was ascribed to be dominated by particulate deposition at neutral pH where magnetite solubility is the highest. Conversely, crystalline build-up induced by electrokinetic activity played an important role at alkaline pH where oxide solubility was the lowest.
[ "ENER", "MATE", "PHYS" ]
[]
[ "2104", "2500", "3106" ]
[ "CRUD is an acronym for Chalk River Unidentified Deposits, although more recently it is used to refer to fouling deposits in the Light Water Reactors (LWRs), typically a mixture of magnetite in Pressurised Water Reactors (PWRs), hematite in Boiling Water Reactors (BWRs), and other phases such as trevorite (NiFe2O4) and NiO [1–3].", "CRUD is responsible for several detrimental effects in LWRs including the CRUD Induced Power Shift (CIPS), where boron is incorporated into CRUD on the fuel rods, which causes an axial power shift in PWRs [2,4,5], in addition to the isotopic transmutation of 59Co into 60Co, that is responsible for issues of radioactivity control for both PWRs and BWRs [6].", "CRUD deposition has also been frequently observed in regions where there are hydrodynamic perturbations of the flow, which induce clogging and localised corrosion issues [7–9].", "For instance, orifices are used as a flowmeter in the secondary circuit of PWRs, and the deposition of CRUD is a major issue that affects the accuracy of the flow rate measurements [7–9].", "In the present manuscript, the focus is on fouling that occurs in regions of restricted flow where the deposit has been reported to be mainly magnetite [7–9].", "Since a wide range of alloys are used as structural materials in PWRs [1,10–14], it is important to understand the role of the substrate material on the deposition of CRUD.", "For instance, steam generators, which account for 65–70% of a reactor’s surface area [10–12], are commonly constructed from nickel-based alloys such as Alloy 600 and Alloy 690.", "Meanwhile, cladding materials for the reactor vessel and fuel rods are generally manufactured from austenitic stainless steels (including type 304 and 316) [15] and zirconium alloys (such as Zirlo and Zircalloy-4) [1,13,14], and finally the tube condenser, located in the secondary circuit of the PWR, is fabricated from titanium alloys (grade 1 and 2) [1,13,14,16].", "CRUD deposits in high-temperature water have been shown to have a double structure, consisting of an outer layer of amorphous and loosely adherent particulate deposit and an inner tightly adherent crystalline deposit [7–9,17].", "CRUD deposit as a function of substrate materials has also been investigated under thermal flux condition in high-temperature water; it was suggested that materials with refraction index coefficient similar to the one of water had the lowest Hamaker’s constant and were more resistant to NiO particle deposition [10].", "However, whilst particulate deposition has been widely investigated [18–21], the magnetite crystalline build-up observed under accelerated flow has received attention only in recent years [8,9,22]; its formation has been ascribed to the divergence of streaming current in regions of accelerated flow [8,9,22,23].", "Specifically, a streaming current is generated by the shear of ions in the diffuse part of the double layer [24] and, under accelerated flow, it increases in magnitude due to increased velocity, resulting in a local unbalance of charges which are compensated by the generation of a wall current.", "The wall current can then induce faradaic reactions such as the oxidation of Fe2+ dissolved in the water to Fe3+ resulting in the deposition of magnetite [7–9].", "Such electrokinetic deposition is expected to occur in accelerated flow when streaming current is positive and the surface zeta potential is negative.", "The streaming current and zeta potential values are dependent on the interaction between the material and the coolant [7–9]; thus electrokinetic deposition is expected to be affected both by the substrate material and by the water chemistry.", "Experimental investigations aiming at elucidating the mechanism that drives CRUD build-up under accelerated flow were mostly carried out at the technical centre of Areva [25,26] and at the University of Manchester [7–9].", "In these investigations, the dual nature between the loosely adherent particulate build-up and the hard-crystalline deposit was highlighted, in particular in the work carried out by Ref. [7] on discs manufactured from of SS304L.", "However, the contribution of electrokinetic deposition to the overall build-up is still unclear, and a comparative ranking of materials in terms of CRUD susceptibility is not presently available.", "Therefore, the aims of this work were: i) to investigate the contribution of the electrokinetic deposition generated by a wall current to the overall CRUD build-up rate, ii) to investigate the dependence between CRUD build-up and substrate materials under accelerated flow conditions as a function of the water chemistry, and iii) to obtain a quantitative correlation between the water chemistry (including dissolved hydrogen, iron and nickel) on the overall CRUD deposition rate and morphology.", "Tests were carried out on the following materials such as SS304, Alloy 690, Titanium, Zirlo that are already commonly used in PWR and a Magnesia stabilised Zirconia (ZrO2–MgO).", "The magnesia stabilised Zirconia is a ceramic material characterised by a mixture of cubic and monoclinic phases, confirmed by the X-ray analysis but not reported in this paper for brevity, which was selected to avoid the possibility of faradaic reactions generated by the deflection of streaming current, thereby allowing discrimination between wall current driven deposition and the particulate build-up.", "The tests were carried out in high-temperature high-pressure water with variable dissolved Li (0–2 ppm, pH230 °C 5.64 to 7.63) and H2 (0–2.5 ppm).", "Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) and conductivity measurements were carried out from the autoclave return line to correlate the amount of iron and nickel in solution with the measured radial build-up.", "Finally, characterisation of the CRUD morphology was carried out by Scanning Electron Microscopy (SEM) and Energy Dispersive X-Ray analysis (EDX).", "The tests were carried out in a 13-L Stainless Steel autoclave vessel that is schematically shown in Fig. 1 and that has been widely used for previous work by the Authors [7–9,27].", "The micro-orifice discs were individually mounted in six flow cells that were fitted inside the autoclave.", "Each flow cell was built using Swagelok fittings comprising a female connector with a 3/8” ISO parallel thread (bore of 9.53 mm in diameter) which housed the disc.", "The sealing between the female connector and the disc was achieved using a copper gasket and by tightening the fitting with a matching face sealing male connector.", "More information about this setup is available in Ref. [7].", "The water was circulated via a volumetric pump (rated up to 35 l/h) and preheated to 230 °C before entering the autoclave.", "After passing through the flow cells, the water was cooled, depressurised, recirculated through a mixed bed of ion exchange resin and re-injected to the 250-L storage feed tank.", "The experiments were carried out at a constant pressure drop (equivalent to a constant flow velocity) [7,9] across the micro-orifice by using two back pressure regulators.", "Specifically, one pressure regulator was at the outlet of the autoclave and the second back pressure regulator was branched off the line between the volumetric pump and the autoclave, used to diverge the excess flow from the pump back into the feed tank, and maintain constant upstream pressure in the flow cell [7].", "The outlet water chemistry was monitored using oxygen (Orbisphere 410/A/P1C00000) and hydrogen sensors (Orbisphere 510B00/T1C0P000), while two sensors measured the inlet and outlet conductivity (ABB AX410/500001).", "The pressure drop across the flow cell was measured using two pressure transducers (Omega model PXM 409, 0–24.5 MPa span, 0.1% full-scale accuracy) and the mass flow rate was measured using two variable area flowmeters (Swagelok model VAF-M2, with a range 0–1.4 cm3s-1 for one and 1.1–11.1 cm3s-1 for the other).", "The flow meters were calibrated prior to the tests and their accuracy was within 1%.", "The specimens consisted of discs containing a micro-orifice.", "The micro-orifice was used to simulate accelerated flow conditions as reported in Refs. [7–9,27–29].", "All the substrate materials were selected because they have practical application in PWRs.", "However, the magnesia stabilised Zirconia was selected to investigate the effect of an insulating material where the wall current, that is believed to be responsible for CRUD deposition according to an electrokinetic driven deposition, was expected to be absent.", "ZrO2 is an insulating oxide [30] however, due to the doping with MgO the electrical resistivity of Zirconia specimens is slightly higher than the pure material.", "Specifically, the Zirconia discs were provided by Morgan Advanced Materials and, from interpolation of the specification provided by the supplier, the electrical resistivity of the disc calculated at 230 °C is expected to be in the order of 107 Ωcm that is one order of magnitude higher than the electrical resistivity of the high purity water at 100 °C (1.274 × 106 Ωcm) [31].", "All discs, except for those manufactured from zirconia, where mechanically drilled to form either a 400 or 300 μm orifice.", "Due to the brittle nature of the zirconia specimen, the micro-orifice was produced via an ultrasonic method, which generated a slightly elliptical orifice, with axis dimension of 423 and 389 μm and a corresponding equivalent hydraulic diameter of 406 μm.", "Prior to the tests, all the discs were ground to 600 grit using a Si–C emery paper.", "Before the start of the tests, the discs were pre-oxidised for 300 h in high pressure (120 bar) high-temperature water (230 °C) in the autoclave using the specific water chemistry adopted during each following test, as presented in Table 1.", "After the CRUD deposition test, the CRUD morphology was characterised by EDX analysis with a Zeiss Sigma FEG-SEM equipped with an Oxford Instruments X-maxN 150 EDX detector.", "The water chemistry was characterised prior to and during the experimental programme with the final aim to relate the amount of dissolved ions to the radial build-up measurements.", "A total of 33 water samples were extracted from the autoclave vessel using a PTFE capillary extraction line that was placed inside a stainless-steel pipe to withstand the pressure.", "The PTFE capillary tube was used to avoid deposition during the sampling as discussed in Ref. [7].", "The water samplings were carried out for each of the three water chemistries investigated specifically: 0 ppm Li 0 ppm H2, 0 ppm Li 2.5 ppm H2 and 2 ppm Li 2.5 ppm H2.", "After sampling, the solution was acidified with concentrated nitric acid and the total mass of iron and nickel present in the coolant were measured using Inductively Coupled Plasma Mass Spectroscopy ICP-MS, a technique that is known to be capable of measuring metal ions concentration at ppb accuracy [32].", "It is worth noting that with this procedure it was not possible to differentiate between the concentration of metal in soluble and particulate form.", "Conductivity measurements from the return line of the autoclave were also taken under variable hydrogen and lithium concentrations.", "The concentration of oxygen was monitored throughout the experimental program and was overall lower than 2 ppb.", "A total of 14 discs were tested in the flow loop to investigate the dependence between the radial CRUD deposition on different materials in 0–2 ppm of Li and 0–2.5 ppm of H2.", "The experiments were carried out at 230 °C and 120 bar.", "The temperature of 230 °C was selected to ensure that the downstream pressure was higher than the water vapour pressure, to avoid two-phase flow and cavitating conditions [7].", "The pressure was selected such as the sum between the imposed pressure of 120 bar and the pressure drop across the specimen was below the maximum working pressure of the autoclave (200 bar) as discussed in Refs. [7–9].", "A summary of the tests performed is presented in Table 1.", "The tests were carried out controlling the pressure drop across the micro-orifice, by using the two back pressure regulators (see schematic in Fig. 1), such that the target flow velocity inside the orifice was ∼32 m/s.", "Three autoclave batch tests were carried out in three different water chemistries.", "The total concentration of iron and nickel in the water was measured using ICP-MS in the three water chemistries investigated and the results are reported in Table 2 as an average value between different measurements carried out along the test program, with the variability between multiple measurements reported as standard deviation.", "No differentiation was made between soluble and particulate in these measures.", "From these measurements, it appears that the alkalinisation, which was obtained by the addition of LiOH (2 ppm of Li) in hydrogenated water (2.5 ppm of H2), caused a reduction in the total content of iron from 15.3 to 1.1 ppb, as well a reduction in the nickel content from 3.9 to 0.3 ppb.", "Room temperature conductivity measurements were carried out on the inlet and outlet of the autoclave.", "An example of the outlet conductivity measurements conducted whilst testing the Zirlo (Zr) and Zirconia (ZrO2) specimens is shown in Fig. 2.", "In high purity de-aerated water, the outlet conductivity was at its lowest value; when 2.5 ppm of H2 was added to the pure water, the outlet conductivity increased by approximately 0.10 μS/cm.", "In 2 ppm of Li and 2.5 ppm of H2, the overall conductivity measured across all the specimens tested ranged between 67 and 72 μS/cm.", "The reduction in micro-orifice diameter over time calculated for all the materials tested is shown in Fig. 3 where light green indicates 2.5 ppm of H2 2 ppm of Li, red indicates 0 ppm of H2 0 ppm Li and blue indicates for 2.5 ppm of H2 0 ppm of Li.", "The reduction in diameter of the micro-orifice over time was dependent on the water chemistry but not on the substrate materials, as indicated by the observation that a similar build-up behaviour was recorded.", "Specifically, the fastest diameter reduction was observed for the discs tested in hydrogenated high purity water (2.5 ppm of H2 and 0 ppm Li).", "Conversely, no variation in diameter was noticed for the discs tested in hydrogenated and lithiated water (2.5 ppm of H2 and 2 ppm of Li), while an intermediate reduction was observed in non-hydrogenated high purity water (0 ppm of H2 and 0 ppm Li).", "The rapid increase in the diameter from 400 to 430 μm observed after 55 h for the SS disc tested in lithiated water chemistry (D400-2.5-2-[SS], Fig. 3) was an experimental error associated with the fluctuation in differential pressure and volumetric flow measurements.", "The radial build-up rate values were then calculated using Eq. (1) and are presented in Fig. 4; the error bars in the radial build-up measurements were introduced taking into consideration the propagation of the error from the pressure drop and the volumetric flow rate measurements as previously discussed in Ref. [7].", "Furthermore, although the tests were carried out at constant flow velocity, a slight variation in pressure drop across the discs sometimes occurred, thus resulting in a velocity variation.", "This was taken into account by introducing error bars in the average value of flow velocity measured, as shown in Fig. 4.", "Fig. 4 also reports the literature data obtained from Ref. [7] for comparison purposes.", "The 400 μm orifices tested in pure water (0 ppm of Li and 0 ppm of H2), Fig. 4-a, had a comparable value of radial build-up to the results obtained by Ref. [7] for SS micro-orifices with an internal diameter of 300 and 600 μm.", "Conversely, for the specimens tested in hydrogenated water chemistry (Fig. 4-b), a higher radial build-up rate was observed compared to the specimens tested in non-hydrogenated water chemistry.", "No reduction in diameter was observed for the discs tested in 2 ppm Li, as shown in Fig. 3.", "The SEM results for Alloy 690 (D400-0-0-[A690]) and Zirconia (D406-0-0-[ZrO2]) were selected to investigate the morphology of the CRUD because they have very different electrical conductivities, with Zirconia being a relatively good insulator whilst passivated Alloy 690 is a good electrical conductor.", "Representative morphologies of the oxide formed on the front face of each orifice are shown in Fig. 5.", "Specifically, CRUD build-up on the front face of the Alloy 690 disc (D400-0-0-[A690]) and the Zirconia specimen (D406-0-0-[ZrO2]) tested in 0 ppm of H2 and 0 ppm of Li are reported in Fig. 5-a and d whilst higher magnification images are shown in Fig. 5 b,c e and f.", "On the Alloy 690 and Zirconia specimens, the CRUD was located across the transition between the front face and the micro-orifice throat.", "Higher magnification images of the Alloy 690 specimen (D400-0-0-[A690]) reported in Fig. 5-b and c showed the formation of crystalline ripples that propagated along the radial direction of the micro-orifice; however these ripples were not visible on the electrically insulated Zirconia sample (D406-2.5-0-[ZrO2]) where all the CRUD was entirely particulate, as shown in Fig. 5-e and f.", "The effect on hydrogen 0–2.5 ppm of H2 on the CRUD morphology was investigated on the Alloy 690 discs (D400-0-0-[A690] and D400-2.5-0-[A690]) and shown in Fig. 6.", "High magnification images of the Alloy 690 specimen tested under 2.5 ppm of H2 (D400-2.5-0-[A690]) showed a more compact CRUD oxide compared to the same material tested in dehydrogenated water (D400-0-0-[A690]).", "Specifically, in non-hydrogenated water chemistry, the CRUD morphology was significantly more porous and dominated by particulate deposition, as shown in Fig. 6-c and f. SEM characterisation was further carried out on all the specimens tested in 2 ppm of Li and 2.5 ppm of H2 as shown in Fig. 7.", "CRUD surface build-up was observed on the front face of titanium, stainless steel and on Alloy 690 specimens (D300-2.5-2-[Ti], D400-2.5-2-[SS] and D400-2.5-2-[A690]) as shown in Fig. 7.", "Conversely, on the Zirlo disc (D400-2.5-2-[Zr]) only a partial deposition was noticed, whilst no deposition occurred on the Zirconia sample (D400-2.5-2-[ZrO2]) as shown in Fig. 7-d and c. Finally, representative high magnification images of the morphology of the oxide obtained in lithiated water chemistry for the Titanium and the Alloy 690 discs (D300-2.5-2-[Ti] and D400-2.5-2-[A690]) are shown in Fig. 8 and Fig. 9.", "Independently on whether the materials investigated was Titanium or Alloy 690, the morphology of the CRUD observed in the proximity of the transition between the front face and the micro-orifice throat consisted of compact elongated needles that propagate along the radial direction of the micro-orifices as shown in Fig. 8-c to e and in Fig. 9-d. Meanwhile, far from the transition amorphous particles were dispersed on the surface as showed in Fig. 8-f for the Alloy 690 specimen.", "SEM-EDX spectrum images of the CRUD oxide deposited on the Titanium specimens (D300-2.5-2-[Ti]) tested in 2 ppm of Li, and 2.5 ppm of H2 are presented in Fig. 10, showing the formation of the iron-rich oxide.", "The solubility of magnetite and nickel ferrite was shown to be dependent on the pH of the solution, which was adjusted by changing the amount of lithium hydroxide.", "Moreover, it was also shown that the oxide solubility also depends on the amount of hydrogen whereby an increase of H2 from 0 to 2.5 ppm increased the total iron content, as shown by the ICP-MS measurements presented in Table 2.", "It could be argued that the deposition is controlled by corrosion phenomena which in turn depend on the electrochemical corrosion potential (ECP).", "However, since the ECP in PWR is dictated by the amount of H2, it becomes difficult to separate the effect of EPC from that of H2.", "Nonetheless, it was shown that increasing H2 (thus decreasing ECP) the CRUD build-up rate increases approximately by a factor of 2, because amount of metal ions in the water was also increased.", "This increment in iron was soluble rather than particulate, as suggested by the increase in the outlet water conductivity measurements.", "This evidence corroborates the theory that, under hydrogenated water chemistry, magnetite undergoes a reductive dissolution as discussed in Ref. [7].", "Conversely, the low amount of Fe and Ni content measured in 2 ppm of Li was due to a reduction in solubility at high pH for magnetite and nickel ferrite [7].", "Another complicating factor is associated with the presence of a small amount of oxygen in the water.", "In fact, it is known that traces of oxygen (even in the ppb level) can dramatically increase the ECP in high-velocity fluid [33].", "However, if it occurred, this increase in the ECP would be confined to micro-orifice because everywhere else in the system the flow velocities are relatively low (<0.5 m/s).", "Therefore, the presence of traces of oxygen in the system would not be expected to directly affect the overall amount of metal ions present in the water.", "Nonetheless, it is not possible to exclude that, due to the presence of traces of oxygen, the ECP at the micro-orifice is higher, thus having an effect on the magnetite deposition mechanism; however more work is required to evaluate this hypothesis.", "In fact, if the magnetite solubility decreased at higher ECP, a lower deposition would be expected in hydrogenated water (lower ECP) [34], but this is not what was found in this paper.", "The results of this work also showed that the material used for the manufacturing of the micro-orifices did not have a significant effect on the radial build-up in pure water (0 ppm of H2 and 0 ppm of Li).", "Moreover, it is evident that specimens with a poor electrical conductivity, such as those manufactured from of Zirconia [30], show a radial build-up rate value that is in the same range as for all the other specimens.", "This result strongly suggests that the electrokinetic deposition, which is driven by the deflection of streaming current and relies on a faradaic wall current [8], is not the dominant mechanism of CRUD deposition in high purity water.", "In fact, poorly conductive materials are unlikely to support Faradaic reactions such as that responsible for the oxidation of soluble Fe2+ to insoluble Fe3+ that is required for the deposition of magnetite.", "The relative insensitivity to magnetite deposition of the different materials tested also suggests that all these materials have high Hamaker constants and are equally “sticky” to magnetite [10].", "However future work is required in order to measure the index of refraction of these materials, which is required for the proper calculation of the Hamaker’s constant [35].", "The radial build-up rate values for the 400 μm micro-orifices was consistent with the results obtained for the 300 and 600 μm specimens reported in Ref. [7] whereby at high flow velocities there is a negative dependence with build-up rate.", "This behaviour can be ascribed to the increase in the shear forces responsible for the mechanical removal of the oxide for high values of Reynolds number [7–9].", "In hydrogenated high purity water (2.5 ppm of H2 and 0 ppm of Li), an increase in the radial build-up rate compared to the specimens tested in non-hydrogenated water (0 ppm of H2 and 0 ppm of Li) was observed for all materials tested.", "This increase in the radial build-up can be rationalised recalling that with higher hydrogen content there is an increase in soluble iron in solution, shown by the ICP-MS and by the conductivity measurements, and considering the fact that the deposition process is controlled by the mass transport for all the specimens investigated during this work.", "SEM characterisation was used to investigate the morphology of the CRUD as a function of the electrical conductivity of the Zirconia and the Alloy 690 specimens (D406-0-0-[ZrO2] and D400-0-0-[A690]).", "The investigation showed that CRUD morphology is a material specific process.", "In particular, crystalline ripples were observed on the front face of the Alloy 690 but not on the Zirconia sample (Fig. 5) which had low electrical conductivity.", "The observed magnetite ripples are in agreement with the postulated mechanism of electrokinetic deposition which is driven by deflection of the streaming current where oxidation of Fe2+ to Fe3+ is occurring on the anodic side of the loop followed by Fe3O4 precipitation [8,9,36].", "However, as discussed previously, the fact that similar values of radial deposition were obtained for different specimens tested suggests that the CRUD deposition induced by wall current generation is not the dominating mechanism of deposition under the investigated water chemistry (0 ppm of Li and 0–2.5 ppm of H2).", "Nevertheless, a partial contribution to the overall deposition was observed for Alloy 690 specimens as shown in Fig. 6.", "The radial build-up was found to be highly dependent on the water chemistry; the highest build-up was observed under high purity hydrogenated water.", "Conversely, after the addition of 2 ppm of lithium responsible for an alkalinisation of the solution (pH25 °C ≈ 10.45 pH 230°C ≈ 7.63), no reduction in diameter over time was observed (Fig. 3).", "The lack of radial deposition observed in 2 ppm of Li can be rationalised considering the reduction in magnetite solubility [8,9] as shown in Table 2.", "Nevertheless, the surface deposition was found to be material specific.", "Specifically, surface build-up was observed on the front face of the discs manufactured from stainless steel, Alloy 690 and titanium (D400-2.5-2-[SS], D400-2.5-2-[A690] and D300-2.5-2-[Ti]).", "Conversely, only a partial surface build-up was observed on the front face of the Zirlo (D400-2.5-2-[Zr]), and no deposit was observed on the front face of the Zirconia (D400-2.5-2-[ZrO2]).", "To understand this behaviour it is necessary to recall that CRUD deposition is a two-step process, namely the transport of particles across the laminar sublayer followed by CRUD attachment to the surface [21].", "Therefore, the lack of surface CRUD observed on the Zirlo and Zirconia specimens indicates that the surface deposition for these two specimens is limited by the attachment rate rather than the transport step since the latter is not material sensitive.", "The limited surface CRUD build-up observed on the Zirlo, and its absence on the Zirconia specimens is not necessarily in disagreement with the findings reported by Dumnernchanvanit et al. [10] who showed that in simulated primary water chemistry, and under sub-cooled boiling conditions, a ZrO2 coating could not prevent CRUD build-up.", "In fact, Dumnernchanvanit et al. observed a correlation between the CRUD build-up, the London contribution of the van der Waals forces and showed that the CRUD morphology was highly amorphous and dominated by particulate deposition.", "Conversely, in this work, which was conducted under isothermal conditions in 2 ppm of Li, the surface CRUD morphology for Alloy 690 and Ti was pseudo-crystalline in the proximity of micro-orifice edge where the formation of needles that were rich in iron was observed (Fig. 8-e and Fig. 10).", "Such crystalline deposit is consistent with a deposition process that is electrokinetically driven [7–9].", "Furthermore, considering that a porous CRUD was observed in non-hydrogenated and non-lithiated water, whilst a more compact deposit was observed in hydrogenated and lithiated water, suggests that the CRUD deposition induced by a wall current plays an important role in deposition in an alkaline environment (2 ppm Li).", "Specifically, at high pH, when the high-temperature corrosion of the oxides is limited, the majority of the iron is present in soluble form in the water rather than as particles and under these conditions, electrokinetic effects are the driving forces for magnetite deposition.", "However, on poorly conductive materials such as zirconia and zirlo, the surface CRUD deposition cannot be sustained by a faradaic reaction thus explaining the absence or limited deposition observed.", "Finally, at neutral pH, when the measured solubility of magnetite is significantly higher than in alkaline environment, colloids and particles are suspended in the water, thus leading to the particulate/amorphous build-up observed.", "The dependence of CRUD build-up on different materials, namely SS304L, Alloy 690, Titanium (Grade 2), Zirlo and magnesia Zirconia was investigated in high-temperature water using micro-orifices, and the following conclusions can be drawn:", "The radial build-up significantly decreased when the pH was increased following the injection of Li (in the form of LiOH) and this effect was ascribed to the large decrease in magnetite solubility at high pH.", "The morphology of the CRUD was also dependent on the pH whereby it was amorphous and particulate at neutral pH and increasingly crystalline at high pH.", "No effect of substrate material was found for the specimens tested under the investigated conditions on the radial CRUD oxide deposition in neutral pH, irrespective of the dissolved hydrogen because the particulate build-up was controlled by mass transport.", "In fact, it was found that hydrogen increased the iron content in the water, and this was reflected by a higher build-up rate, but it did not affect the CRUD morphology.", "A significant effect of the material substrate was found on the surface build-up of the specimens at high pH where the deposit, when formed, was crystalline.", "In this case, the deposition mechanism is likely to be driven by an electrokinetic process, which requires an electrically conductive surface.", "In fact, zirconia, which has a very low electrical conductivity, and Zirlo, which forms a poorly electrically conductive oxide, showed the lowest build-up rate, due to the suppression of the faradaic reactions required for the deposition of the oxide." ]
[ "Presence of lithium reduces solubility of magnetite and CRUD build-up rate.", "CRUD morphology is pH dependent: particles dominated at neutral pH and crystalline at alkaline pH (2 ppm of Li).", "The radial CRUD build-up is independent of substrate material at neutral pH.", "The surface CRUD deposition depends on the electrical conductivity of the substrate material at alkaline pH.", "Electrokinetic activity drives the deposition of magnetite at alkaline pH." ]
Personalised Ecology
The field of ecology has focused on understanding characteristics of natural systems in a manner as free as possible from biases of human observers. However, demand is growing for knowledge of human–nature interactions at the level of individual people. This is particularly driven by concerns around human health consequences due to changes in positive and negative interactions. This requires attention to the biased ways in which people encounter and experience other organisms. Here we define such a ‘personalised ecology’ and discuss its connections to other aspects of the field. We propose a framework of focal research topics, shaped by whether the unit of analysis is a single person, a single population, or multiple populations, and whether a human or nature perspective is foremost.
[ "AGRI" ]
[ "ecosystem services", "extinction of experience", "human–nature interactions", "nature–health interactions", "observer bias", "urbanisation" ]
[ "1105" ]
[ "Ecology has been defined as the study of the abundance and distribution of organisms and the interactions that determine these [1].", "As such, it has been important to measure what those abundances and distributions actually are, or at least to have well behaved and characterised proxies, and to limit the influence of the human observer on these estimations.", "A vast and rich literature has developed particularly around the forms of biases in the human detection of individual organisms, the factors that influence those biases (individual and species characteristics, species richness, habitat, season, weather, observer skills, etc.), and the strengths and weaknesses of approaches to their reduction (e.g., [2–5]).", "Indeed, a major theme of the history of ecology as a discipline has been progressive improvement in documenting the real abundances and distributions of organisms and their respective dynamics.", "By contrast, there has been little consideration of the converse need to understand the interactions that occur between human observers and nature.", "Nonetheless, a demand has arisen from several quarters to focus on the very effects that traditionally ecologists have sought to minimise or control in their studies.", "First, and perhaps foremost, it has become apparent that people derive a wide array of health and well-being benefits from their personal interactions with nature (reviewed in [6]).", "This is particularly so in urban areas, which are epicentres for chronic and noncommunicable physical and mental health conditions [7] and where opportunities for nature experiences may be less prevalent.", "These health and well-being benefits include components of mental, physical, and social health [6,8,9].", "Key to determining how these benefits are achieved is a better understanding of the form, frequency, and duration of people’s interactions with nature [10].", "Second, there is growing evidence of a progressive reduction in positive human–nature interactions, particularly in more westernised societies, especially during childhood [11].", "This so-called ‘extinction of experience’ (see Glossary) [12] results from a combination of local and regional losses of biodiversity, growth of sedentary pastimes, and perceived safety concerns that limit children’s independent activities.", "This may have profound consequences because the loss of human–nature interactions limits the associated health and well-being benefits.", "There is also evidence that it results in a reduction in emotional affinity toward nature and pro-environmental attitudes and behaviour [11].", "Ongoing extinction of experience could thus imply a cycle of disaffection toward nature, and ultimately constitute one of the greatest challenges to conservation policies and management actions aimed at slowing or halting the biodiversity crisis [13].", "Again, better understanding the actual nature experiences that people have and how these compare with those that are available is key to addressing these issues.", "Third, there is much discussion and debate around human–wildlife conflict, and hence negative human–nature interactions (e.g., [14,15]).", "One form of this conflict concerns direct interactions between people and wildlife.", "In the extreme, for example, attacks on humans by large predators appear to be on the rise [16], likely as a consequence of some combination of reduction in available natural undisturbed habitat, increases in ecotourism to previously remote locations, growing familiarity of these animals with people, and inappropriate behaviour of people toward them (possibly in itself evidence of the growing extinction of experience).", "Other conflicts resulting from direct interactions are doubtless rife, with consequences that range from severe (e.g., emerging infectious diseases, snake bites, vector-borne disease transmission; e.g., [17,18]) to inconvenient (e.g., noise nuisance, mess and mild aggression; e.g., [19,20]).", "Management of these interactions are often improved by better understanding how they arise, and with what regularity.", "To address this demand in a more coherent manner, we propose the need for a ‘personalised ecology’ that is distinguished by its focus on the direct interactions between individual people and nature.", "In this opinion article, we offer a definition of personalised ecology, suggest a framework of research topics on which personalised ecology should focus, and highlight the connections of personalised ecology to other aspects of ecology.", "We define personalised ecology as the investigation of the direct interactions between individual people and nature and their ecological dimensions.", "We define nature to span individual living organisms to ecosystems, but to exclude organisms that are not self-sustained (e.g., crops, house plants, zoo and domesticated animals); we acknowledge that whilst a broadly understood distinction between these two groups is achievable, a precise and uniformly agreed one is challenging.", "A human–nature interaction is then a particular instance of an individual person being present in the ‘same space’ as nature or perceiving a stimulus from nature (through sight, sound, smell, taste or touch; although in practice, sight and sound tend to predominate).", "This might be the ecosystems that they experience, the species that they encounter, or the individual organisms they see or hear.", "Such an interaction could occur intentionally or unintentionally and consciously or unconsciously.", "To a greater or lesser extent, unconscious experiences are likely to be occurring for much of the time that people are outdoors.", "A definition of this breadth allows inclusion of a wide range of types of human–nature interactions, such as visiting urban greenspaces or national parks, viewing trees through a window, listening to bird song, and being bitten by mosquitoes.", "It excludes interactions with nature through the media (e.g., through books, television, websites), albeit these interactions can have positive outcomes for humans (e.g., [21,22]).", "The focus of personalised ecology is on the ecological dimensions of human–nature interactions, recognising that other important dimensions are not ecological and more relevant to other fields (e.g., medicine, public health, environmental education).", "We will also exclude for present purposes consideration of organisms that live on or in people, whilst recognising this can be a legitimate topic of ecological enquiry.", "One can view personalised ecology from two perspectives; first, from that of the person, and second, from that of nature.", "Whilst the fundamental unit of study remains the individual person, one can consider both of these perspectives at the level of a single person, a population of people, or across multiple human populations (Figure 1).", "We will address each of these six combinations in turn.", "Arguably at its most reductionist, personalised ecology considers the nature that is experienced by a single individual person over a defined period.", "The vast majority of studies to date have simply assumed that characterisation of the environs in which people live, or of the places that they visit (e.g., public parks, protected areas), captures their experience [23].", "In the main, even this has been done crudely, typically using measures of the extent of green landcover (e.g., [24,25]), although some studies have sought to characterise the abundance or diversity of taxa in these environs or places (e.g., [26–28]).", "Undoubtedly, the actual nature interactions of people may be very different from what has typically been measured (e.g., [20,29]).", "A key research focus of personalised ecology will need to be on understanding how (e.g., passively or actively) and what type of nature people are experiencing, how these experiences are influenced by personal characteristics (e.g., gender, age, observer knowledge, skills and behavioural preferences), and by the physical/environmental conditions under which these nature interactions occur (e.g., time of day, seasonality, weather).", "Whilst some of these factors (particularly observer skills) have been investigated in an attempt to understand the impacts on biodiversity monitoring schemes, the extension of these studies to a much broader cross-section of people and factors has been limited [27,30].", "Nonetheless, it has, for example, been shown that ecological knowledge can be important in shaping people’s nature experiences (e.g., [31]).", "The continuing rapid advancement of personal monitoring devices (e.g., eye-tracking glasses, GPS trackers, electroencephalography, acoustic recorders) will enable much improved characterisation of the nature that people encounter and how this varies.", "If we know which components of nature an individual person is interacting with, then we can ask how these relate to the nature that is potentially available for such experiences.", "The occurrence and relative frequency of interactions will almost invariably be a non-random subset of those available.", "For example, abundances of bird species apparent even to a trained observer will often be far less than those actually present (Figure 2).", "The numbers of birds that untrained people see and hear as they move around the landscape is likely to be significantly lower [32].", "Such differences can arise for a diverse array of reasons, the unpicking of which may be important.", "These will include the actual distribution and abundance of species, their appearance and behaviour, their response to people (e.g., flight initiation distances, changes in calls), the timings of activities (e.g., daily and seasonal activity patterns, annual migration), and perceptions of where individuals are.", "Most obviously, people are more likely to interact with species that are common, diurnal, apparent (e.g., large, active, vocal), accustomed to people, and that can be attracted to their vicinity (e.g., through resources such as bird feeders, nest boxes).", "Within a human population, nature experiences will vary between individuals in their composition, frequency, and duration.", "Particularly in towns and cities, those having regular nature experiences, or ones of long duration, tend to be rare.", "A study in the UK found that three-quarters of direct nature interactions (instances where people were present in nature) were experienced by just one-third of an urban population [33].", "As more detailed data on the nature experiences of individual people becomes easier to collect, then so too will comparisons between people.", "Two major sets of factors have been proposed to influence the frequency and duration of human–nature interactions.", "The first is the opportunity to experience nature, which is particularly shaped by the ease of access to greenspace within the local environs [34].", "This can depend heavily on people’s socioeconomic circumstances.", "These strongly determine the kinds and location of the properties that they inhabit, and hence the availability and biodiversity of associated greenspaces [35–37], whether they can invest in green infrastructure [38] and activities to attract wildlife to those environs [39], and also whether they can engage in ecotourism elsewhere.", "The second influence on the frequency and duration of human–nature interactions is the orientation (or preferences) of people toward exploiting these opportunities.", "Although more attention has been paid to opportunity in discussions of the design of urban green infrastructure, there is evidence that orientation may be more important in shaping nature experiences [40].", "These two tend to be correlated, with people living in greener areas having increased opportunity to experience everyday nature, also having a greater orientation toward doing so [41].", "Different areas and different individual organisms will contribute very differently to the nature experiences of a given human population.", "Some areas will be visited by many people, others by few or none.", "This issue is presently best understood with regard to urban greenspaces and protected areas, where human footfall has been measured and associated with their ecological (e.g., [42]) or geographical (e.g., [40]) features.", "However, it remains challenging to disentangle the influence of the wide array of possible features that may determine whether areas are visited, how often, for how long, and with what consequences for nature experiences and for the management of sites (e.g., to encourage or direct access both to enhance nature experiences and mitigate impacts on wildlife).", "These include the sizes of areas, their accessibility, their vegetational complexity (e.g., evidence that people prefer ‘savannah-like’ natural spaces), the presence or absence of key species (e.g., large mammals), and the occurrence of wildlife spectacles.", "The numbers of people visiting an area will impact their individual nature experiences, due to an increase in numbers of observers (and hence what wildlife is located) and in the disturbance resulting from their activities.", "Equally, there will be great variation in how species and individual organisms interact with the human population.", "Some individual organisms will interact with many people, others with few or none [e.g., for many years a single black-winged stilt Himantopus himantopus, resident on a protected area in Norfolk, UK, was held to have been watched by more people than any other bird in the country; Eele, P. (2015) Gone but never forgotten.", "https://ww2.rspb.org.uk/community/placestovisit/titchwellmarsh/b/titchwellmarsh-blog/archive/2015/05/21/gone-but-never-forgotten.aspx].", "These experiences will be further influenced by interactions between species, which increase the probability that the organisms will encounter people or provide a more interesting spectacle.", "Improvements in remote sensing data and tracking technology have begun to enable evaluation of how individual organisms contribute to nature experiences [43].", "In urban areas in particular, those mobile individuals that move between a greater number of greenspaces, are likely to be seen by more people (Figure 3).", "Similarly, individuals of those stationary organisms (e.g., trees) that are readily visible, such as beside roadsides, will be experienced by more people than others of the same or similar species.", "There will inevitably be differences in nature experiences of people in different populations, such as, different villages, towns, and cities.", "What will be particularly important to understand is the macroecology of such variation; how the frequency, duration and composition of interactions change over large spatial and temporal scales.", "As with variation within single populations, opportunity and orientation will be significant, with cultural, socioeconomic, and environmental differences likely to play profound roles in shaping how people in different populations use their natural environment (e.g., [44]).", "However, little is known about these patterns, with the majority of studies limited to westernised countries (e.g., [45]), and so the findings may have limited generality.", "For example, whilst in these usually temperate zones, vegetation around the home is often seen as associated with human well-being benefits and to be encouraged (at least where it does not pose a fire risk), in many tropical areas it can harbour species dangerous to human health, and is often cleared.", "Even focusing on narrow issues, approaches to nature experiences may be very different across the world.", "This is well illustrated with regard to attitudes toward providing supplementary food for birds and mammals in urban areas.", "In some parts of Europe and North America, the practice, often to increase the likelihood of viewing them, is widespread, and indeed is the basis for a substantial industry (e.g., [45]).", "In Australia, it is much less favoured, in part because it is seen to encourage alien or unwelcome species (e.g., [46]).", "In much of the rest of the world, such feeding activities are virtually unknown [47].", "When contrasting the nature experiences of multiple human populations, it seems logical to ask to what extent it is the same or analogous components of nature (e.g., the same species or species that have similar traits and ecologies) that are contributing.", "Such studies will be akin to those in urban ecology that have attempted to characterise the similarities and dissimilarities of species assemblages found in different towns and cities, albeit in this case without explicit reference to their contribution to human–nature experiences (e.g., [48]).", "In the main, it seems likely that species, or groups of species, that occupy similar niches in different cities will provide similar kinds of nature experiences to people.", "However, there are clear cases where different species fulfil the same role, with, for example, urban bird feeding tending to focus in some regions on granivorous species, and in others on nectivorous ones [47].", "When looking across human populations one can start to map the spatial distribution of nature interactions, which will often be different from the underlying distributions of the species concerned.", "The distribution across Britain of the Magpie Pica pica, as recorded by citizen scientists is, for example, very different from that documented by formal ornithological mapping schemes (Figure 4).", "Unsurprisingly, the former highlights encounters along major transport routes and in major centres of population, as these are the places in which the vast majority of nature experiences actually occur; while the latter reveals many areas in which the species occur, but interactions are more limited.", "Obviously, personalised ecology is not divorced from a number of other topics of focal interest in ecology.", "In addition to those already observed above to motivate the need for such an agenda, these include the following which are addressed in turn.", "While biodiversity monitoring has been focused principally on understanding the relationship between the actual abundances and distributions of species, and what expert observers detect, personalised ecology is less concerned with these actual quantities and more with the abundances and distributions experienced by people, and with a focus on ‘ordinary’ people (i.e., non-experts, and often with a limited knowledge of ecology), and experiences during everyday activities.", "The growing use of citizen science in biodiversity monitoring makes the concerns of personalised ecology increasingly relevant.", "Whilst the topic of ecosystem services is explicitly concerned with the benefits that people gain from ecosystems [51], rather than emphasising personal nature interactions in the main this is approached in a generic sense of community or societal benefits (e.g., from agricultural production, pollination, carbon sequestration, waste decomposition).", "The two approaches are obviously complementary, with the ecosystem benefits to individual people often becoming very apparent in terms of cultural ecosystem services (e.g., recreational, sense of place, aesthetic, educational and therapeutic values).", "The bulk of urban ecology research remains focused on a traditional understanding of the determinants of the abundance and distribution of species, and the interactions that determine these, albeit in urban areas [52].", "Nonetheless, there have been repeated calls for, and important contributions toward, broader approaches (e.g., [53,54]), and particularly those that address the complex interplay between people and urban ecosystems.", "Personalised ecology would clearly contribute to such an agenda.", "The field of human ecology studies the relationship between humans and their environment, and typically has a strong emphasis on the anthropological, social, or political dimensions to this interaction [55].", "Personalised ecology would again serve to add an important dimension to such investigation, by strengthening the links to more conventional ecological concerns.", "A well-developed understanding of personalised ecology would have major practical consequences in two primary arenas.", "First, it would improve the ability to design policy and management for people’s access to nature in such a way that their benefits (i.e., the positive interactions), were enhanced, and their costs (i.e., the negative interactions), were reduced.", "Second, and more importantly in the face of a global biodiversity crisis, well developed understanding of personalised ecology would improve the ability to determine policy and management of people’s interactions with nature in such a way that the benefits to nature were also increased, and the costs minimised.", "Of course, these two arenas interact, and what is presently lacking is a strong evidence-based approach for encouraging the positive engagement of people with nature, whilst promoting the conservation of populations and ecosystems.", "The global human population is continuing to grow rapidly and become more urbanised, with people less likely to experience regular positive interactions with nature.", "At the same time, the importance of those interactions to human well-being is becoming increasingly apparent.", "It thus seems vitally important that ecologists develop a much more comprehensive and detailed understanding of those interactions, their composition, and temporal and spatial dynamics (see Outstanding Questions).", "Such a ‘personalised ecology’ constitutes a challenging agenda, and one that has thus far lagged far behind others in the field of ecology.", "How can the suite of direct interactions between individual people and nature, and their strengths, most effectively be determined?", "What are the differences between the direct interactions between people and nature that could occur, that do occur, and that the people concerned perceive to occur?", "Which components of nature interactions provide the different kinds of benefits and costs to people, and how do these vary with cultural, socioeconomic, and environmental circumstances?", "What role does personal nature experiences play in shaping people’s attitudes and behaviour toward biodiversity conservation?" ]
[ "Traditionally, ecologists have focused on understanding the ecological world unbiased by how human observers interact with it.", "However, both the positive and negative nature interactions that people experience are the result of these biases.", "The nature interactions people experience will be heavily dependent on the ecology of species, as well as their own opportunities and behaviour.", "Scientists and policymakers need to determine and account for this ‘personalised ecology’, to better understand and balance the benefits that people gain from the natural world, whilst limiting negative impacts upon it." ]
A critical review of CO2 photoconversion: Catalysts and reactors
Photocatalytic conversion of CO2 to either a renewable fuel or valuable chemicals, using solar energy has attracted more and more attention, due to the great potential to provide an alternative clean fuel and solve the problems related to the global warming. This review covers the current progress of photocatalytic conversion of CO2 by photocatalysis over the metal oxides. A brief overview of the fundamental aspects for artificial photosynthesis has been given and the development of novel photocatalysts for CO2 photoreduction has been discussed. Several key factors for high-efficiency CO2 photoreduction and the recent development of photocatalytic reactor design for this artificial photosynthesis have also been highlighted. © 2013 Elsevier B.V.
[ "CENG", "CHEM" ]
[ "Carbon dioxide", "Photoreduction", "Reactor", "Semiconductor photocatalysts" ]
[ "1503", "1600" ]
[ "Global warming is considered to be one of the major environmental concerns that humankind is facing [1].", "Carbon dioxide (CO2) contributes largely to the global climate change because it is one of the main greenhouse gases that are present in the atmosphere.", "CO2 takes part in raising the global temperature through absorption of infrared light and re-emitting it.", "International Panel on Climate Change (IPCC) predicted that atmospheric CO2 level could reach up to 590 ppm by 2100 and the global mean temperature would rise by 1.9 °C [2].", "The impact of greenhouse effect will be global and serious in many different aspects, such as ice melting at the Earth's pole, fast rising sea level and increasing precipitation across the globe [3].", "Energy generation by fossil fuel combustion dominates CO2 emission and fossil fuel will be inevitably depleting.", "Therefore it is urgent for the scientists to find a renewable energy resource to mitigate the effect of global warming as well as meet the increasing energy demand [4].", "In principle, there are at least three routes of reducing the amount of CO2 in the atmosphere, including direct reduction of CO2 emission, CO2 capture and storage (CCS), and CO2 utilization [5–7].", "Despite the increasing utilization efficiency of fossil fuels, to dramatically lower the CO2 emission seems difficult due to the increasing population and demand for high quality of life.", "The capacity of CCS technology is also limited due to the environmental risk of leakage and the energy requirement for gas compression and transportation.", "During the past decade, growing concerns have driven research activities toward the artificial conversion of CO2 into fuels or valuable chemicals, through thermochemical, biological, electrochemical or photocatalytic methods [8–10].", "In the long term, artificial photosynthesis, photocatalytic conversion of CO2 using solar energy is the most attractive route for the transformation of CO2 [11,12].", "The interest in such field has been aroused dramatically after several examples which demonstrated photoelectrocatalytic reduction of CO2 to organic compounds in 1970s [13].", "Especially, the pace has recently increased enormously, because of the promoting effect of advanced technologies (e.g. nanotechnology and in-site advanced characterization) on the development of novel photocatalysts.", "This review covers the approaches and opportunities of the artificial photosynthesis driven by solar energy using CO2 as the raw material, including both material design and reactor engineering.", "The basic processes for the photocatalytic synthesis will be presented first.", "A significant proportion of the review focuses on the rational design of metal oxide photocatalysts with enhanced photocatalytic activity and the critical factors for high-efficient photoconversion of CO2.", "The recent development of photocatalytic reactors for the artificial photosynthesis is also highlighted.", "Photocatalytic CO2 conversion makes use of semiconductors to promote reactions in the presence of light irradiation which is known as artificial photosynthesis.", "This review covers the artificial photosynthesis by metal oxide photocatalysts.", "The basic process can be summarized into three steps: (1) generation of charge carriers (electron–hole pairs) upon absorption of photons with suitable energy from light irradiation, (2) charge carrier separation and transportation, (3) chemical reactions between surface species and charge carriers [14,15].", "Photocatalytic CO2 conversion is a complicated combination of photophysical and photochemical processes.", "The redox reaction is initiated by photoexcitation when the energy of photons equal to or greater than the band gap of a semiconductor is received by a photocatalyst.", "Then the electrons are excited from the valence band (VB) to the conduction band (CB).", "VB is the highest energy band occupied by electrons and CB is the lowest in which there is no electron at the ground state [16].", "As shown in Fig. 1, the electrons and holes undergo intra-band transitions.", "They can travel to the surface, combine at the trap sites (recombination process) through radiative or non-radiative pathways.", "Alternatively these electrons can travel to the surface of semiconductor and react with surface adsorbed species (CO2 in this case), if recombination happens slower than the reactions during transitions [17].", "However, not all the electrons reaching the surface can reduce CO2 which is a thermodynamically inert and very stable compound.", "Compared with most of the reduction methods mentioned above which require high-energy input, either at high-temperature and/or under high pressure [18], photosynthesis does not require extra energy except solar irradiation.", "Photogenerated electrons at higher reduction potential level can offer driving force (also called over-potential) for the expected chemical reactions.", "The reduction potential measures the capability of a chemical specie to gain electrons.", "Species with a lower (more positive) reduction potential will gain electrons (i.e. be reduced) and those with a higher (more negative) reduction potential will lose electrons (i.e. be oxidized) [19].", "In order to reduce CO2 into carbon monoxide or hydrocarbons, electrons in the semiconductor are required to have more negative chemical potential, while for water oxidation, holes need to lie on more positive potential level.", "Eqs. (1)–(8) illustrate the pathways for the generation of solar fuels and the related potentials at pH = 7 [20].", "From a thermodynamic point of view, formation of methane and methanol are more favorable in CO2 reduction, since these reactions take place at lower potentials.", "However, the kinetic drawback makes methane and methanol formation more difficult than carbon monoxide, formaldehyde and formic acid because more electrons are required for the former reactions [21].", "Moreover, the 2–8 electrons and protons reactions to obtain the desired products are extremely difficult.", "Due to the complicated nature of the inorganic photocatalyst surface, the interaction between photocatalyst and absorbed species may undergo a series of one-electron processes instead of a multi-electron, multi-proton process.", "Thus the actual redox potential required is determined by the reaction pathway.", "For example, if CO2 reduction is initiated by single electron reduction of CO2 to CO2−, the potential is around −1.9 V vs NHE.", "With such concern, being able to discharge multiple electrons with protons at a time is important to improve reaction efficiency.", "Thus, generating sufficient electron–hole pairs, separating charges efficiently and providing active catalytic sides are the paramount factors for CO2 photoreduction.", "Although photoreduction of CO2 shows great potential [22,23], at present one of the greatest drawbacks is the low conversion efficiency.", "Herein, some of the key factors which limit the efficiency are listed: (1) mismatching between the absorption ability of semiconductor and the solar spectrum; (2) poor charge carrier separation efficiency; (3) low solubility of CO2 molecule in water (approximately 33 μmol in 1 ml of water at 100 kPa and room temperature); (4) back reactions during reduction of CO2; and (5) competition reaction of water reduction to hydrogen [24].", "UV radiation only contributes less than 4% to the whole solar spectrum and 43% of the solar energy lies in the visible light region.", "To find a photocatalyst which can absorb visible light meanwhile has high enough CB position is one of the main goals of the research.", "An overpotential is necessary as a driving force for charge carrier transport and reactions, thus practical requirement for CO2 conversion is usually greater than the theoretical energy required to produce the desired products [25].", "Although directly matching the band gap of a semiconductor to the solar spectrum is challenging, several strategies have been used to improve the absorption ability of an inorganic photocatalyst.", "Doping with elements has been pursued to sensitize photocatalyst with a wide band gap, toward visible light absorption.", "As a broad and active topic, doping of photocatalysts with metal ions (Fe3+, Zn2+, W6+, etc.) and non-metal ions (C, N, S, B, etc.) have already been widely studied in several review papers [26–30].", "Therefore, a brief and general introduction is mentioned in this review.", "Doping does not only retard the fast charge recombination, but also introduce defect states (interband states or mid-gap levels) [31].", "For example, it was reported that the narrowed band gap of a semiconductor after doping with non-metal ions (e.g. N or C) is ascribed to the mixing of p states of the dopants with O 2p states to form a new valance band [32].", "However, the function of doping on CO2 conversion is still arguable.", "Formation of semiconductor heterostructures is another effective way to enhance the light absorption and charge separation.", "Due to the band alignment, the band bending induces a built-in field, which drives the photogenerated electrons and holes to move in the opposite direction [33].", "Semiconductor quantum dots (QDs) are also considered as an ideal choice for the coupled component in the heterostructures.", "In the presence of QDs, the visible light response of the photocatalysts is easily adjusted.", "In addition, QDs can also utilize hot electrons to generate multiple charge carriers when excited by a single high energetic photon, leading to an increased amount of the charge carriers [34].", "Similarly, organic dyes are often used as sensitizers to enhance the visible light absorption of a semiconductor.", "Under irradiation, dyes can inject photoexcited electrons into the conduction band of the semiconductor.", "However, the electron transfer efficiency between the dye sensitizer and the semiconductor depends on many factors, such as the LUMO level of the dye and the conduction band edge of semiconductor [35].", "When CO2 is reduced by the photogenerated electrons, utilization of an equal number of photogenerated holes should also be considered.", "Otherwise, the accumulation of the holes in a photocatalyst will increase the probability of charge recombination and shorten the lifetime of electrons.", "In addition, it is believed that holes could play a negative role in the photocatalytic reaction if they cannot be used efficiently, such as photocorrosion of the photocatalysts.", "The use of artificial electron donor to scavenge the holes is the most popular solution [36].", "However, the process and energy used to synthesize the artificial electron donor needs to be taken into account since it may cause more CO2 emission.", "Water is considered to be the ideal electron donor.", "Nevertheless, the large water oxidation potential is the main drawback, only very few photocatalysts that can reduce CO2 and oxidize water simultaneously have been reported [37,38].", "Competition from water reduction process by photogenerated electrons is also problematic when water is used as an electron donor.", "In comparison to most of the CO2 reduction routes, reducing water is a relative easy process in term of kinetics and thermodynamics.", "In thermodynamics aspect, the reduction potential of water to hydrogen is 0.0 V (pH=0) which is more positive than CO2 reduction to CO, formic acid and formaldehyde.", "In kinetics aspect, water reduction is a 2-electrons process, it is more facile than most of the CO2 reduction which required 4–8 electrons.", "While CO2 reduction is also limited by its low solubility in water, the water reduction does not suffer from the similar problem, thus the chance for electrons to meet and react with water is much higher than with CO2.", "Although very little investigation has been conducted to address this problem, it is generally agreed that the reaction selectivity can be controlled by modifying photocatalysts’ morphology, changing the exposed facets and introducing new reaction sides.", "It is believed that particular atom arrangement on the surface can be more favorable to absorb CO2 molecule than water molecule on the surface.", "Two different morphologies of Cu2O have been found to have dramatic difference in products’ selectivity [39].", "Co-catalyst loading has also claimed to be able to vary the reduction products’ selectivity, Ag and Cu are commonly used as co-catalysts for CO2 reduction whereas the loading of Pt or Au is more favorable for hydrogen production.", "Inoue et al. demonstrated CO2 photoreduction over various semiconductors under 500 W Xe or Hg lamp in the presence of electrical bias in 1979 [40].", "The one or more converted products such as formaldehyde, formic acid, methanol and methane were detected on different materials.", "Since then, there have been some reports on CO2 photoconversion.", "Compared to the material development for the analog process, photocatalytic water splitting, fewer materials have been developed for photoreduction of CO2.", "Many photocatalysts that are acknowledged as good candidates for water splitting cannot reduce CO2 due to the kinetic difficulty.", "Although some sulfides and nitrides have been reported to be active for CO2 conversion, the metal oxides are more preferable [41,42], due to their potential advantages over sulfides and nitrides, e.g. relatively safe to handle, fairly low cost and considerably stable, thus particular attention is paid to photoreduction of CO2 using metal oxide photocatalysts below.", "TiO2 is the most investigated photocatalyst for artificial photosynthesis.", "Pure titanium oxide has three common mineral phases, including anatase, brookite and rutile.", "Photocatalytic conversion of CO2 over single-phase or mix-phase TiO2 has been widely investigated.", "The phase structure and surface properties of TiO2 have a significant influence on the efficiency of photoreduction.", "Liu et al. compared the photoactivity for CO2 photoreduction to CO and CH4 over TiO2 in three different phases [43].", "It was found the brookite had the highest CO and CH4 yield.", "Although rutile showed smallest band gap which had the strongest visible light absorption, it was the least active photocatalyst.", "The application of mix-phase TiO2 was more attractive.", "It has demonstrated an enhanced visible light harvesting ability and the anatase-dominated mixed phase was benefited to CO2 photoreduction [44].", "The enhancement was also attributed to the junction effect between rutile and anatase resulting an efficient charge separation.", "Morphology of TiO2 has significant influences on the photoactivity of CO2 reduction.", "TiO2 nanorods and nanotubes have attracted much attention due to their large surface area, reduced grain boundaries and facile charge transport paths of 1-D nanomaterials [45].", "For example, mixed-valence state Pt dispersed TiO2 nanotube (TNT) was used for CO2 photoreduction [46].", "The Pt nanoparticles were uniformly distributed on TiO2 nanotube (TNT) through deposition of Pt complex.", "Due to the synergetic effect of tubular morphology and the mixed valence Pt nanoparticles, CO2 adsorption ability of Pt/TNT was greatly improved.", "The in situ FT-IR study also showed that Pt/TNT was extremely active toward the hydrogenation of CO2 to methane at 100 °C.", "Zhang et al. further optimized the Pt loading amount, reaction temperature and the ratio of H2O/CO2 in the reaction.", "The optimal condition for CO2 reduction was determined to be: 0.15 wt% Pt-loaded TNT with H2O/CO2 molar ratio of 0.9:1 at 343 K [47].", "The influence of annealing temperature on the specific photocatalytic reactions was also studied by other groups.", "Vijayan et al. reported that TNT calcined at 400 °C had the best photocatalytic performance for converting CO2 to methane [48].", "On the other hand, Schulte et al. showed that low-temperature calcination (550 °C) resulted in the lowest production (0.26 μmol m−2 h−1) whereas samples fabricated at 680 °C had the highest production rate of 0.79 μmol m−2 h−1 owing to the increased visible light harvesting [49].", "The results are not aligned which may be due to different reaction conditions in different labs and raises a serious question how to reasonably compare the activity of a photocatalyst reported by different groups.", "Varghese et al. carried out an outdoor experiment over nitrogen-doped TNT arrays under actual sunlight.", "52% of nanotube surface was covered by Cu and the rest was coated by Pt.", "A series of hydrocarbons (methane, olefin branded paraffin and other alkane) and CO were detected.", "Their formation rates were found superior to the TNT without co-catalyst or TNT with single co-catalyst.", "The maximum production rate was reported to be 111 ppm cm−2 h−1 [50].", "Most recently, Zhang et al. reported four-fold increase of CO2 conversion rate by filling Cu–Pt binary co-catalyst inside the nanotube cavity, the SEM images of which are shown in Fig. 2.", "When Cu0.33–Pt0.67/TNT was used to photoreduce diluted CO2 (1% in N2), hydrocarbons (CH4, C2H4, and C2H6) production rate of 6.1 mmol m−2 h−1 was achieved under AM 1.5 illumination [51].", "Copper decorated TiO2 nanorod films have also been used for CO2 photoreduction.", "With addition of Cu nanoparticles, the CH4 production rate (2.91 ppm g−1 h−1) was about two time higher than that of bare titania films, this might be due to the enhanced electrons and holes’ separation or surface plasmonic effect induced by the metal nanoparticles [52].", "The unique pore structure and large surface area of mesoporous materials have let their application toward CO2 photoreduction rather attractive.", "Noble metals (Pt, Au, and Ag) loaded nitrogen doped mesoporous TiO2 have been used for converting CO2 into methane under visible light irradiation.", "Their photocatalytic activities followed the descended order of Pt/TiO2 > Au/TiO2 > Ag/TiO2.", "The reason for this was ascribed to the higher working function of Pt, which facilitated the transfer of photogenerated electrons from TiO2 to noble metal particles [53].", "The optimum loading amount of Pt was found to be 0.2 wt%, with methane yield of 2.9 μmol m−2 h−1.", "Dispersion of photocatalysts is noted to be another important factor for efficient photoreduction of CO2.", "Photocatalysts immobilized in the zeolite or silicate frameworks are usually highly dispersed, which offers unique pore structure and ion exchange capacity for reactions [54,55].", "It was reported that titania anchored on zeolite had high selectivity for methanol formation.", "Selectivity of reaction products could be controlled by the addition of the co-catalysts, such as Pt is beneficial for formation of methane rather than methanol [56].", "Ti-containing porous SiO2 films could convert CO2 into CH4 and CH3OH with the quantum yield of 0.28%, which was superior to dense phase TiO2 particles.", "The enhanced efficiency was ascribed to the ligand-to-metal charge transfer, which was caused by the excitation of isolated Ti centers by UV light [57].", "Furthermore, Ulagappam et al. used Ti silicate molecular sieves to convert CO2 into formic acid, acetic acid and CO and the possible reaction paths were investigated by FT-IR measurements [55].", "The photoreduction of CO2 over TiMCM-41 molecular sieves was carried out under laser light.", "The double electron transfer of CO2 generated carbon monoxide, which was directly proportional to the power of the laser [58].", "Since the field of artificial photosynthesis is advancing fast, the family of non-titanium photocatalysts for CO2 reduction is in parallel developing and updating in a rapid speed.", "Several types of metal oxide and mixed metal oxide semiconductors have been reported, including ZrO2, Ga2O3, Ta2O5, SrTiO3, CaFe2O4, NaNbO3, ZnGa2O4, Zn2GeO4 and BaLa4Ti4O15, etc. [59–61].", "As most of them have a large band gap, which can provide great overpotential for the reaction, these metal oxides usually are UV active.", "ZrO2 is a popular photocatalyst owing to its high CB position, its photogenerated electrons have large driving force for CO2 reduction.", "Sayama et al. investigated 1% Cu loaded ZrO2 to reduce CO2 using NaHCO3 aqueous solution as CO2 source.", "The selectivity for CO was about 10% and H2 was the main reduction product indicating that electrons were tended to reduce protons in water [62].", "Kohno et al. and Lo et al. both investigated photoconversion of CO2 over ZrO2 in the presence of H2 under UV illumination.", "CO was found to be the sole product of the reaction, with the yield of 0.70 μmol g−1 h−1 and 0.51 μmol g−1 h−1 respectively [63–65].", "Kohno et al. further investigated the reaction mechanism.", "As reported, ZrO2 reduces CO2 molecules into CO2− anion radicals, which subsequently reacts with H2 to form formate (HCOO−) on the surface.", "The reaction between formate and another CO2 molecule led to the formation of CO [66].", "Similar results were also achieved when CH4 was used instead of H2, except the formation of extra carbonaceous residue (CH3COO−) in the reaction [67].", "When H2 was used as the reductant, the CO production rate was about 0.17 μmol h−1, followed by 3.3 μmol of CO additionally released upon heating.", "In contrast, the CO yield was about 0.14 μmol h−1 in the presence of CH4 reductant and further 1.5 μmol CO, 0.2 μmol H2 and 0.2 μmol CH4 were collected upon heating [68].", "ALa4Ti4O15 (A = Ca, Sr, and Ba), with a layered perovskite structure and band gap of 3.79–3.85 eV, have been used for CO2 photoreduction.", "Iizuka et al. found that ALa4Ti4O15 can reduce water without additional reductant.", "However, hydrogen was the preferential reduction product [69].", "BaLa4Ti4O15 was determined to be the most active photocatalyst and its selectivity was shifted toward CO2 reduction after introducing Ag metals on the surface.", "The effect of co-catalyst loading methods on the photoactivity has also been investigated.", "The photoactivity has been found in descending order: Ag production using liquid-phase reduction by NaPH2O2 > impregnation and subsequent H2 reduction > impregnation > in situ photodeposition.", "The advanced performance was ascribed to the small particle size of Ag metals and the uniform distribution of co-catalysts on the surface.", "The optimal loading amount was determined to be 2.0 wt% Ag-loaded BaLa4Ti4O15, with maximum H2, O2, CO and HCOOH yields of 10 μmol h−1, 16 μmol h−1, 22 μmol h−1 and 0.7 μmol h−1 [69].", "For most of the other photocatalysts, O2 usually cannot be detected while in the absence of artificial electron donor, which raises concerns about the role of holes in the photocatalytic reaction.", "Xie et al. employed self-doped SrTiO3−δ for CO2 photoreduction under visible light.", "SrTiO3−δ was prepared through a carbon-free combustion method followed by the heat treatment in argon, which could create sufficient oxygen deficiencies and high spin Ti3+ in the material.", "Because of the enhanced CO2 adsorption and the reduced band gap, methane production rate over 0.3 wt% Pt-loaded SrTiO3−δ reached 0.25 μmol m−2 h−1.", "The photogenerated holes were thought to be consumed by the oxidization of photocatalyst from Ti3+ to Ti4+.", "The assumption was partially backed up by the factor that photoactivity of SrTiO3−δ dramatically decreased after 10 h.", "In order to regenerate Ti3+, the photocatalyst needed to be recovered by heating at 1200 °C in argon [70].", "Although oxidation of photocatalysts was seldom mentioned in the other reports, which raises a serious issue about the photocatalyst stability, decrease of photocatalyst performance was frequently observed with prolonged reaction time.", "Several non-titanium metal oxides nanomaterials with special morphologies have also been developed for photoreduction of CO2.", "NaNbO3 nanowire was fabricated through the hydrothermal synthesis followed by the heat treatment.", "The CH4 evolution rate of nanowires reached 653 ppm g−1 h−1, much higher than that of bulk NaNbO3 particle fabricated through solid-state reaction (22 ppm g−1 h−1) [71].", "Yan et al. studied the photoreduction of CO2 into CH4 over ZnGa2O4.", "The CH4 production rate of mesoporous ZnGa2O4 (5.3 ppm h−1) was superior to ZnGa2O4 obtained by solid-state reaction (trace amount).", "The CH4 yield was further increased to 50.4 ppm h after loading 1 wt% RuO2 on mesoporous ZnGa2O4 [72].", "Liu et al. compared the photoactivities of Zn2GeO4 nanoribbon and bulk Zn2GeO4 prepared through solid-state synthesis.", "In the presence of 1 wt% RuO2 and 1 wt% Pt, the optimal CH4 yield of Zn2GeO4 nanoribbon was 25 μmol g−1 h−1, which is much superior to bulk Zn2GeO4 (only trace amount of CH4 detected) [73].", "Although the overall water photooxidation and photoreduction by pristine Zn2GeO4 and RuO2-Pt-loaded Zn2GeO4 have been observed during photocatalytic water splitting, the yields of H2 and O2 were not determined in the report [74].", "The photoactivity of highly porous Ga2O3 for CO2 reduction was four times higher than that of commercial bulk Ga2O3, without the addition of co-catalyst and sacrificial reagent [75].", "It was reported that the direct conversion from CO2 to CH4 was realized in the absence of CO intermediates, while holes were consumed by water to produce H+ and OH•.", "The enhanced performance of porous Ga2O3 was attributed to the two-fold increase of surface area and the tripled CO2 adsorption ability.", "Tanaka et al. carried out the photoreduction of CO2 over bulk Ga2O3 in the absence of H2O, using H2 as reductant.", "CO was formed instead of CH4, with the yield of 10.96 μmol after 2 days.", "It was noticed that about 7.3% of surface adsorbed CO2 was converted [76].", "Subsequently, photoreduction of CO2 over ATaO3 (A = Li, Na and K) was investigated under similar conditions.", "LiTaO3 showed the highest activity for CO production (0.42 μmol g−1 in 24 h), while KTaO3 presented the lowest yield [77].", "KTaO3 have the smallest band gap among those three tantalate compounds and it have a suitable VB that can oxidize water [78].", "It can potentially reduce CO2 using water as hole scavenger.", "To enhance its photoactivity, KTaO3 nanoplate were synthesized in solvothermal reaction [79].", "CO, H2 and O2 were detected without electron donor or electric bias in our reaction.", "A seven times increase in CO yield over KTaO3 nanoplate compared to the conventional KTaO3 was achieved.", "The increase was ascribed to the short electron diffusion distance to the surface in the thin plate-like particle.", "Matsumoto et al. reported the conversion of CO2 into methanol and formaldehyde over p-type CaFe2O4 in the solution of sodium hydroxide [80].", "Developing visible-light-driven semiconductors for artificial photosynthesis is one topic of great interest with practical importance.", "Besides the self-doped SrTiO3−δ, unfortunately, only a few candidates with both visible light response and acceptable photoactivity have been reported even in the presence of electron donor, such as Cu2O, BiVO4, LaCoO3 InTaO4 and N-doped Ta2O5.", "Since Punchihewa discovered Cu2O for photocatalytic reduction of CO2 in 1989, Cu2O has been an attractive visible-light-driven photocatalyst for artificial photosynthesis [81].", "However, stability of Cu2O is still the biggest drawback to its application in photosynthesis, as Cu2O can be easily oxidized or reduced by photogenerated charge carriers.", "Punchihewa et al. found that formation of formaldehyde and methanol reached maximum concentrations in 30 min and 45 min respectively.", "After that, the product concentrations declined, because the back reaction by photogenerated holes became faster than the reduction of CO2.", "More recently, photocatalytic reduction of CO2 into methanol over Cu2O/SiC was rendered by Li et al. [82].", "The photocatalytic performance of SiC nanoparticles was improved by the modification with Cu2O.", "As a result, the yield of methanol increased from 153 to 191 μmol g−1.", "Although a linear increase of methanol yields over Cu2O/SiC was shown, the chemical state of Cu2O after the reaction was not mentioned.", "Thus, the problem of stability could not be excluded, as the enhancement could be ascribed to Cu metal or thin layer of CuO on SiC.", "For the practical application of Cu2O in the field of solar energy conversion, new strategies to form heterostructured protection layer are highly desirable.", "For example, with the protection of Al-doped ZnO and TiO2 nanolayers, the stability of Cu2O electrodes were significantly improved for water reduction even if it was not tested for CO2 conversion [83].", "It was also reported that the existence of robust carbon layer could improve the stability of Cu2O [84].", "In our previous research, it was found that improved CO conversion yield could be achieved by coupling Cu2O with RuO2, forming a heterojunction which retarded charge recombination [39].", "Zou et al. first reported the application of Ni-doped InTaO4 for overall water splitting in the absence of scavenger under visible light irradiation.", "With the aid of RuO2 or NiOx as a co-catalyst, H2 and O2 production in the stoichiometric ratio was achieved.", "The quantum yield at 402 nm was reported to be 0.66% [85].", "Later, Pan et al. used NiOx/InTaO4 to convert CO2 into methanol.", "The role of NiOx was ascribed to attract electrons and to provide active reaction sites.", "With a proper deposition method (reduction followed by oxidation), the maximum methanol yield was 1.394 μmol g−1 h−1 [86].", "It should be noted that methanol was the only product in the reaction.", "Other carbohydrates, CO, hydrogen or oxygen were not mentioned.", "Wu et al. prepared InTaO3 nanoparticles with the size of 17.7 nm by sol–gel method.", "Compared to InTaO3 fabricated by solid-state synthesis, 37.5% increase was achieved during the conversion of CO2 into methanol [87].", "The conversion efficiency of CO2 into ethanol over monoclinic BiVO4 was found to be 17 times higher than tetragonal BiVO4.", "The enhancement was ascribed to the binding of CO32− to the Bi3+ sites by BiO bonds [88].", "Alcohols are widely used as hole scavengers, which can efficiently extract the photoinduced holes, subsequently oxidize themselves into CO2, CO, etc., therefore there is an argument about the detected products which may be produced by the surface organic contaminants absorbed during photocatalyst synthesis.", "Jia et al. studied the photoreduction of CO2 over C and Fe co-doped LaCoO3 under 125 W Xe lamp with 400 nm long pass filter.", "For C-LaCo0.95Fe0.05O3, formaldehyde and formic acid were collected with the maximum yield of 19 μmol g−1 h−1 and 128 μmol g−1 h−1 [89].", "Doping is an efficient way to enhance the visible light response of photocatalysts.", "Visible light driven CO2 conversion into formic acid was realized over a wide bandgap material (Ta2O5) when modified by nitrogen doping.", "In the presence of ruthenium complex, hybrid photocatalysts showed the quantum yield of 1.9% under visible light (405 nm) [90].", "Suzuki et al. synthesized nitrogen doped mesoporous Ta2O5 spheres, which exhibited enhanced visible light response than N-doped Ta2O5 fine particles [91].", "In the past few years, graphene-based semiconductor photocatalysts have attracted a wide interest in the field of solar fuel production.", "It has been reported that coupling metal oxide with graphene can not only enhances the visible light absorption, but also improves the efficiency of charge separation, which are crucial factors for visible-light-driven photocatalytic reduction of CO2.", "The conversion rate of CO2 into methanol over NiOx loaded Ta2O5/1.0 wt% graphene composites reached 0.5 μmol g−1 h−1, which was 3.4 times higher than the corresponding photocatalyst without graphene.", "The enhanced conversion efficiency was attributed to the superior conductivity of graphene and the facilitated charge transfer from Ta2O5 to active site of co-catalysts [92].", "In situ simultaneous reduction-hydrolysis technique was developed to fabricate TiO2/graphene hybrid nanosheets.", "The synergistic effect of the surface-Ti3+ abundant TiO2 and graphene favors the generation of C2H6, and the yield of the C2H6 increases with the content of incorporated graphene [93].", "Hollow spheres consisting of titania nanosheets and graphene nanosheets were fabricated by a layer-by-layer assembly method.", "As shown in Fig. 3, the sufficiently compact stacking of ultrathin Ti0.91O2 with graphene nanosheets resulted in the nine times improvement [94].", "However, scientific research is highly desirable to understand the underlying mechanism of graphene-based photocatalysts for CO2 reduction, as several aspects showed significant influence on the efficiency of composites.", "For example, Liang et al. found that nanocomposites based on the less defective solvent-exfoliated graphene exhibited a significantly larger enhancement during the photoreduction of CO2 to CH4, compared to solvent-reduced graphene oxide [95].", "The fabrication of high quality graphene-based composites is still challenging and more efficient strategies would need to be developed to address these issues.", "It is difficult to compare the activities of different photocatalysts reported by different groups by only considering the conversion rate or quantum efficiency due to the differences among preparation methods and in particular test conditions.", "However, it is generally agreed that several aspects can be considered to improve the photocatalytic performance of semiconductors.", "The particle size of photocatalyst has great effect on the efficiency of CO2 photoreduction.", "Decreasing particle size can often results in the larger surface area of the photocatalysts, which serves more active reaction sites for CO2 adsorption.", "The photocatalysts with smaller particle size also benefit from the shorter transfer pathway for charge carriers to reach its surface.", "However, the smaller the particle size is, the more the particles’ boundary, which may influence the communication between these particles and thus leads to a lower activity.", "Ye et al. prepared NaNbO3 by hydrothermal method and polymerized complex method separately.", "NaNbO3 nanoparticles fabricated by the polymerized complex method exhibited much larger surface area than the hydrothermal products, which were 38 m2 g−1 and 1.7 m2 g−1 respectively.", "As a result, treble and sextuple enhancements of O2 and H2 production rate were achieved [96].", "Several kinds of nanomaterials with typical morphologies show great impact on the efficiency of CO2 reduction, such as one-dimensional nanorods and nanotubes with unique charge transfer paths, mesoporous nanostructures with considerable large surface area.", "Besides particle size, controlling the facets of photocatalysts has been proved to be an efficient way to improve the activity of photocatalysts.", "Our preliminary results showed that the photocatalytic reduction preference shifts from H2 (water splitting) to CO (CO2 reduction) by controlling the exposed facet of Cu2O.", "The low index facets {100} exhibit higher activity for CO2 photoreduction than high index facets {111} [39].", "Loading co-catalysts on the surface of photocatalysts is another commonly used procedure to achieve substantial enhancement on both conversion efficiency and products’ selectivity.", "It is generally known that the photoreduction reaction can be improved by loading noble metal nanoparticles.", "On the other hand, metal oxide nanoparticles are found to favor the oxidation reaction.", "The choice of co-catalyst is crucial.", "Some noble metals are particular active toward water reduction while some are more suitable for CO2 reduction.", "This is due to the co-catalysts providing active catalytic sites for reduction of different absorbed species.", "The co-catalysts also extract the photogenerated electrons or holes to prolong the lifetime of the charge carriers.", "Although metal oxide does not provide reduction catalytic reaction side, loading it on photocatalyst may still increase the reduction products’ yield, since it will extract hole and extend the lifetime of electrons for reduction reaction (e.g., CO2 reduction over Cu–RuOx).", "In addition, electron accumulation on co-catalyst allows the discharge of more than one electron at a time, facilitating the multi-electron CO2 reduction processes.", "The choice of the co-catalysts varies from different photocatalysts for reaching their best photoactivity.", "To date, the co-catalyst application strategy for CO2 reduction is mainly focused on CO2 reduction co-catalysts.", "However, there are other strategies used in photocatalytic or photoelectrocatalytic (PEC) water splitting system, such as (1) oxidation co-catalysts for hole removal from photocatalyst and (2) binary co-catalysts system for both reduction and oxidation reactions.", "Noble metals Pt, Cu and Ag are the three most commonly used co-catalyst for CO2 photoreduction.", "Loading Pt co-catalyst generally benefits the formation of CH4 whereas CO is more preferable in the presence of Cu or Ag.", "Yamakata et al. reported that the recombination of electrons and holes was greatly retarded when Pt was loaded on photocatalysts [97].", "Pt loaded TiO2 and Zn2GeO4 have been used for photocatalytic CO2 reduction, CH4 was found to be the only reduction product [73,98].", "However.", "Pt is also a well-known co-catalyst for water reduction.", "In one of our experiments, Pt loaded KTaO3 was used to photoreduce CO2 under UV irradiation.", "It was found the hydrogen yield was dramatically increased compared to pristine KTaO3, while only trace amount of CH4 was produced [79].", "Thus, how to controlling the competition reaction of water reduction is important, as Pt may facilitate the activation of H2O.", "To overcome the problem, Zhai et al. used a Pt/Cu2O binary co-catalyst with a core/shell structure for CO2 reduction over TiO2.", "H2, CO, CH4 and O2 were formed and the presence of Cu2O shell attributed to the higher yield of CO.", "Pt co-catalyst decreased the selectivity of CO2 reduction, whereas the selectivity increased from 60 to 80% by loading Cu2O shell.", "It was proposed that the Cu2O shell provided the preferential reaction sites for CO2 conversion while the Pt core was served as electron sink to “collect” the photogenerated electrons from TiO2.", "Therefore, the deposition of a Cu2O shell on Pt markedly suppresses the reduction of H2O to H2, a competitive reaction with the reduction of CO2 [99].", "Copper and silver have been widely used as co-catalysts for CO2 reduction.", "Owing to the effect of Cu as electron trapper to prohibit the recombination of electron–hole pairs, the loading of Cu to BaLa4Ti4O15, ZrO2, SiC and TiO2 could efficiently enhance their photoactivities [100–103].", "Peterson et al. investigated the mechanism of Cu as co-catalyst for CO2 reduction.", "They found the weak bonding of CO to copper surface prevented the formation of adsorbed CO to CHO, which was a crucial intermediate for the hydrocarbon [104].", "Ag-loaded BaLa4Ti4O15, SrTiO3 and TiO2 have been utilized for CO2 photoreduction [105,106].", "It is worth to point out that the addition of Ag, Cu and NiOx to BaLa4Ti4O15 as a co-catalyst resulted in the photoreduction of CO2, whereas only H2 and O2 were detected for pristine BaLa4Ti4O15.", "Enhancements in the production of H2 and O2 were also observed when Ru and Au were loaded, but CO could not be detected.", "These results indicate that both Cu and Ag can function as reaction sites for CO2 reduction and electron trapper.", "Although noble metal co-catalysts are well-known for improving photocatalytic activity, development of low-cost co-catalyst seems extremely important.", "To date, several types of non-noble-metal co-catalysts, such as NiO, Co–Pi, MoS2 and WS2, have been engaged in photocatalytic reactions [107–111].", "Kudo et al. first reported the overall water splitting using Ni/NiOx-loaded La doped NaTaO3 in 2000.", "Later, NiO was employed as a co-catalyst on InTaO3, Sr2Nb2O7 and Sr2Ta2O7, etc.", "Co–Pi was first reported for boosting water oxidation in 2008, which could significantly lower the overpotential required for water oxidation.", "Recently, Durrant et al. studied the dynamics of photogenerated charge carriers in Co–Pi loaded Fe2O3 by transient absorption spectroscopy.", "The improvement was ascribed to the increased life time of photogenerated holes by at least 3 orders of magnitude [112].", "Recently, Wang et al. reported the addition of MgO on TiO2 to enhance the chemisorption of CO2 onto the catalyst surface greatly accelerated the photocatalytic reduction of CO2 to CH4 [113].", "Molybdenum and tungsten sulphides have also been reported for water reduction over CdS, which may serve as potential non-noble metal co-catalysts for CO2 reduction [114,115].", "Enhancement of overall water splitting was also found over NaTaO3 coupled with [Mo3S4]4+ [116].", "Nevertheless, the stability and safety issues of metal sulfide compounds may limit their practical applications.", "In conclusion, several factors are important for a good photocatalyst, e.g. small particle size, high surface area and right exposing facets besides long lived charge carriers.", "Furthermore, even though the photocatalyst is suitable for CO2 reduction, the photogenerated electron and hole may recombine if there are no suitable active/reaction sites available on the surface of the light harvesting semiconductor.", "Therefore, it is essential to introduce active catalytic sites for CO2 reduction on the surface of semiconductor by loading co-catalysts.", "Photoreactor development is an engineering approach to enhance efficiency of CO2 conversion.", "It involves advancing the light harvesting technique, reducing loss of photon, improving products separation and influencing charges carrier recombination as well as the reactive surface area of a photocatalyst.", "The reactor system can be grouped into two categories: fluidised bed reactor and fixed bed reactor.", "Fig. 4 shows a fluidised bed batch reactor system, it is the most commonly used system.", "Fluidised bed reactor is operated as a batch process in two-phase heterogeneous system.", "The photocatalysts are suspended in a fluid-like state providing highly dispersed photocatalysts with agitation by magnetic stirrer to prevent catalyst sedimentation.", "Reaction starts with filling CO2 into the sealed reactor (some may firstly remove air by vacuum to improve the purity), then light irradiates from the top of the reactor.", "The sample is taken in a fix time interval by gastight syringe or on-line automatic sampling system.", "It should be noted the types of lamp, reactor size and amount of water and photocatalyst used may vary in different systems.", "The light harvesting efficiency can be increased by changing the lamp position.", "As shown in Fig. 5, in an inner irradiation cell, the lamp is placed inside the reactor.", "Hence the light loss by reflection is minimized.", "Wu et al. also developed fiber reactor to reduce the loss of light.", "CO2 photoreduction was performed where photocatalyst was coated on optical fiber (the fixed bed) and the fluid was guided to form a plug-flow-state [105].", "As shown in Fig. 6, the fiber served as a medium to deliver light effectively and uniformly to the surface of a photocatalyst.", "When light travels inside the fiber, part of the light is reflected and transmitted along the fiber and the rest of the light penetrates and excites the TiO2 layer at the interface.", "NiOx-loaded InTaO4 photocatalyst has been used to compare fiber reactor and conventional reactor for CO2 photoreduction.", "The methanol yield in optical fiber reactor was 14 times higher than the yield in the conventional batch reactor [60].", "In another report, CO2 photoreduction was conducted over Pd and Rh-loaded TiO2 in an optical fiber reactor.", "Quantum efficiency (0.049%) achieved in the optical fiber reactor is higher than that in a conventional batch reactor (0.002%) [118].", "Lo and co-workers reported a circulation photocatalytic reaction system using a fixed bed for CO2 conversion, which is shown in Fig. 7.", "The circulation reactor has advantages over the conventional batch reactor in providing a high uniformity of the gas concentrations distribution inside the reactor and shortening the reaction time.", "In addition, the immobilized photocatalyst offers high specific reactive surface area.", "Ichikawa and Doi reduced CO2 using a Nafion film as a proton separator coated by TiO2 and ZnO/Cu on different sides in the presence of an external bias [119].", "As shown in Fig. 8, the system consists of a thin film TiO2 and an electrocatalyst which were coatedon a Nafion film.", "The reactor separated the production of oxidation and reduction products to avoid further reactions between products as well as reduce energy required in subsequent purification.", "Furthermore, the use of proton membrane reduced the chance of the back reaction between proton and O2.", "Recently, Wu et al. adapted a similar reactor design for CO2 photoreduction using WO3 as an oxidation catalyst and CuAlGaO4 or Rh doped SrTiO3 as a reduction photocatalyst [120].", "CO2 photoreduction over ruthenium dye-sensitized TiO2-based catalysts under concentrated sunlight has been rendered by Nguyen et al. [121].", "A solar concentrator was used to provide the concentrated sunlight.", "It consisted of a series of reflecting surface which gathered large area light into a focus zone (Fig. 9).", "As sunlight is the preferable energy provider for this technology, the solar concentrator is beneficial for boosting the technology further.", "In summary, the fluidised batch photoreactor is still the most commonly used photoreactor.", "Both the top and inner illuminated photoreactor are equally popular.", "The actual design in different research group may be varied slightly, for example, some equip an inferred filter to remove the heat, others use water bath to maintain the reactor temperature.", "Although advancing reactor design has demonstrated an alternative way to optimize the photoconversion efficiency, this field has not been extensively investigated.", "Only limited literatures on reactor design have been published so far.", "It seems an efficient and appropriate reactor's design is still at its fancy stage.", "On the other hand, a fluidised batch photoreactor can provide a simple environment to scan different photocatalysts in practice.", "Tackling global warming and fuel crisis is one of the key challenges in this century.", "Amongst all the techniques to be used for carbon dioxide reduction, photocatalytic CO2 conversion has its unique advantages that it solely utilises the most abundant solar energy which should not increase CO2 emission.", "This technology not only provides an alternative way to produce the sustainable fuels, but also convert the waste CO2 into valuable chemicals, which is important for keeping our environment clean and development sustainable.", "There are limitations in the process of CO2 photoreduction and various strategies have been developed to overcome them.", "However, more efforts are required to improve the efficiency of the photoreduction reaction.", "The development of the novel heterostructured photocatalysts with considerable activity, high reaction selectivity for CO2 reduction and stability is extremely urgent.", "The crucial role of co-catalysts should be further investigated and the mechanism of the photochemical process is little understood, which limits the reaction efficiency and requires large efforts.", "In parallel, the efficient photoreactor and reaction system engineering are important but with limited success up to now.", "In total the technology is rather challenging, thus huge amount of effort should be put into searching a low-cost and robust artificial photocatalyst, understanding the underlying chemical processes and designing an efficient reaction system." ]
[ "This review covers the current progress of photocatalytic conversion of CO2 by inorganic photocatalysis.", "A brief overview of some fundamental aspects for artificial photosynthesis has been given.", "Several key factors for high-efficiency CO2 photoreduction are discussed.", "The recent developments of photocatalytic reactors for artificial photosynthesis are highlighted." ]
Experimentally measuring an isolated branch of Nonlinear normal modes
Nonlinear normal modes (NNMs) are a key tool for investigating the behaviour of nonlinear dynamic systems. Previous work has shown that branches of NNMs can be isolated from other NNM responses, in a similar manner to isolated, or detached, resonance curves in the forced responses. Their isolated nature poses a significant challenge for the prediction and measurement of these NNM branches. This paper illustrates how isolated NNMs may exist in two-degree-of-freedom systems, with cubic nonlinearities, that exhibit a 1:3 resonance. This is first introduced using a general two-mass oscillator, before considering a two-mode reduced-order model of a continuous cross-beam structure that exhibits a coupling between its primary bending and torsional modes. In both cases, a combination of analytical and numerical techniques is used to show how the isolated NNM branch may evolve from a set of bifurcating NNM branches. A nonlinear force appropriation technique is used to experimentally measure the NNMs of the cross-beam structure. By comparing these measurements to the numerical studies, it is shown that some of these NNMs are on the isolated branch, representing the first experimental confirmation of isolated NNM branches.
[ "ENGI", "PHYS" ]
[ "Experimental methods", "Force appropriation", "Isolas", "Modal interactions", "Nonlinear normal modes", "Phase-locking" ]
[ "2210", "2211", "3102", "3104" ]
[ "When considering the responses of harmonically-forced nonlinear systems, it is well-known that multiple solutions can be observed at specific frequencies [1].", "For certain systems, regions of forced responses are disconnected from other solutions [2–4].", "These isolated regions, known as isolated resonance curves, detached resonance curves or isolas, have been observed experimentally in structures such as spring-mass systems [5,6] and beams [7].", "As they are disconnected from the primary solution branches, these isolas prove challenging to compute, but often represent critical responses [8].", "Nonlinear Normal Modes (NNMs) are used to interpret the responses of nonlinear systems and represent a nonlinear analogue of linear normal modes.", "NNMs were first defined by Rosenberg [9] and later expanded by Vakakis [10], Kerschen [11] and Haller [12].", "These later works define an NNM as any periodic oscillation of the underlying conservative equations of motion – the definition that is used throughout the current paper.", "Numerous approaches for computing such NNMs have been proposed [13].", "It has been shown that NNMs can be related to the forced responses of these systems using an energy-based method [14].", "Of specific interest to the current work, the NNMs can be used to predict the onset of isolated resonance curves – as demonstrated in Refs. [3,15].", "This approach requires that the NNM branches (i.e. the loci of NNM responses) of the system are known before the energy-based method is used to predict where the isolas cross the NNM branches.", "Along with isolated forced responses, nonlinear systems may exhibit isolated NNM branches – i.e. branches of periodic solutions of the underlying conservative system that are not connected to other branches, or to any zero-amplitude solutions.", "Note that this represents a modification to the definitions that consider NNMs to be extensions of the underlying linear responses, such as that by Shaw and Pierre [16,17]; however, as shown in this work, these NNM branches can correspond to significant features in the forced responses.", "Isolated NNM branches have received less attention than isolated forced responses and have yet to be observed experimentally.", "It is the existence of these isolated NNM branches, measured experimentally, that is the main subject of the current paper.", "It has been theoretically shown that isolated NNM branches may exist in a mass-spring oscillator [15], and that these may evolve from non-isolated branches as the symmetry of the system is broken.", "In Ref. [15], the NNMs are computed analytically, and hence the isolated NNM branches are computed in the same manner as the primary branches.", "However, for more complex systems where analytical techniques are impractical, computing isolated NNM branches poses a significant challenge.", "This challenge is discussed in the current work, and it is shown that the isolated NNM branches may correspond to significant features in the forced responses.", "Following on from Ref. [15], Section 2 of the current paper begins by showing that isolated NNM branches may exist in a simple two-mode system, representative of an asymmetric two-mass oscillator.", "This system exhibits a 1:3 resonance between the two modes (i.e. the second mode responds at three times the frequency of the first) and hence differs from the oscillator considered in Ref. [15] which exhibits a 1:1 resonance.", "It is then shown how this isolated NNM branch evolves from a set of bifurcating branches, as a nonlinear parameter of the system is varied, but the asymmetry is retained.", "These results, using a system that has been studied by numerous authors, provide a basis for interpreting the results presented in the remainder of the paper.", "Section 3 introduces an experimental cross-beam structure, consisting of a clamped-clamped beam with an additional cross-beam that introduces a torsional component of vibration.", "The torsional mode is tuned such that this system also exhibits a 1:3 modal interaction.", "A similar structure has been considered in detail in Ref. [18]; however, the structure was tuned to exhibit a 1:1 resonance, and no isolated NNM branch was seen.", "In Section 3, a nonlinear reduced-order model is formulated using the implicit condensation method1 [20,21], and it is shown that this model is equivalent to the simple two-mode system considered in Section 2.", "This model exhibits an isolated NNM branch, which is shown to evolve from a bifurcated set of branches as the symmetry of the structure is varied.", "Compared to the experimental realisation, this numerical model provides greater flexibility for investigating the dynamic behaviour of the structure, offering further context for the experimental results.", "Finally, in Section 4, the NNMs of the structure are found experimentally using nonlinear force appropriation [22].", "It is shown that there is a very good qualitative agreement between the experimentally-measured NNM branches, and those of the numerical reduced-order model.", "Not only are NNMs on an isolated branch measured experimentally, but it is shown that, for certain forcing amplitudes, responses on the isolated branch are inevitable and hence represent significant features of the response.", "In this section, isolated NNM branches are investigated using a two-mode system with cubic stiffness nonlinearities – arguably the simplest system that may exhibit isolated NNM branches.", "The system considered here is representative of an asymmetric two-mass oscillator, and in Section 3 it is shown that it may be extended to a continuous structure.", "The natural frequencies and nonlinear parameters considered in this section are given in Table 1.", "These are representative of an asymmetric two-mass oscillator with cubic nonlinear springs, as described in Appendix B, where the physical parameters of the system are also provided.", "Fig. 1 shows the NNM branches of the two-mode system, computed using the harmonic balance method.", "In Panels (a–c), the dark-blue lines represent the NNMs where the modes are in-phase, i.e. where p = + 1, and the light-blue lines represent the antiphase NNMs, i.e. where p = −1.", "These are shown in terms of the initial displacements of the modes, q1(0) and q2(0), where the initial displacement of q1 is defined as its maximum positive value.", "As such, when the two modes are in-phase the initial displacement of the second mode, q2(0), is positive, whilst an antiphase response gives a negative q2(0) value.", "Although Panel (b) appears to show the in-phase and antiphase NNM branches crossing, the 3-D plot in Panel (a) reveals that this is due to the projection and, in fact, the antiphase NNM branch is isolated.", "This illustrates that, for the parameters considered here, an isolated NNM branch does exist in a simple two-mode system, with cubic stiffness terms, and a 1:3 resonance between the underlying linear modes.", "Panels (d–g) show four different NNM responses in the projection of time against the modal amplitudes, q1(t) and q2(t).", "These illustrate how the phase between the two modes changes between the in-phase (dark blue) branch, shown in (d) and (e), and the antiphase (light blue) branch, shown in (f) and (g).", "Note that additional NNM branches may exist for this system.", "For example, the primary branch emerging from the second linear natural frequency, which can be found using the harmonic balance method by setting the frequency ratio of the assumed solutions, Eq. (3), to 1:1.", "Furthermore, the NNMs are not necessarily limited to cases where the modes are in-phase or in antiphase, and additional phase relationships may exist [23]; however, the behaviour of interest here is captured by these phase relationships, hence the current study is limited to these cases.", "In Ref. [15] it is shown that a two-mass oscillator may exhibit an isolated NNM branch with a 1:1 resonance between the modes.", "This 1:1 branch is shown to evolve from a set of NNM branches, of a symmetric system, that exhibit a bifurcation.", "By breaking this symmetry, an imperfect bifurcation is formed, i.e. the bifurcation connecting the two branches splits to form two separate, disconnected, branches, one of which is an isolated NNM branch [24].", "It is now shown that the 1:3 isolated NNM branch, presented in Fig. 1, can also evolve from a set of bifurcating NNM branches, as this bifurcation becomes imperfect.", "Here, however, it is shown that this is not due to a symmetry-breaking of the system, and response may also exhibit a bifurcation when the system is asymmetric.", "The nonlinear parameter γ2 may be set to zero by varying the physical parameters of the two-mass oscillator represented by this model, as detailed in Appendix C.", "Here, the nonlinear parameter of the spring connecting the masses, α2, is varied such that γ2 = 0.", "This leads to the modal parameters given in Table 2, where α2 = α2,I denotes the value of α2 used previously, and α2 = α2,B denotes the value that gives γ2 = 0.", "Note that α2,I is positive, hence corresponding to a spring with a hardening nonlinearity, whilst α2,B is negative, hence corresponding to a spring with a softening nonlinearity.", "The case where Δ = 90% is shown in Fig. 2(c).", "Again, this shows little movement in the lower portion of the primary branch (green circle), or the upper portion of the isolated branch (red square); however, the upper portion of the primary branch (orange triangle) has now moved above the isolated branch (red square).", "A detailed region shows that the antiphase (light-blue) NNM is still isolated.", "Finally, the case where Δ = 100%, representing α2 = α2,B, is shown in Fig. 2(d).", "In this Panel, the black line represents NNMs that only contain the first mode, and the purple line represents NNMs containing both modes.", "The purple dot represents the bifurcation between these two branches, hence connecting the mixed-mode (purple), to the single-mode (black) NNM branch.", "As such, the mixed-mode branch is not isolated.", "Comparing Panel (d) to Panels (a–c), it can be seen that the upper portions of the primary and isolated branches (orange triangle and red square, respectively) have merged to form the multi-mode (purple) branch, whilst the single-mode (black) branch is composed of the lower portion of the isolated branch (purple triangle).", "This shows that the 1:3 isolated NNM branches can evolve from a set of bifurcating branches and represents an imperfect bifurcation.", "The physical system considered here is asymmetric for all configurations represented in Fig. 2, and hence this evolution is not driven by a breaking of the symmetry of the physical system.", "In Fig. 2(d), the multi-mode branch is shown in purple (rather than light- or dark-blue, as previously) as the phase between the modes cannot be determined.", "This is due to the lack of phase-dependent terms in Eq. (6); i.e. Eq. (6) do not contain the phase-difference parameter p (defined in Eq. (5)).", "As such, the multi-mode branch is phase-unlocked [25].", "As described in Ref. [26], phase-unlocked NNMs do not strongly attract forced responses; hence, it is not anticipated that a forced response following the single-mode branch would bifurcate and follow the multi-mode branch [25].", "If a small γ2 were introduced (such as in Fig. 2(c)) the phase of the backbone curves can once again be specified.", "However, a sufficiently small γ2 will not significantly alter the structure of the system, and the phase-dependent terms in Eq. (6) will remain small, hence it can be assumed that the phase-locking mechanism will be weak.", "This suggests that a forced response following the lower portion of the primary NNM branch (green circles in Fig. 2) will not be strongly attracted to the upper portion of the primary branch (orange triangle), or the upper portion of the isolated branch (red square), as both of these are closely-related to a phase-unlocked solution.", "It therefore follows that we expect a forced response to be drawn to the lower portion of the isolated branch (purple triangle), despite its isolated nature.", "This concept is revisited in Section 4, where experimental results are discussed.", "Fig. 2 illustrates how isolated NNM branches may evolve from a set of bifurcating branches in a simple, two-mode system that is representative of a two-mass oscillator.", "In this section it is observed that a similar two-mode model, which exhibits isolated NNM branches, may be used to describe a continuous structure.", "It is shown that these isolated branches evolve from a set of bifurcating branches as the symmetry of the structure is changed.", "The cross-beam structure, considered for the remainder of this paper, is illustrated in Fig. 3, where Panel (a) shows a schematic of the structure and Panel (b) shows an experimental realisation.", "This section is dedicated to numerical analysis of this structure, whilst experimental results are considered in Section 4.", "The structure consists of a 960 mm long rectangular beam, clamped at both ends, with two cross-beams.", "The circular cross-beam, shown in red in Fig. 3(a), is of length 220 mm and has two adjustable masses, shown in blue, that may be moved along the length of the cross-beam.", "The rectangular cross-beam, shown in green in Fig. 3(a), is used as a means of applying a torsional force in an experiment.", "Adjusting the movable masses allows the natural frequencies of the structure to be tuned and allows its symmetry to be adjusted.", "When the structure is symmetric, its first two modes consist of a flexural bending of the main beam, and a torsional motion.", "If the masses are moved to break the symmetry, these modes change such that a small torsional component is introduced to the bending-dominated mode, and a small bending component is introduced to the torsion-dominated mode.", "Further details about this structure, and how it may be tuned, can be found in Ref. [18].", "Note that the structure described in Ref. [18] is a modified version of that shown in Fig. 3, where the natural frequencies of the bending and torsion modes are approximately equal; as such the structure exhibits a 1:1 resonance.", "However, here, the masses are tuned such that the natural frequency of the torsion-dominated mode is approximately three times that of the bending-dominated mode.", "As such, this structure exhibits a 1:3 resonance (as seen in the two-mass oscillator considered in Section 2).", "Details of the experimental procedure are given later in Section 4.", "When the cross-beam structure undergoes large deflections, the axial stretching along the length of the main beam leads to a nonlinear stiffening effect.", "To capture the effect of this nonlinearity on the bending- and torsion-dominated modes, a two-mode nonlinear reduced-order model (ROM) is derived using the implicit condensation and expansion method (ICE), as described in Refs. [20,21].", "The ICE method captures the nonlinear coupling between these two modes and any membrane-type modes, described in a finite-element model of the structure.", "Here, this finite-element model has been constructed using ABAQUSⓇ and consists of a total of 288 B31 beam elements (with 6 DOFs at each node [27]).", "Additionally, linear axial springs are added to mimic the boundary conditions, as statically measured in Ref. [18].", "Note that the finite-element model has not been updated to match the experimental results; however, the model is sufficiently close that the same qualitative behaviour is seen – as discussed later in Section 4.", "The ICE method generates a ROM described by equations of motion that are identical in form2 to the two-mode equations of motion considered in Section 2 – Eq. (1).", "Two different configurations of the structure are considered in this paper: one in which the masses are arranged symmetrically, and one in which they are asymmetric.", "The resulting values of the parameters of the nonlinear ROM are given in Table 3.", "Note that the asymmetric case has been tuned such that the linear natural frequencies, ω1 and ω2, are approximately equal to the symmetric case (and retaining an approximate 1:3 ratio).", "Due to the approximate nature of the ICE method, the symmetric case cannot be precisely represented by the ROM.", "However, near symmetry the nonlinear parameters γ2 and γ4 are close to zero, relative to γ1, γ3 and γ5; as such, they have artificially been set to zero in Table 3 to reflect the truly symmetric case.", "Note that the parameters of the symmetric configuration are comparable to those of the two-mode system for the case where α2 = α2,B, shown in Table 2; however, for the cross-beam structure, γ4 is also equal to zero.", "The NNM branches of the nonlinear ROMs are computed using the numerical continuation algorithm NNMcont [28].", "Unlike the harmonic balance method, used in Section 2, this numerical approach does not require an assumed form of the response, and thus is expected to give greater accuracy.", "However, the disadvantage of numerical continuation is that it requires an initial solution to compute an NNM branch.", "For a primary NNM branch, this solution may simply be the low-amplitude linear solution [28]; however, the isolated NNM branches require an NNM that is outside of the linear regime for an initial solution.", "Here, the harmonic balance method is used to compute approximate NNMs on the isolated branch.", "One of these approximate solutions is then used as an initial solution for continuation.", "This combination of both techniques ensures that all relevant NNM branches are computed to a high degree of accuracy [15].", "The NNM branches of the symmetric ROM are shown in Fig. 4.", "Panels (a–c) show the normalised response frequency against the initial accelerations of the two modes.3", "The black line, shown in Panels (a–c) represents the NNMs consisting of a response in just the first linear mode.", "This is analogous to the black line shown for the bifurcating case in the two-mode system, Fig. 2(c).", "The dark- and light-blue lines represent NNMs containing both modes, which is analogous to the purple line4 shown in Fig. 2(c).", "A blue dot in Fig. 2(a–c) denotes the pitchfork-like bifurcation that connects these two branches to the primary branch [29] – as such, these branches are not isolated.", "Fig. 5 clearly shows that the antiphase (light-blue) NNM branch is isolated, showing that isolated NNM branches may exist in a conceptually-simple, continuous structure.", "Comparing Figs. 4 and 5 it can be seen that the isolated NNM branch arises from the breaking of the pitchfork-like bifurcation, to form an imperfect bifurcation.", "This is analogous to the evolution of the isolated NNM branch of the two-mode system, shown in Fig. 2.", "As discussed previously, a known solution on the isolated branch has been used to initiate the continuation, which has been computed using the harmonic balance method.", "If a continuation scheme was initiated on the primary (in-phase) branch, without a priori knowledge of the existence of the isolated branch, it is unlikely its presence would be detected.", "Additionally, the isolated NNM branch is only connected to the primary branch when the system is purely symmetric.", "This is a special case, which could not be found precisely using the parameters from the ICE method, due to its approximate nature, further complicating any attempt to find the isolated branch by continuation.", "This section considers an experimental realisation of the cross-beam structure introduced in Section 3.1.", "A brief overview of the experimental procedure is provided before results are shown.", "For further details of the experimental procedure see Ref. [18].", "The experimental realisation of the cross-beam structure was shown previously in Fig. 3(b).", "It can be seen that a shaker is attached to the rectangular cross-beam – shown in green in the schematic in Fig. 3(a).", "As the shaker is offset from centreline of the main beam, this provides a small torsional component of excitation.", "Six accelerometers are attached to the structure to measure its dynamic response and the excitation force is measured using a force transducer.", "The adjustable masses, shown in blue in Fig. 3(a), are tuned such that the ratio between the linear natural frequencies is slightly higher than 1:3 (measured as 1:3.09, with the bending-dominated mode at the lower frequency).", "Note that if the ratio is less than 1:3, the isolated NNM branch will not be seen.5", "The masses are also positioned slightly asymmetrically, i.e. they are at slightly different distances from the centre line of the beam.", "As a result, the beam is qualitatively similar to the asymmetric structure considered previously in Section 3.", "Nonlinear normal modes represent the periodic responses of an unforced and undamped structure.", "Although the damping in the structure under consideration is light, its presence prevents an NNM motion from being reached precisely [30].", "For the structure to exhibit a response that is close to that of an NNM, the forcing must negate the damping – a method known as force appropriation [22,31].", "This is achieved by tuning the excitation force until it is in quadrature with the response (either displacement or acceleration).", "Here, force appropriation is performed using a single shaker and multiple excitation frequencies, where the excitation amplitudes are tuned manually with respect to the phase of the input force and two accelerometers (near the root and on the cross beam) [22].", "Once this is achieved for one excitation frequency, and the response is recorded, the excitation frequency is adjusted, and the process is repeated.", "Fig. 6 shows the results of the experimental measurements of the NNM branches using force appropriation.", "This is achieved by tuning the first and third harmonics of the force such that they are in quadrature with the corresponding harmonics of the displacement, to within 5° [22].", "Panels (a) and (b) show the branches in the projection of the normalised response frequency against the amplitude of acceleration.", "In this case, the amplitude of acceleration represents the Fourier coefficient with the largest amplitude, and the response frequency corresponds to the frequency of this coefficient.", "For the first mode, the response frequency is normalised to the first linear natural frequency, ωn1, and for the second mode the response frequency is normalised to three times the first linear natural frequency, 3ωn1.", "In keeping with previous figures, the acceleration amplitudes in Fig. 6(b) have been adjusted to illustrate the phase between the modal accelerations – i.e. the light-blue, antiphase, NNM branch has a negative acceleration amplitude.", "The phase between the modes is also shown in Fig. 6(c–f), which illustrate that the modes are in antiphase for the responses represented by the purple triangle and red dot.", "Initially, at low amplitudes, the experimentally-measured NNMs follow the lower portion of the primary branch, with one point on this branch marked with a green dot in Fig. 6.", "This is analogous to the response highlighted with a green dot in Fig. 5.", "Note that, at these low amplitudes, the NNM branch appears to soften before exhibiting a hardening behaviour.", "A similar phenomenon has been observed by Londoño et al. [32], and reported to be due to the effects of dry friction.", "At a higher frequency, the response transitions from the lower to the upper portion of the primary branch (denoted by an orange triangle).", "Again, this corresponds to the orange triangle in Fig. 5.", "As the frequency is increased, the sign of the forcing of the third harmonic is reversed, and the response then jumps from the upper portion of the primary branch to the lower portion of the isolated branch (denoted by a purple triangle).", "Note that, as with previous figures, this branch does not appear to be isolated when viewed in the projection of the first modal coordinate; however, the second modal coordinate clearly shows that this branch is isolated and responding with a different phase.", "At higher frequencies, the responses then follow the upper portion of the isolated branch (red square).", "The transition from the primary to the isolated response branch does not require any specific change to the excitation applied to the system, but rather it is a natural behaviour.", "Whilst it may seem counter-intuitive that a response should jump from one NNM branch to another, it is to be expected if the phase-locking conditions of the NNM branches are considered.", "As discussed previously, in Section 2.3, the phase-locking in the upper portion of the primary branch, and the lower portion of the isolated branch, is anticipated to be weak.", "As such, the forced response is not expected to follow these portions of the branch,6 as observed in the experiment.", "This illustrates the importance of this isolated NNM branch, as it represents behaviour that is highly-likely to be observed when the system is forced.", "There is a clear qualitative similarity between the NNM branches of the experimental system, in Fig. 6, and those of the asymmetric ROM, shown in Fig. 5.", "As discussed in Section 3.1, the finite-element model has not been updated to quantitatively match the experimental results.", "As such, to compare the numerical and experimental NNM branches, the experimentally-measured NNMs are scaled and superimposed in Fig. 7.", "This figure further highlights the similarity between these branches and confirms that the antiphase NNMs belong to an isolated branch.", "Nonlinear normal modes are an important tool for understanding the dynamics of nonlinear systems, and for finding isolated forced response branches.", "However, in this paper, it has been shown that NNM branches may, themselves, be isolated.", "This has been illustrated using both a simple two-mode system, representative of an asymmetric physical system, and a nonlinear reduced-order model of a continuous cross-beam structure.", "The isolated NNM branches have also been observed experimentally in the cross-beam structure.", "In both systems, it has been shown that the isolated NNM branches may evolve from bifurcated branches by varying the physical parameters of the system.", "In the cross-beam structure, this evolution is driven by a breaking of the symmetry of the physical system; whilst the simple, two-mode system shows that a similar evolution may also occur in an asymmetric system.", "As NNM branches are often computed using numerical continuation methods, isolated NNM branches pose a significant challenge as, without a priori knowledge of their existence, these branches may not be computed.", "Here, this problem is resolved by first computing the NNM branches using an analytical technique, which is unaffected by the isolated nature of the branches.", "The isolated NNM branches may correspond to significant responses when the system is forced; for example, in the cross-beam structure, the isolated branch was found to attract forced responses more strongly than the high-amplitude NNMs of the primary branch.", "This can be understood by considering the strength of the phase-locking in the NNMs of these branches." ]
[]
Modeling healthy male white matter and myelin development: 3 through 60months of age
An emerging hypothesis in developmental and behavioral disorders is that they arise from disorganized brain messaging or reduced connectivity. Given the importance of myelin to efficient brain communication, characterization of myelin development in infancy and childhood may provide salient information related to early connectivity deficits. In this work, we investigate regional and whole brain growth trajectories of the myelin water fraction, a quantitative magnetic resonance imaging measure sensitive and specific to myelin content, in data acquired from 122 healthy male children from 3 to 60. months of age. We examine common growth functions to find the most representative model of myelin maturation and subsequently use the best of these models to develop a continuous population-averaged, four-dimensional model of normative myelination. Through comparisons with an independent sample of 63 male children across the same age span, we show that the developed model is representative of this population. This work contributes to understanding the trajectory of myelination in healthy infants and toddlers, furthering our knowledge of early brain development, and provides a model that may be useful for identifying developmental abnormalities. © 2013 The Authors.
[ "NEUR" ]
[ "Brain development", "Infant imaging", "Myelin maturation", "Myelin water fraction", "White matter development" ]
[ "2805", "2808" ]
[ "The elaboration of the myelin sheath around neuronal axons, and the associated white matter maturation, is a cornerstone of human neurodevelopment.", "Myelinated white matter forms efficient communication pathways that shape the integrated neural systems responsible for higher order functioning (Fornari et al., 2007; Johnson and Munakata, 2005).", "Given myelin's critical role in brain communication, processes that disrupt its development may result in reduced brain connectivity and inefficient interneuronal communication.", "In turn, this may lead to altered neuronal functioning, and may contribute to some neurodevelopmental and psychiatric disorders, including autism and attention deficit and hyperactivity disorder (Courchesne, 2004; Haroutunian and Davis, 2007; Konrad and Eickhoff, 2010).", "Myelination during the first five years of life is a rapid and dynamic process.", "Prior histological studies have established that myelination begins in the cerebellum and brainstem in utero (Barkovich et al., 1988; Flechsig, 1901; Paus et al., 2001; Yakovlev and Lecours, 1967).", "Following birth, myelination proceeds caudocranially from the splenium of the corpus callosum, optic radiations and internal capsule by 3–4 months; occipital and parietal lobes by 5–6 months; temporal and frontal lobes by 9–11 months (Flechsig, 1901; Yakovlev and Lecours, 1967); and continues into the second decade of life (Barnea-Goraly et al., 2005; Bartzokis et al., 2010).", "However, while retrospective histological studies provide the most faithful characterization of myelin development, they suffer significant limitations.", "They are i) inherently cross-sectional; ii) difficult to combine into a single temporal timeline, owing to differences in staining techniques and inconsistent brain coverage; iii) difficult to obtain from large specimen pools spanning the infant age-range; iv) preclude investigation of underlying structure–function relationships; and v) labor intensive.", "Further, they may not necessarily reflect healthy development as these studies are conducted post-mortem.", "Recent in vivo magnetic resonance imaging (MRI) techniques, including conventional T1- and T2-weighted structural imaging, diffusion tensor (DT)-MRI, and magnetization transfer imaging (MTI), have become popular for investigating early brain development (Giedd and Rapoport, 2010; Giedd et al., 1999; Knickmeyer et al., 2008) and, especially, white matter maturation (Geng et al., 2012; Lebel et al., 2012; van Buchem et al., 2001).", "These non-invasive techniques provide detailed anatomical tissue contrast and micro-structural insight that affords a more sensitive and direct means of examining white matter development.", "However, these methods also have their disadvantages.", "While conventional MRI (T1- and T2-weighting) have shown alterations in the gray/white matter contrast (Huang et al., 2006; Paus et al., 2001) temporally mirroring myelination, these qualitative observations are influenced by a variety of micro-structural and biochemical elements (MacKay et al., 2009).", "DT-MRI offers a quantitative approach, with metrics including fractional anisotropy (FA), mean diffusivity (MD), and axonal and radial diffusivity (AD and RD, respectively).", "Changes in these metrics during development have often been associated with myelination, however these measures are also associated with changes in the local tissue architecture (Jones et al., 2013; Mädler et al., 2008).", "Many of these measures are also derived directly from the tensor model of diffusion that does not apply to all brain voxels, making the interpretation difficult (Wheeler-Kingshott and Cercignani, 2009).", "Similarly, while the magnetization transfer ratio (MTR) has been shown to correlate strongly with myelin content (Moll et al., 2011; van Buchem et al., 2001; Zaaraoui et al., 2008), the MTR is also influenced by other processes including edema and/or inflammation (Gareau et al., 2000; Vavasour et al., 2011).", "Multi-component analysis of relaxation time data, also termed multi-component relaxometry (MCR), may provide a more sensitive measure of myelin content.", "MCR decomposes the measured MRI signal into contributions from distinct micro-structural water compartments.", "Prior MCR studies have consistently reported at least two water compartments: a fast-relaxing water pool attributed to water trapped between the myelin-lipid bilayers; and a slower-relaxing water pool attributed to intra-/extra-cellular water (MacKay et al., 2006; Whittall et al., 1997).", "Quantification of the signal from the myelin-bound water, termed the myelin water volume fraction (MWF), has been shown to strongly correlate with histological assessments of myelin content (Gareau et al., 2000; Laule et al., 2006, 2008; Odrobina et al., 2005; Webb et al., 2003) and provide improved myelin specificity compared to DT-MRI measures or MTR (Gareau et al., 2000; Mädler et al., 2008; Vavasour et al., 2005).", "While MCR has traditionally been performed using multi-echo T2 decay data, a more recent approach, termed mcDESPOT (multi-component driven equilibrium single pulse observation of T1 and T2), has been proposed (Deoni et al., 2008).", "Though at the expense of a more complicated signal model that must include the effects of water exchange, mcDESPOT offers the potential advantages of improved SNR, reduced acquisition times, and increased spatial resolution and volumetric coverage compared to the established T2 approach.", "While comparison of multi-echo T2 and mcDESPOT MWF values present a known discrepancy, with mcDESPOT values being consistently larger (Zhang et al., 2013), they do, however, correspond qualitatively with histological myelin content measures in a Shaking Pup model of dysmyelination (Hurley et al., 2010), and have been used to investigate structure–function impairment in MS (Kitzler et al., 2012; Kolind et al., 2012) and other demyelinating disorders (Kolind et al., 2013).", "More recently, the mcDESPOT has been applied to the study of white matter maturation and healthy infant neurodevelopment (Deoni et al., 2011, 2012), revealing a strong consistency with the known spatial–temporal pattern of myelination.", "A continuous and probabilistic model of myelination could alleviate these concerns.", "Derivation of an appropriate growth model would allow estimation of the typical mean MWF, and variance, for any age.", "This work sought to develop such a model by comparing common growth functions fit to measured MWF data.", "The most appropriate model was then used to generate a continuous, four-dimensional “atlas” of healthy MWF development, allowing calculation of the ‘typical’ average and standard deviation MWF maps at any desired age.", "As proof-of-concept, individual and group-averaged MWF maps were statistically compared to the growth model derived MWF maps, with no significant differences found.", "The developed atlas, therefore, represents the first continuous model of myelin maturation in healthy male infants; provides an important step for understanding the typical myelination trajectory; and provides a framework from which to identify the earliest of white matter changes.", "Regional MWF trajectories for each investigated brain region are shown in Fig. 2.", "All trajectories follow the expected sigmoidal shape, with a period of lagged growth extending from birth through approx. 90–150 days, depending on the brain region, followed by rapid, exponential growth to approx. 400 days of age.", "Beyond 400 days, the trajectory begins to appear logarithmic in nature.", "We found transformed MWF values to be highly correlated with native-space values, with Pearson's r correlation coefficient ranging from 0.9225 to 0.9974 (Fig. 3).", "Thus, the normalization did not result in inhomogeneous changes to the MWF maps and consequently was found to be reliable for aligning individual MWF maps.", "A representative sample of the best-fit results for each model to data from the body of the corpus callosum is shown in Fig. 4.", "A summary of the entire fitting results, including free parameter estimates and BIC values, for each region is shown in Table 4.", "Based on BIC measure comparisons, we found that the modified Gompertz model consistently provided the most faithful characterization of the MWF data.", "Thus, this function was subsequently used to construct the 4-D developmental atlas by fitting this function to each individual voxel.", "The predictive validity of the model is shown in Figs. 5 and 6.", "Fig. 5 contains results from the comparison of in vivo group-averaged MWF maps and model-derived maps at the same ages.", "Qualitatively, the in vivo and model-derived MWF maps appear similar.", "Results from the paired t-test analysis yielded few statistical differences (p < 0.05 uncorrected for multiple comparisons) between the acquired and model derived MWF maps for each age group.", "We also investigated how well the atlas predicted individual MWF estimates at variable ages by comparing derived MWF maps with individually acquired MWF data.", "Representative results of these analyses are shown in Fig. 6, which shows images from the acquired maps, model-derived images, and the Z-statistic maps.", "Z-statistic values are indicated at each image voxel of the representative axial slice.", "A small number of regions of significant difference (|Z| < 1.96, corresponding to p < 0.05) were found between the acquired and model-derived MWF maps in the examined subjects.", "Bootstrap analysis of the regional MWF trajectories for the genu, body and splenium of the corpus callosum was performed to construct 95% confidence intervals using the modified Gompertz model.", "The mean modified Gompertz fit with the 95% confidence intervals are shown overlaid on the trajectories of the three regions in Fig. 7.", "Residual histograms (Fig. 7) illustrate the distribution of the residuals across the trajectories of these three regions.", "As expected, bootstrapping the parametric fit of these regional trajectories resulted in approximately 95% of the individual measurements to be contained within the bounds of the confidence intervals and the residuals normally distributed (Fig. 7).", "This work sought to model white matter myelination throughout the brain across the first five years of life in healthy male infants and toddlers.", "Investigating the overall shape of the myelination trajectories in the investigated regions revealed a non-linear sigmoidal developmental trajectory: with a lag period (0–150 days) preceding exponential growth (150–400 days) followed by a period of reduced growth thereafter.", "This growth trajectory is consistent with previous studies investigating this age-range (Deoni et al., 2012, 2013; Hermoye et al., 2006).", "Of the sigmoidal models investigated, we found a modified version of the Gompertz function to provide the best characterization of the regional MWF developmental data.", "This result is also consistent with a prior study that used the traditional, 3 parameter Gompertz function to model DT-MRI metrics across a younger age-range (Sadeghi et al., 2012).", "However, this study did not investigate alternative functions, used data from only 3 age points and focused on a more restricted age range (under 2 years of age).", "Furthermore, while the traditional Gompertz function may be best to describe the underlying growth trajectory at an early age, this function approaches an asymptote as age increases, and thus limiting its ability to account for the continued myelination that occurs throughout childhood and early adulthood (Kumar et al., 2011; Lebel and Beaulieu, 2011).", "The inclusion of an additional linear term inside the exponent of the Gompertz function corrects for the asymptotic behavior of the Gompertz function, allowing the model to account for continued growth.", "The biological interpretation of the modified Gompertz function's free parameters may provide important insight into the developmental process.", "Fig. 1B illustrates the role that each parameter has on the overall shape of this function.", "The first parameter, α, sets the overall scale of the function's trajectory and corresponds to the amplitude of the model at the transition from rapid, exponential development to reduced, continued growth.", "In terms of the current model, this parameter corresponds to the MWF value at which the developmental trajectory transitions from rapid, exponential growth to slow steady growth.", "The second parameter, β, describes the initial lag period of the model that corresponds to the time before rapid myelination.", "The third parameter, γ, describes the growth rate of the rapid exponential developmental period; while the fourth parameter, δ, corresponds to the growth rate of the slower, continued myelination observed to take place after 24 months of age.", "These measures are descriptive of the underlying maturational process and may be useful for future investigations of intrinsic growth patterns of white matter microstructure.", "For example, investigating the growth rate among brain regions would simply entail comparing the parameter γ of the interested regions.", "Furthermore, the age at which the MWF trajectory transitions from rapid, exponential growth to slow, continued development (characterized by α) may represent a pivotal time of the neurodevelopmental process (i.e. transition from infancy to toddler).", "The development of a parametric growth model of myelination is also important for understanding linkages between evolving neurodevelopment and malbehavior, as well as clinical-relevance in premature infants, infants diagnosed with multiple sclerosis, among other complications.", "For example, children with autism have been reported to exhibit widespread atypical patterns of early brain growth, including accelerated maturation of white matter (Ben Bashat et al., 2007).", "MWF growth trajectories of children with autism could be directly compared to trajectories generated by the developed model in order to see where, when and how the growth trajectory deviates from the normative population.", "While additional work is required to evaluate comparisons with such populations, the robustness of the method demonstrated here strongly intimates its feasibility.", "Although model MWF maps may be reconstructed after performing a single whole brain fit of the in vivo MWF data, the resulting fitting parameters contain no information regarding their uncertainty.", "To obtain information about the fitting parameter distribution, we utilized wild bootstrap resampling of the residuals.", "Bootstrap resampling is not new to MR imaging, as it has been used to estimate the uncertainty of fiber orientations from DT-MRI (Haroon et al., 2009; Jones, 2008; Yuan et al., 2008) and uncertainty of neural brain activity from functional MRI (fMRI) (Darki and Oghabian, 2009; Kirson et al., 2008).", "This non-parametric, model-based resampling method is advantageous because it does not require redundant data (i.e. multiple image acquisitions) in order to estimate standard errors and is sufficient to use in cases of homoscedasticity (uniform variance among data) and heteroscedasticity (non-uniform variance among data) (Chung et al., 2006).", "One potential caveat to this approach is that the model of choice (in this context, the modified Gompertz growth model) needs to appropriately characterize the measured data.", "However, our initial analysis of comparing a wide variety of growth functions, using BIC parsimony measures, examined this issue.", "We consistently found the modified Gompertz growth model to provide a better representation of the regional MWF developmental trajectories and therefore concluded that this model best represents the underling developmental pattern.", "MWF imaging has a long history in the field of known demyelinating disorders, such as multiple sclerosis (MacKay et al., 2009), however, its use in examining structural and functional development is new.", "Further, mcDESPOT differs from the conventional and established techniques that have been verified histologically (Laule et al., 2006; Webb et al., 2003).", "Similar verification of mcDESPOT has been limited to histological comparisons in the Shaking Pup model of dysmyelination (Hurley et al., 2010) and indirectly through comparison with the known histological time-course of myelination in human infants (Deoni et al., 2011, 2012), and demyelination studies in MS (Kitzler et al., 2012; Kolind et al., 2012).", "Thus, the specificity of mcDESPOT MWF measures as a reflection solely of myelin may be questioned.", "However, animal and in-vivo results garnered so far give confidence that if not specific to myelin, mcDESPOT provides novel information regarding white matter microstructure, and offers enhanced sensitivity to myelin changes relative to T1 and T2 relaxation times, or other measures.", "Outliers, caused by intra-scan motion and other confounds, have the potential of influencing the calculation of the quantitative imaging maps (i.e. MWF maps) and therefore affecting the sum of squares fitting, which could lead to erroneous results (Chang et al., 2005).", "Further, these outliers may unreasonably inflate variability when performing wild bootstrapping.", "Motion artifacts were minimized as the scanning was performed during natural, non-sedated sleep.", "Images were further assessed for motion artifacts and none were found to be present.", "Other potential imaging-related confounds, such as B0 and B1 inhomogeneity, are accounted for within the mcDESPOT processing pipeline by mapping the B1 field and removing off-resonance effects by phase-cycling the bSSFP data (Deoni, 2010).", "A 3 mm full-width-at-half-maximum 3D Gaussian kernel was additionally used to spatially smooth the MWF maps of each participant.", "Although spatially smoothing the data reduces its effective spatial resolution, the kernel size chosen was reasonably conservative relative to comparable MRI studies, preserving image detail and minimally impacting MWF values.", "Qualitative inspection of the regional trajectories in Fig. 2 as well as the 95% confidence intervals and residual distributions constructed for the genu, body and splenium of the corpus callosum (Fig. 7) suggests that individual MWF measurements are well contained.", "Nonetheless, smoothing filter size has been shown to augment statistical neuroimaging findings (Jones et al., 2005) and therefore this parameter has an impact on the findings.", "Quantifying the uncertainty of each parameter represents a critical step in the development of a model that describes MWF maturation.", "Mean and standard deviation estimates of each parameter can then be used to compute representative average and standard deviation MWF maps, for a given age between 70 and 1809 days, which can then be used to statistically compare against other typically developing and at-risk children in this age range.", "This aspect of the developed model was investigated using longitudinal measurements acquired in four subjects.", "The variation of the Z-statistic values observed in Fig. 6 suggests that there is individual deviation along the overall developmental trajectory.", "However, while this individual variation is known to exist (Giedd and Rapoport, 2010; Zilles and Amunts, 2013) and is therefore expected to be observed, a small number of these inter-individual deviations were found to be significant (|Z| < 1.96).", "In particular, examination of the first scan of subject 2 (Fig. 6) reveals areas of higher MWF in right hemispheric frontal and bilateral temporal white matter within the in vivo MWF map than the model-derived map.", "These results suggest that, at this point in time, the individual deviates from the normal myelination trajectory in these regions.", "The individual Z-statistic results suggest the developed MWF model to be representative of myelin maturation and sensitive to individual variation along the typical myelination trajectory.", "Moreover, although the paired t-test analysis of group averaged in vivo and model-derived MWF maps yielded few significant differences (p < 0.05, uncorrected), these differences did not remain significant after correcting for multiple comparisons using a cluster correction technique (FSL, cluster threshold of 1.96) in a post-hoc examination of these statistical results, suggesting the model-derived MWF maps to be representative of this normative population.", "This represents a significant new direction for investigating early neurodevelopment.", "Importantly it provides a means to identify where and when myelination deviates from the typical trajectory.", "In this work, we have restricted ourselves to single gender data across a restricted age range.", "This was to ensure a homogeneous sample with which to explore the modeling functions as well as avoid potential gender-based variations in development.", "It should be noted, however, that while the goal of this work was not to identify potential gender differences in myelination, developmental trajectories have been reported to differ between males and females.", "For example, Lenroot et al. (2007) observed males and females to have distinct patterns of brain growth, specifically noting males to have an accelerated rate of white matter volume maturation.", "While volumetric measures, such as white matter volume, reflect broad microstructural composition, the observed gender differences are likely to be influenced by changes in myelin content.", "Hence, we would expect to find similar gender differences in the trajectories of MWF and a future study investigating the role that gender has on MWF development is of great interest.", "Another aspect of the developed model not investigated in this work is generalizability of the model to data acquired from other imaging centers and scanners.", "Indeed, in order for model-derived MWF maps to have broad applicability and provide meaningful comparisons across data from different groups, values should be consistent across imaging centers and scanners.", "While the mcDESPOT imaging technique has been reported to have high intra- and inter-site reproducibility (Deoni et al., 2009), none of the data analyzed in this work has been separately acquired at a different imaging facility nor has any participant been imaged on a different MRI scanner.", "Scanning individuals at different imaging facilities as well as on multiple scanners would not only be of significant interest but necessary to investigate this aspect of the developed model.", "In this work, we have outlined a framework that can be used to model myelination trajectories that are derived from MWF measurements in children under the age of 5 years.", "We have shown that a modified Gompertz growth model provides the most accurate representation of myelin maturation using the BIC parsimony metric and have developed the first 4D atlas of myelin maturation in healthy males under 5 years of age.", "This work demonstrates the ability to accurately model early myelin maturation trajectories and provides a normative template of typical myelination in healthy male infants across a large age span (between the age of 70 and 1809 days) from which atypical myelin development can be assessed.", "The resulting model thus provides an important step for understanding ‘typical’ myelin development as well as providing the ability to identify when and where white matter abnormalities occur in neurodevelopmental disorders.", "MRI data analyzed in this work are part of an ongoing longitudinal study investigating white matter maturation in healthy, typically developing children and its relationship to behavioral development (Deoni et al., 2012).", "Informed parental consent was obtained in accordance to ethics approval from the Institutional Review Board of the host institution.", "Enrolled infants met the following inclusion criteria: uncomplicated single birth between 37 and 42 weeks; no exposure to alcohol or illicit drugs during pregnancy; no familial history of major psychiatric or depressive illness; no diagnosis of major psychiatric, depressive or learning disorder in participant; and no pre-existing neurological conditions or major head trauma.", "In total, 122 healthy male infants and toddlers between 70 and 1809 days of age (mean = 690.14 days, corrected for a 40-week gestation) were analyzed.", "Table 1 provides an age-group break down of these participants.", "Whole-brain MWF maps were acquired using the rapid mcDESPOT (Deoni et al., 2008) imaging technique.", "Imaging protocols for this age-range, using acoustically-muffled sequences, have been presented previously (Deoni et al., 2011, 2012), and comprise 8 T1-weighted spoiled gradient echo images (SPGR or spoiled FLASH), 2 inversion-prepared (IR)-SPGR images and 16 T1/T2-weighted steady-state free precession (SSFP or TrueFISP) images.", "MWF maps were calculated from these data using a three-pool signal model estimating intra/extra-axonal water; myelin-associated water; and a non-exchanging free water pool (Deoni et al., 2013).", "Corrections for flip angle (B1) and off-resonance (B0) inhomogeneities were also performed (Deoni, 2010).", "Total imaging times ranged from 19 min for the youngest infants to 24 min for older and larger children.", "Children under 4 years of age were scanned during natural, non-sedated, sleep; while children over this age were able to watch a favorite TV show or movie.", "All data was acquired on a 3 T Siemens Tim Trio scanner equipped with a 12 channel head RF array.", "To minimize intra-scan motion, children were swaddled with an appropriately sized pediatric MedVac vacuum immobilization bag (CFI Medical Solutions, USA) and foam cushions were placed around their head.", "Scanner noise was reduced by limiting the peak gradient amplitudes and slew-rates to 25 mT/m/s.", "A noise-insulating insert (Quiet Barrier HD Composite, UltraBarrier, USA) was also fitted to the inside of the scanner bore.", "MiniMuff pediatric ear covers and electrodynamic headphones (MR Confon, Germany) were used for all scanned children, while a pediatric pulse-oximetry system and infrared camera were used to continuously monitor the sleeping infants during scanning.", "After acquisition, image data was assessed for motion artifacts (blurring, ghosting, etc).", "Following calculation of the 122 MWF maps, all maps were non-linearly aligned to a study specific template (Deoni et al., 2012) using the Advanced Normalization Tools software package (Avants et al., 2008) and smoothed with a 3 mm Gaussian kernel.", "Non-brain parenchyma was removed using FSL's brain extraction tool (BET) (Smith, 2002).", "Regional masks for bilateral frontal, temporal, parietal, and occipital lobes, cingulum, and cerebellar white matter, as well as the genu, splenium and body of the corpus callosum were derived from the MNI adult template (Mazziotta et al., 2001), co-registered to the study template, and superimposed upon each infant's MWF maps (Deoni et al., 2012).", "Mean MWF values for each mask were obtained for each infant and plotted against the infant's gestational-corrected age.", "To examine whether the nonlinear transformation affected the quantitative values, mean MWF values were also extracted from native space after applying the inverse warp transformation to the regional masks.", "These native-space values were then compared to the standard space values.", "To the plotted data, the growth models of Gompertz (1825), Stannard et al. (1985), Richards (1959), Bleasdale and Nelder (1960), as well as more generic functions such as the logistic model, and hyperbolic tangent were fit.", "In addition to these, a modified form of the Gompertz model was also included in the group of growth models.", "Although diverse, each of these sigmoidal functions shares similar characteristics: 1) a lag or period of slow growth; 2) a period of rapid exponential growth; and 3) a period of reduced growth-rate (Fig. 1A).", "From these functions, biologically relevant metrics, such as lag period, growth rate, and maximum size, may be derived (Fig. 1A).", "Non-linear regression via a Levenberg–Marquardt non-linear least squares (Levenberg, 1944) algorithm was used to determine the best fit for the free-parameters of each sigmoidal function (see Table 2).", "Fits of each model were compared using the Bayesian Information Criterion (BIC, Schwarz, 1978), a parsimony metric that compares the fit residuals while penalizing for the number of model parameters.", "The model that provided the lowest BIC measure consistently across the investigated brain regions was defined as the most representative.", "Using the BIC-selected ‘best’ model, voxel-wise fitting using wild-bootstrap with residual resampling (Efron, 1979) was performed to generate whole-brain, three-dimensional maps of the mean model parameters and their associated uncertainty.", "The resampling was performed 5000 times to provide accurate estimations of each parameter's distribution.", "To illustrate the similarity between model-derived and in vivo acquired MWF maps, two main analyses were performed on an independent sample of 63 male children.", "Subjects for these analyses were not included in the group of 122 participants used in generating the model and met the same inclusion/exclusion criteria as mentioned previously.", "Table 3 provides supplemental information on these additional subjects.", "First, we examined the model's ability to examine average group differences.", "In vivo comparison data was broken into 9 different age groups (Table 3) and compared with age-matched model-derived mean MWF and δMWF maps.", "In vivo mean and standard deviation MWF maps were generated by calculating the mean and standard deviation of the MWF for each age group (Table 3).", "Paired t-tests were performed using FSL's Randomise tool (www.fmrib.ox.ac.uk/fsl/) to compare these in vivo and model-derived data, with significant differences defined as p < 0.05, uncorrected for multiple comparisons.", "Permutation testing was restricted to a custom white matter mask for each group.", "This custom white matter mask was created using the mean in vivo MWF map (for each group) by thresholding voxels with a MWF value below 0.02.", "To further demonstrate that the selected model best characterizes the underlying growth trajectory of the MWF, 95% confidence intervals were constructed for the regional trajectories of the genu, body, and splenium of the corpus callosum.", "Non-linear fitting using the wild-bootstrap with residual resampling was performed on these three regions to generate mean and uncertainty measures for the parameters of the growth model from 5000 resamples.", "From these mean and uncertainty measures, 95% confidence intervals of the growth trajectory were constructed.", "Regional trajectories of the independent sample of 63 additional subjects previously mentioned (Table 3) were also compared to these normative model fits and confidence intervals.", "Residuals between the predicted (mean model) and measured (in vivo) MWF values were then calculated for each region." ]
[ "Proposes various growth models for modeling MWF trajectories", "Demonstrates modeling of MWF trajectories of 122 male infants under 5 years", "Statistically compares tested models using Bayesian information criterion", "Generates first four-dimensional model of whole-brain myelination of early childhood", "Discusses the utility of developed model" ]
Sustainable manufacturing of consumer appliances: Reducing life cycle environmental impacts and costs of domestic ovens
Electric ovens are among the least energy efficient appliances, with the efficiency of only 10%-12%. With new policy instruments in Europe requiring energy reduction, manufactures are seeking to develop more efficient domestic appliances. The aim of this paper is to aid sustainable manufacturing of an innovative, highly-efficient oven (HEO) by evaluating its life cycle environmental impacts and costs in comparison to conventional ovens. The results suggest that the HEO has 9%-62% lower environmental impacts than conventional ovens with the equivalent savings in the life cycle costs ranging from 25% to 61%. Replacement of conventional ovens by HEO in Europe (EU28) would save 0.5-5.2 Mt of CO2 eq. and the life cycle costs would be lower by €0.5-1.96 billion (109) per year. At the household level, energy consumption would be reduced by up to 30% and consumer costs by 25%-50%. These results suggest that policy measures should be put in place to encourage the uptake of energy efficient ovens by consumers.
[ "ENER", "ENGI", "ENVI" ]
[ "Domestic ovens", "Life cycle assessment", "Life cycle costs", "Sustainable manufacturing" ]
[ "2105", "2209", "2304", "2305" ]
[ "It is estimated that there were 185.3 million cooking appliance units globally in 2015 (Global Industry Analyst, 2011) with the market value expected to reach $231 bn by 2018 (Data Monitor Research, 2014).", "In Europe, around 12 million electric ovens are sold each year (Eurostat, 2009).", "Electric ovens are among the least energy efficient appliances, with the efficiency of only 10%–12%.", "Given that they consume 100–300 kWh per year (Fonseca et al., 2009) and that 61% of 213.8 million households in the European Union (EU28) have electric ovens (Bertoldi et al., 2001), this amounts to around 26 TWh of electricity per year.", "If their efficiency increased by only 20%, that would mean a saving of around 5 TWh of electricity annually.", "In an attempt to stimulate reduction of energy use by domestic appliances and particularly ovens, the EU has adopted several policy instruments, including the Energy Labelling Directive (EC, 2013a).", "The Directive, which for ovens came into force in January 2015, classifies ovens into seven categories, from A+++ to D, based on the energy efficiency of the oven cavity.", "The Directive requires manufacturers and retailers to display on a label the energy consumption by the oven (expressed in kWh per cycle) based on a standard load.", "In anticipation of the Directive, manufactures have been seeking to develop more efficient appliances.", "This paper considers sustainable manufacturing of a new highly-efficient oven (HEO), being developed by Whirlpool, one of the largest oven manufacturers in the world.", "The primary aim of developing the HEO is to increase the energy efficiency of domestic ovens during use by around 30% relative to conventional ovens.", "A further aim is to improve energy efficiency of oven manufacturing.", "These improvements are also expected to reduce the costs to consumers and the manufacturer as well as to lead to substantial savings in environmental impacts.", "To quantify these potential benefits, HEO is compared here to conventional ovens using life cycle assessment (LCA) and life cycle costing (LCC).", "To our knowledge, this is the first study of its kind for domestic ovens.", "The following section details the methodology, followed by the discussion of the results in Section 3 and conclusions in Section 4.", "The main goal of the study is to assess the life cycle environmental impacts and life cycle costs of the HEO and quantify the environmental and economic benefits relative to conventional ovens.", "As described further below, the only difference in the design of the two oven types is the cavity, so that the study considers only this part of the oven.", "As illustrated in Fig. 1, the system boundary includes production of the raw materials used to manufacture the cavity, the manufacturing process, use of the oven and end-of-life waste management.", "Transport is excluded as it contributes less than 0.1% to the impacts and costs.", "Both ovens have the same volume of the cavity (73 litres) but they are made from different materials: low-carbon steel and enamel are used for the conventional oven, while the HEO cavity is made using stainless steel and sol–gel (Fig. 2).", "Stainless steel is used for the HEO because of its high reflectivity (Fig. 3) and ease of cleaning, while the sol–gel coating prevents loss of reflectivity owing to metal oxidation which occurs at high temperatures, a common problem in conventional enamel-based oven cavities.", "The manufacturing process for both ovens is the same except for the enamelling process for the conventional oven and application of sol–gel for the HEO.", "Therefore, only the enamelling and application of sol–gel are considered, respectively, in the manufacturing stage.", "The substrate material for the enamel layer in the conventional oven is a low-carbon enamelling grade steel formed into a cavity.", "The stainless steel substrate for the HEO cavity is produced at supplier in the form of coil.", "A protective film is then applied to the coil, which is then rolled and shipped to the in-house post-coating line.", "The coil is unwound and cut into appropriate panel dimensions after which the protective film is removed and the panels degreased.", "The first sol–gel coating is applied in a liquid-spray coating stage, dried, cured and allowed to cool down.", "Subsequently, the second sol–gel layer is applied again in another liquid-spray coating stage, dried, cured and allowed to cool down.", "The coated panels are then sent to the manufacturing line after application of a protective film.", "The use stage includes electricity consumed and oven cleaning over its lifetime.", "The conventional oven can be cleaned either by using chemicals (aerosol oven cleaners or traditional dish-washing detergents) or a built-in pyrolytic self-cleaning cycle in which the oven is heated to over 400 °C to reduce any deposits to a thin layer of ash, which can then be cleaned away easily.", "The HEO, on the other hand, can be cleaned using traditional dish-washing detergents.", "The unit of analysis (functional unit) of the study is defined as the ‘manufacture of 1 domestic electric oven cavity and its use over a lifetime of 19 years’.", "This lifetime is based on the average lifetime of ovens estimated by Mudgal et al. (2011); however, a shorter lifetime is considered as part of a sensitivity analysis.", "The oven is manufactured in Italy and assumed to be used in the EU28 region.", "Tables 1–3 summarise the data used in the study and their sources.", "As indicated in the tables, primary production data have been sourced from Whirlpool Europe while the secondary data have been obtained from LCA databases and the literature.", "For the use stage, 110 use cycles are assumed annually over 19 years for both types of oven.", "The conventional oven consumes 0.69 kWh of electricity per cycle as measured by the manufacturer in accordance with the standards CSA C358-03 (R2008) (CSA, 2008), IEC 60350-1:2011 (IEC, 2011) and EN 50304 and 60350 (BSI, 2009).", "For the pyrolytic cleaning of the conventional oven, 10 cleaning cycles have been assumed annually, based on a consumer survey carried out by Whirlpool.", "The short cleaning cycle consumes 3.5 kWh of electricity and lasts for 75 min, while the long cycle takes 120 min and uses 6.5 kWh; both are carried out at 440 °C.", "For the HEO, three options are considered for electricity usage per cycle, including oven pre-heating (155 °C over 13 min as stipulated by IEC 60350-1:2011 IEC, 2011): 0.63 kWh (HEO1), 0.59 kWh (HEO2) and 0.49 kWh/cycle (HEO3), all measured on the pilot line, also in accordance with the above-mentioned standards.", "These options have evolved over the course of the oven development process and each has been assessed for the environmental and economic sustainability relative to the conventional oven to help improve energy efficiency of the next design.", "This has led not only to a 30% increase in the efficiency in the use stage relative to the conventional oven, but also a two times higher efficiency of oven manufacturing (Table 2); the latter is due to the lower temperature required for curing the sol–gel coating compared to the conventional enamelling process.", "The price of electricity for industrial use in Italy is estimated at €0.167 per kWh (EC, 2013b), including market price, transmission through networks, administrative charges, non-recoverable taxes and duties.", "The average domestic electricity prices in the EU28 have been estimated at €0.176 per kWh (EC, 2013b).", "In the base case, it is assumed that domestic electricity prices remain constant over time; however, the effect of increasing energy prices over time is explored within the sensitivity analysis.", "For end-of-life waste management, it is assumed that 80% of oven steel is recycled (ELCD and ESA, 2011) and 20% landfilled, while all the enamel is assumed to be landfilled (Table 3).", "The system has been credited for the recycled materials.", "To assess the uncertainty in the data and results, a data quality assessment has been carried out following the CCaLC methodology (CCaLC, 2014) which is described in Supplementary Information S1 (see Appendix A).", "Based on the five criteria considered (data age, geographical origin, source, completeness and reproducibility, reliability and consistency), the quality of both the LCA and LCC data is estimated to be high.", "Therefore, the results can be considered to have high certainty.", "For full details on the data quality assessment, see Supplementary Information (see Appendix A).", "The next sections first present the results for the environmental impacts, followed by a discussion of the life cycle costs.", "The influence on the results of different parameters and assumptions is explored subsequently in the sensitivity analysis in Section 3.3.", "Finally, the potential implications for the environmental impacts and life cycle costs at the EU28 level are considered in Section 3.4.", "As can be observed in Fig. 4, the total GWP for the conventional oven ranges from 812–1478 kg CO2 eq. over the lifetime of 19 years, depending on the cleaning method assumed, with the long pyrolytic cleaning having the highest impact and the use of oven cleaner the lowest.", "For the HEO, the lifetime impact ranges from 738 kg CO2 eq. for HEO1 to 576 kg CO2 eq. per oven for HEO3.", "Therefore, the GWP of HEO relative to the conventional oven is reduced from 9%–61%, depending on the assumptions.", "As also shown in Fig. 4, the main contributor to the impact for the conventional oven is the use stage, owing to the energy used over the lifetime of the oven.", "The contribution of end-of-life waste management, which is included within the use stage in Fig. 4, is negligible (∼1%).", "Pyrolytic cleaning is the second most important contributor to the GWP, also because of the energy consumption (see Table 2).", "The contribution of the raw materials is also negligible (∼1%).", "For the HEO, the energy use accounts for the large majority of the impact (97%).", "Stainless steel adds a further 2%, sol–gel around 1% and manufacturing of the oven cavity 0.2%.", "As for the conventional oven, the end-of-life waste management contributes less than 1% to the impacts across all the HEO options considered.", "The other life cycle impacts are also lower for the HEO than for the conventional oven (Fig. 5).", "For example, relative to the conventional oven with the long pyrolytic cycle, the environmental savings for HEO1 range from 48% (photochemical smog) to 51% (eutrophication, ozone layer depletion and human toxicity) and for HEO3, from 58% to 62%.", "If the use of oven cleaner is assumed for the conventional oven instead of pyrolytic cleaning, then the savings for the best option, HEO3, are between 24% and 42%.", "Like the GWP, this is largely due to the improvements in the energy efficiency of the HEO.", "The life cycle costs, shown in Fig. 6, range from €320 to €479 over the lifetime of the conventional oven (assuming constant prices over time).", "The main cost driver is the energy use, contributing 53%–79% of the total LCC, depending on the cleaning option.", "If used, pyrolytic cleaning accounts for 31%–45% of the total costs because of the electricity used during the process (see Table 2).", "The costs of the raw materials contribute the remaining 1.5%–2% while the contribution of manufacturing is negligible.", "For the conventional oven using oven cleaner, the use stage is responsible for 79% of the total costs, while the oven cleaner and raw materials account for 18% and 2%, respectively.", "The LCC of HEO1 range from €247 for HEO1 to €194 for HEO3.", "The cost of energy in the use stage is the dominant contributor, accounting for 94% of the total LCC so that the cost to the consumer is equivalent to €232 for HEO1, €217 for HEO2 and €180 for HEO3 over the lifetime of 19 years.", "For the best option, HEO3, this represents a saving of 41%–61% over the lifetime, depending on the cleaning option assumed for the conventional oven.", "Even for the worst HEO option (HEO1), 25%–50% of the lifetime costs would still be saved by the consumer.", "Like the environmental impacts, the end-of-life waste disposal costs are negligible, accounting for less than 1% of the total LCC for all the options over the lifetime of 19 years.", "This section explores the effect of three key variables on the impacts and costs: the lifetime of ovens, electricity consumption by the oven over its lifetime and household electricity prices.", "The trends in the results for the GWP and the other impacts are similar so that only the GWP is considered here, alongside the LCC.", "In addition to the originally assumed 19-year lifetime of the ovens, a shorter lifetime of 10 years is considered here.", "The results are compared in Fig. 7 for both the GWP and LCC.", "As can be inferred from the figure, the GWP savings of HEO relative to the conventional oven are greater over 10 than over 19 years.", "For example, the saving in the impact for HEO3 compared to the conventional model cleaned using the long pyrolytic cycle is 72% over 10 years; the corresponding saving over 19 years is 61%.", "This is because of the high contribution of energy for cleaning the oven, which is proportionally lower over 10 than 19 years.", "This benefit disappears if an oven cleaner is used instead of the pyrolytic cleaning so that the relative savings in the GWP over 10 and 19 years are the same (e.g. 29% for HEO3).", "On the other hand, there is little change in the relative difference between the LCC for the 10 and 19-year lifetimes, exhibiting the opposite trend to the GWP: the cost savings are greater over the longer lifetimes, but only by 1%–2% across the above options.", "As can also be seen in Fig. 7, as expected, the total GWP and LCC are lower over the shorter lifetime as less energy is consumed.", "However, this does not take into account that the consumer would have to buy a new oven after 10 years so that the total impacts and costs would be much higher.", "This aspect is beyond the scope of the study since only the cavity is considered rather than the whole oven.", "To examine the impact of electricity consumption in the use stage, different scenarios for electricity consumption are considered and these results are shown in Figs. 8 and 9 for the GWP and LCC, respectively.", "The savings in the GWP and costs are expressed as a function of ‘delta energy’ which represents the difference in electricity consumption per cycle between the conventional oven and HEO.", "These findings have been used to guide improvements in the design of HEO during the development process.", "As indicated in the figures, the GWP and cost savings increase linearly with the increase in ‘delta energy’ between the two types of oven.", "For example, for every 0.1 kWh increase in energy efficiency of HEO, 115 kg CO2 eq. and €37 are saved over the oven’s lifetime of 19 years.", "While per single oven this may not appear to be significant, at the European level, the savings are much more substantial; this is discussed further in Section 3.4, but prior to, we examine the effect on the LCC of rising the household electricity prices over time.", "The impact of electricity prices on the LCC is compared to the base case for the conventional oven and the HEO for both the 19 and 10-year lifetimes.", "A year-on-year annual increase of 1.4% in household electricity prices is assumed (European Commission, 2009).", "As the focus is on energy prices, the costs of cleaning are excluded for both types of oven.", "The results in Fig. 10 indicate that increasing household electricity prices would lead to 12% higher costs over the lifetime of 19 years and a 6% higher costs over 10 years, for both the conventional oven and HEO.", "Therefore, while the effect of rising the costs of electricity are significant for the consumer, the relative difference between the conventional oven and HEO remains the same.", "This section considers the climate change and cost impacts of a potential replacement of conventional ovens by the HEO in Europe, considering 28 EU countries.", "The analysis is based on the number of households in EU28, estimated at 213.8 million in 2013 (Eurostat, 2014).", "It is assumed that 61% of EU28 households (130.4 million) use electric ovens (Bertoldi et al., 2001).", "The assumptions for the energy consumption by the conventional ovens and HEO are the same as previously, with 110 use cycles and 10 cleaning cycles annually (see Table 2).", "The results are presented in Figs. 11 and 12.", "Assuming the worst case whereby all households with conventional ovens use long pyrolytic cleaning cycles, the total annual GWP is estimated at 10.1 Mt of CO2 eq. and the LCC at €3.29 bn (see Fig. 11).", "If HEO1 were to replace all of the ovens, 50% or 5.08 Mt of CO2 eq. would be saved in total and the LCC would be reduced by 61% or €1.6 bn.", "Most of the latter would be direct consumer savings because of the reduced energy consumption.", "The corresponding savings for the best HEO option (HEO3) are higher still, estimated at 6.23 Mt CO2 eq. and €1.96 bn, respectively.", "If, on the other hand, it is assumed that all consumers used only detergent for cleaning the conventional ovens, the GWP savings range from 9%–30% and LCC from 23%–39% for the HEO1 and HEO3, respectively.", "Assuming an uptake rate of 5% per annum starting in 2015, it would take 20 years, to 2034, to achieve these benefits (see Fig. 12(a) and (b)).", "At a 10% penetration of HEO per year, these savings would be realised by 2025 while at a slower uptake rate of, for example 3%, it would take up to 2046 before all the conventional ovens are replaced by HEO.", "However, it should be noted that these results would vary among the individual EU28 member states because of different electricity mixes, number of households that use electric ovens and consumer behaviour.", "The results of the analysis demonstrate that replacement of conventional domestic electric ovens by highly-efficient models would be beneficial from both the environmental and economic perspectives.", "However, to promote the uptake of HEO, the use of fiscal instruments may have to be considered.", "A number of options have been suggested by the European Committee of Domestic Equipment Manufacturers (CECED) as effective for promoting the uptake of energy efficient household appliances including tax credits granted directly to consumers, consumer purchase rebate or cash-back schemes and tax credits to consumers coupled with tax credits to manufacturers (CECED, 2007; GAP, 2012).", "However, any such initiatives would have associated cost implications which should be considered carefully to avoid unintended consequences, often associated with fiscal instruments.", "Financial incentives should also be accompanied by a wide-ranging awareness raising among consumers as most are not aware that cooking, and particularly ovens, are significant energy consumers (Hoolohan and McLachlan, 2015) and that they could save money by switching to more efficient appliances.", "An example of a successful awareness raising campaign in the EU, accompanied by financial incentives as well as legislation, are light bulbs.", "To phase out energy-inefficient types, a concerted campaign involving awareness raising and free low-energy bulbs was rolled out across Europe, leading to a much faster uptake than would have happened otherwise.", "In addition, some types have been banned, notably 100 W incandescent bulbs.", "As it is going to be much more difficult to convince the consumer to replace household appliances than bulbs, similar ‘choice editing’ may be needed to help phase out energy-inefficient models more rapidly.", "This study has considered life cycle environmental and economic impacts of conventional and novel highly-efficient ovens.", "The GWP of the former ranges from 812–1478 kg CO2 eq. and of the latter between 576–738 kg CO2 eq. over the lifetime of 19 years.", "Therefore, HEO ovens have a potential to save up to 30% of energy and between 9% and 61% of the GWP, depending on the assumptions for the cleaning options for the conventional oven as well as on the amount of electricity used per cycle by HEO.", "Most of the GWP for both oven types is generated during the use stage, with the electricity contributing 53%–97% to the total.", "The raw materials contribute around 1%–2%, while the manufacture of the oven cavity accounts for less than 1% of the total impact.", "The other environmental impacts are reduced by 24%–62%.", "The LCC of HEO are also lower than for the conventional oven, ranging between €194–247 per oven over its lifetime, compared to €320–479 for the conventional oven.", "In the best case (HEO3), the consumer could save 41%–61% over the lifetime of the oven, depending on the cleaning option assumed for the conventional oven.", "Even for the worst HEO option (HEO1), 25%–50% of the lifetime costs would still be saved by the consumer.", "At the EU28 level, the results suggest that replacement of conventional domestic electric ovens by highly-efficient models would lead to significant environmental and cost savings ranging from 0.5–5.2 Mt CO2 eq./yr and €0.5–1.96 bn/yr, respectively.", "Most of the latter would be direct consumer savings because of lower energy consumption.", "Assuming an uptake rate of 5% per annum, it would take 20 years to achieve these benefits at the EU28 level.", "At 10% annual uptake per year, these savings would be realised in half the time while at 3% it would take 33 years.", "Therefore, policy makers should consider measures to encourage the uptake of energy efficient ovens, including financial incentives and ‘choice editing’ through legislation." ]
[ "An innovative, highly-efficient domestic oven (HEO) has been developed.", "HEO has 9%–62% lower environmental impacts than conventional ovens.", "The equivalent savings in life cycle costs range from 25% to 61%.", "Replacement of conventional ovens by HEO in EU28 would save 0.5–5.2 Mt of CO2 eq.", "The life cycle costs would be lower by €0.5–1.96 billion (109) per year." ]
Bending angle prediction and control of soft pneumatic actuators with embedded flex sensors – A data-driven approach
In this paper, a purely data-driven modelling approach is presented for predicting and controlling the free bending angle response of a typical soft pneumatic actuator (SPA), embedded with a resistive flex sensor. An experimental setup was constructed to test the SPA at different input pressure values and orientations, while recording the resulting feedback from the embedded flex sensor and on-board pressure sensor. A calibrated high speed camera captures image frames during the actuation, which are then analysed using an image processing program to calculate the actual bending angle and synchronise it with the recorded sensory feedback. Empirical models were derived based on the generated experimental data using two common data-driven modelling techniques; regression analysis and artificial neural networks. Both techniques were validated using a new dataset at untrained operating conditions to evaluate their prediction accuracy. Furthermore, the derived empirical model was used as part of a closed-loop PID controller to estimate and control the bending angle of the tested SPA based on the real-time sensory feedback generated. The tuned PID controller allowed the bending SPA to accurately follow stepped and sinusoidal reference signals, even in the presence of pressure leaks in the pneumatic supply. This work demonstrates how purely data-driven models can be effectively used in controlling the bending of SPAs under different operating conditions, avoiding the need for complex analytical modelling and material characterisation. Ultimately, the aim is to create more controllable soft grippers based on such SPAs with embedded sensing capabilities, to be used in applications requiring both a 'soft touch’ as well as a more controllable object manipulation.
[ "COMP", "ENGI" ]
[ "Artificial neural networks", "PID control", "Regression analysis", "Soft grippers", "Soft pneumatic actuators" ]
[ "1706", "2208", "2210" ]
[ "Soft pneumatic actuators (SPAs) with internal fluidic channels (commonly referred to as PneuNets) are made of highly stretchable elastomer materials, which deform upon the pressurisation of the internal channels to create a predefined motion [1].", "The response of this type of actuators is governed by its morphology, which is defined by the geometry of the internal fluidic channels and the properties of the materials used in fabrication.", "Inserting a flexible but inextensible strain limiting layer, in the form of a paper or fabric, at the base of the SPA prevents it from elongating and forces it to generate a bending motion that is analogous to that of a human finger.", "Hence, this class of bending actuators is being adopted as compliant soft gripper fingers, which are able to passively conform to objects of complex geometries and adapt to dimensional variations and location uncertainty [2,3].", "In addition, the soft nature of the elastomer materials used to create these soft gripper fingers, allows grasping of delicate objects safely without damaging their surface [4].", "On the other hand, the complex deformation exhibited by the non-linear elastomer materials, commonly used to create the SPA based fingers, are difficult to model and control accurately [5].", "Some examples of recent work addressing the modelling and characterisation of bending SPAs include; an experimental characterisation of the geometry of bending and rotary SPAs [6,7], finite element analysis (FEA) of cylindrical SPAs for surgical applications [8], theoretical modelling of a soft snake robot based on the bending SPAs [9], and a detailed analytical and finite element modelling of a single chamber fibre-reinforced bending SPA [10].", "One of the main challenges associated with the analytical and FEA modelling approaches is the need for accurate material models and relevant material coefficients, which can accurately describe the nonlinear behaviour of the hyperelastic materials used.", "This becomes even more complex when SPAs are made of combinations of different materials, or when equipped with external reinforcements or embedded components.", "Furthermore, there is some uncertainty in the manual process commonly followed in fabricating SPAs due to human error.", "This could result in variations in the geometry or material properties of fabricated SPA samples, which would influence their bending response.", "Therefore, it would be interesting to investigate a simpler approach for predicting and controlling bending SPAs, based on experimental data that implicitly accounts for the effects of uncontrollable variations in the morphology.", "The main contribution of this paper is in the proposition of a purely data-driven modelling approach that utilises feedback from inexpensive commercially available sensors, to derive empirical models that can be used for predicting and controlling the free bending response of soft actuators.", "This data-driven modelling approach was initially introduced in our previous work [11], and is further extended here by utilising the derived empirical models for controlling the free bending response of SPAs based on real-time sensory feedback.", "Recent relevant work demonstrated how the free bending angle can be accurately controlled using a feed-forward controller, which relies on detailed analytical models describing the physical behaviour of fiber-reinforced bending actuators specifically [10].", "Yet, the data-driven approach presented here is not constrained to a specific actuator morphology or input actuation pressure, since it is entirely based on the generated experimental data.", "Thus, this approach not only avoids the need for deriving precise physical and material models that could be difficult to achieve in some cases, but also the experimental data generated from real tests implicitly accounts for variations that are otherwise difficult to model mathematically.", "The primary requirement of this approach however, is to generate sufficient experimental data that describes the behaviour of the modelled SPA under different operating conditions, so that the derived models can be further generalised to new untrained scenarios.", "Hence, equipping SPAs with reliable sensing capabilities becomes essential to generate the required sensory feedback.", "The paper proceeds with a review on relevant work addressing the modelling and control of bending SPAs aided by different techniques for embedding sensory feedback.", "This is followed by a summary of a common fabrication process that can be followed to create typical bending SPAs, while embedding a flex sensor inside.", "Afterwards, in Section 4, the platform involving the use of pneumatic control board and a high-speed imaging system is presented, explaining how the SPAs are actuated under different operating conditions to collect the required experimental data.", "In Section 5, the data-driven modelling of the relation between the acquired sensory feedback and the bending angle measured using the vision system is derived using regression analysis and neural networks.", "The results obtained using both techniques are presented, comparing their prediction accuracy when tested with a new dataset acquired at untrained operating conditions.", "Moreover, in Section 6, the derived empirical model is utilised as part of a closed-loop PID controller to control the bending of the SPA to a desired target value.", "Finally, the paper ends with some conclusions regarding the outcomes of the proposed data-driven approach, highlighting the planned future work.", "Despite the fact that the passive compliance of SPA based soft fingers is desired for adapting to sources of variations and uncertainties without the need for expensive sensing and complex control, it also has the drawback of limiting their application to simple pick and place tasks that do not require controlled manipulation and feedback about the grasp quality.", "The absence of active sensing also means that the orientation of a grasped object with respect to the soft gripper would be unknown, since the grasp was achieved passively.", "Hence, accurate object positioning would be difficult to achieve, which is required in applications such as assembly tasks for example.", "Thus, equipping SPA based soft fingers with some level of sensing capabilities that do not hinder their desired softness and compliance would result in more controllable SPAs with enhanced functionality and wider application involving more complex manipulation tasks.", "The primary controllable input parameter that can be varied during the actuation of soft actuators is the pressure of the pneumatic supply, which in turn controls the input pneumatic flow rate.", "The internal pressure response can be easily measured using common pressure sensors connected to the pneumatic supply tubes, and can be used to control the bending of a SPA if the model relating the input pressure to the bending angle response is known in advance.", "This has been demonstrated for a soft fibre-reinforced actuator, which used an angle filter to estimate the bending angle based on the pressure measurements and a PID controller to meet the target bending angle [10].", "The main challenge in sensing however is to directly measure the bending motion of SPAs as they curve towards their base, without actually hindering this passive behaviour.", "Hence, a flexible sensor is required that can be embedded at the base layer of an SPA, where extension is restricted by the constraint layer, to provide a measurable change in a physical parameter that can be directly related to the witnessed bending motion.", "This is one of several applications motivating research over the past few years into developing new concepts for flexible and stretchable sensors, which can be integrated with soft bodies in general [12].", "The main soft sensing techniques that could be smoothly integrated with soft gripper fingers specifically for measuring and controlling their bending angle can be classified into three main approaches as follows:", "The first approach is adding different forms of carbon content into an elastomer material, in order to make it conductive and hence becoming a soft sensing element that changes in resistance when strained.", "This type of conductive elastomer sensor has been incorporated with a two-fingered soft gripper design that is actuated using linear displacements [13], to detect grasped objects and recognise their different sizes using an adaptive neuro-fuzzy controller [14].", "The main challenge with conductive elastomer sensors is the difficulty in producing sensors with consistent electrical properties, since repeated deformation may affect the distribution of carbon particles within the elastomer material.", "Also, stable electrical connections are difficult to achieve and may be a source of additional fluctuations in the sensory readings.", "Yet, the process of fabricating conductive elastomer sensors is potentially scalable through a customised 3D printing process of carbon grease inside a silicone elastomer reservoir [15].", "A popular soft sensing approach now is achieved by filling patterns of micro-channels imprinted within an elastomer body with a conductive liquid metal (EGaIn).", "Different physical parameters can be measured depending on the geometry and pattern of the conductive channels [16].", "Previous work demonstrated the use of this sensing approach to measure parameters such as: Multi-axis forces [17], strain [18], curvature [19], and pressure [20].", "The concept was integrated with a SPA based gripper to achieve accurate position and force control using feed-forward models in conjunction with a PID controller [21].", "It was also used to control the bending of soft beams actuated by an antagonistic pair of SMAs [22], and was integrated with a soft gripper to detect the presence of an object while grasping [23].", "However, the process of creating the embedded micro-channels and injecting conductive liquid metal is still a manual multi-stage process that is not easily repeatable.", "In addition, the conductive EGaIn material is quite expensive, though usually needed only in small quantities.", "The strain feedback from this approach was reported to be mostly linear and highly repeatable, but suffered from some hysteresis at higher strain rates as the EGaIn material is allowed to refill the micro-channels [18].", "An alternative soft sensing approach is achieved by simply embedding commercial resistive flex sensors within the strain limiting layer of bending SPAs.", "The flex sensors are made of thin films that can easily bend and change in resistance upon bending [24].", "This has been adopted with SPA based gripper fingers for haptic identification, by clustering the Readings from the embedded flex sensors so that a trained algorithm can identify the grasped objects [25].", "Another attempt for embedding flex sensors within soft gripper fingers was presented by [26], were the feedback was used to control the shape of the soft fingers actuated using antagonistic shape memory alloys.", "The main advantage of this approach compared to using conductive silicone rubber or conductive EGaIn channels, is the fact that it relies on simple and inexpensive commercially available sensors that can be easily wired and embedded within the strain limiting layer of SPAs.", "Additionally, the response of the sensor was found to be repeatable at different input pressures once encapsulated within the SPA body [11].", "However, achieving a consistent sensory response for different SPA samples cannot be guaranteed, since the process of embedding the sensor is still manual and the response of different sensor samples is not identical.", "Hence, each fabricated SPA with an embedded flex sensor still needs to be individually calibrated.", "The work presented here follows the third sensing approach for measuring the bending angle of a typical SPA based soft finger design.", "This is achieved by correlating the readings from the embedded flex sensors in conjunction with the internal pressure readings from on-board pressure sensors, to the actual bending angle measured using a developed vision system.", "This combination of multi-sensory feedback enables better estimations of the bending angle, without the need for deriving accurate physical and material models.", "Feeding the internal pressure to the model allows reliable predictions at varying input pressures during both the actuation and retraction phases, even if the system is disturbed with some pressure losses.", "In addition, the static bending due to gravitational forces is accounted for, by including the initial bending angle as an additional variable in the model.", "This allows for more accurate predictions of the absolute bending angles at different orientations of the soft actuator, which would be necessary when used as part of a robotic gripper.", "The conventional technique for fabricating SPAs relies on moulding silicone rubbers into the required shape, using 3D printed moulds with the negative of the features to imprint, followed by bonding the parts together after curing to create the final shape of the actuator [1].", "A soft finger based on a standard bending SPA design with ribbed channel morphology (shown in Fig. 1), was fabricated from a common silicone rubber material (Ecoflex-501).", "The dimensions of the soft finger were based on the results of previous work characterising the bending response and force generation of a set of soft fingers with variable internal channel dimensions [7].", "To fabricate the soft finger, the body is divided it into three main parts (labelled on Fig. 1): (1) The main body moulded from Ecoflex-50 with the imprinted fluidic channel pattern, (2) the bottom base made also from EcoFlex-50 or a stiffer elastomer if desired, which seals the internal channels, (3) and a strain limiting layer in a form of a sheet of paper between those two parts.", "This layer is necessary to prevent the finger from extending, allowing only a bending motion towards its base.", "Here, we also attach a flexible resistive sensor2 to the strain limiting layer, in order to change in resistance as the soft finger bends.", "The sensor is thin and flexible, so it does not hinder the desired bending response, but causes a small increase in the overall stiffness of the soft finger.", "The procedure involved for fabricating the soft bending fingers with embedded sensing can be summarised in the following steps:", "Printing moulds: The moulds with the negative of the geometry featured required to be imprinted are designed and 3D printed from ABS filament using a Lulzbot TAZ5 printer.", "Mixing and degasing: EcoFlex-50 is prepared by mixing equal volumes of the provided components and stirred well for a maximum of 2 min.", "The mixed material is then quickly placed in a vacuum chamber for degassing at 900 mbar for about 5 min.", "This extracts trapped air bubbles that could create weak points in the stretchable soft body.", "Moulding: the mixed material is then carefully poured in the 3D printed moulds to create the two parts of the soft finger with imprinted features, and then left to cure.", "The curing process can be accelerated by placing the moulds in an oven at 50° for around an hour.", "Strain limiting layer: two pieces of paper are cut into the required dimensions to create the strain limiting layer which will fit between the two parts of the soft finger.", "Embedding flex sensor: The flex sensor is positioned between those two sheets of paper and glued together to form one flexible but not extendable layer, which is embedded at the interface between the two moulded parts as shown in Fig. 2 below.", "Demoulding and bonding: both parts of the soft finger are demoulded and dipped in a freshly mixed EcoFlex material at their joining faces, to act as a bonding agent.", "The prepared strain limiting layer is placed on top of the base part and the main body of the finger is then placed and aligned on top.", "Pneumatic connection: once the bonding layer cures, a needle is inserted at the base of the finger to penetrate through the internal channels, which will be pneumatically actuated.", "A tube is then connected to the other end of the needle to deliver the controlled pneumatic supply.", "The outlined fabrication process uses inexpensive materials and requires fairly simple equipment to implement.", "However, the manual nature of the process could introduce variations during different stages of the fabrication process including: the material preparation, mould printing, sensor placement, and manual bonding.", "This uncertainty in the actuator dimensions and material properties is one of the factors limiting the accuracy of analytical models that are derived based on theoretical values, which are difficult to guarantee.", "Hence, the data-driven modelling approach considered here is encouraged, as variations in the fabrication process due to human error will be implicitly accounted for within the experimental data.", "Moreover, the focus of the work presented here is on modelling and controlling the free bending response of individual soft fingers with embedded sensing, which can be then connected to a gripper for controlled grasping applications.", "Fig. 3 shows a proposed soft gripper prototype utilising four bending SPAs with embedded flex sensors, acting as controllable soft finger that are easily interchangeable.", "A customisable 3D printed interface secures the individual soft fingers in the desired configuration and holds the pneumatic tubes and sensors’ wiring in place.", "This results in a modular soft gripper design that can be reconfigured to modify the number and location of individual soft finger modules according to the application needs.", "The aim is ultimately to achieve accurate positioning in complex manipulation tasks using such simple and inexpensive hardware.", "An experimental setup was constructed to systematically test fabricated SPA samples, embedded with the flex sensor at variable operating conditions.", "This includes controlling the pressure and duration of the input pneumatic supply, as well as setting the initial orientation of the actuator.", "The pneumatic supply flows through a 1.6 mm diameter needle attached to the end of the supply tube.", "The needle passes through a locating hole inside the 3D printed fixture, which guides it to pierce the actuator at the base of its internal channels.", "This allows easy and fast switching between different SPA samples, without the need for bonding the pneumatic tubes directly to the actuator body.", "The initial orientation ‘ϕ’ of the soft actuator can be varied by simply rotating a 3D printed fixture that securely holds the soft actuator to a fixed frame, and is measured from the positive x-axis as illustrated in Fig. 4.", "As for varying the pressure of the pneumatic supply, this is achieved through high speed valves on a pneumatic control board that was based on the design proposed by the soft robotics toolkit3.", "The board includes solenoid valves (SMC-VQ110U-5 M) controlling the flow of pneumatic supply, pressure sensors (Honeywell-ASDXAVX100PGAA5) measuring the resulting internal pressure, and an Arduino Mega board that is programmed to control the timing of the actuation and the effective pressure supply.", "The on-board pressure sensors and the embedded flex sensors are interfaced with the Arduino board to feedback the resulting sensory readings at 100 Hz for each actuation test conducted.", "The resulting sensory feedback from the embedded flex sensor and the measured pressure response, is recorded and synchronised with measurements for the actual bending angle for each actuation.", "The input pressure to the tested soft actuators can be effectively varied through high speed valve switching, controlled by a pulse width modulated (PWM) signal at 60 Hz from the Arduino board.", "A fixed input regulated pressure line can be thus effectively reduced to a desired value according to the duty cycle of the generated PWM signal.", "This provides a fairly simple and inexpensive method for controlling the pressure supply to the soft actuator.", "However, it has the drawback of introducing noise to the measured internal pressure response because of the mechanical switching of the valves.", "Having noisy sensory feedback will limit the accuracy of any predictive models or controllers that rely on such data.", "One way of overcoming this problem is the use of a pneumatic tank in the form of fixed volume syringe and a pneumatic resistance in the form of a porous plug [27].", "Fig. 5 shows a schematic diagram for the pneumatic circuit including a 4 ml syringe and a 20 mm pneumatic resistor between the valve and the pressure sensor, while Fig. 6 shows how this results in significantly damping the oscillations in the pressure response measured at 50% duty cycle.", "It can be observed from Fig. 6 that introducing the pneumatic tank and resistor has minimal effect on the measured internal pressure, but in return results in a significant improvement in noise reduction.", "It is important however to avoid changing the size of the pneumatic tank and resistor after collecting the experimental data required for the data-driven modelling, since this would affect the prediction accuracy of the derived models as the measured pressure changes slightly.", "Furthermore, a moving average can be applied to further smoothen remaining oscillations in the signal, yet this would come at the expense of an added delay to the response of the system depending on the window size.", "The final smoothed pressure response shown in Fig. 6 is the combined outcome of adding the pneumatic tank and resistor as well as applying a two-point moving average, which would introduce a delay of around 20 ms.", "A high-speed camera was used to capture the gradual deformation of the tested SPA upon actuation by recording image frames at a rate of 130 Fps.", "Such a fast frame rate is required to allow capturing sufficient number of images within the actuation durations that could be as short as 500 ms. The camera is fixed to the same frame that holds the actuator, to ensure that it remains in the same location with respect to the actuator.", "Calibration for the intrinsic and extrinsic camera parameters was conducted to allow measurements in real-world coordinates for the tip trajectory of the tested soft actuators with a mean calibration error of 0.003 mm.", "The measurements are done automatically for each image frame by an image processing program that was developed using Halcon library4 without the use of any external markers that could alter the bending response.", "To achieve this, the program segments the deforming SPA body using automatic thresholding aided by a dark background.", "Contours defining the circumference of the segmented blob representing the actuator body are then extracted and the position of the tip is located within each image frame in real-word coordinates.", "Afterwards, the bending angle ‘θ’ can be calculated with respect to the axis passing through the base of the soft actuator, as illustrated in the sample output from the program shown in Fig. 7.", "During a typical actuation test, the camera is externally triggered via the Arduino microcontroller, so that the bending angle value calculated for each captured image frame can be synchronised with the corresponding sensory readings recorded simultaneously.", "The primary sensory feedback of interest in this work, is the change in resistance of the embedded flex sensor due to the bending motion of the actuator, which is converted to voltage and measured through the analog input port of the Arduino board.", "This provides a direct measurement that can be correlated to the actual bending angle of the actuator, allowing accurate prediction and closed-loop control of such actuators.", "In order to evaluate the quality and repeatability of the feedback from the embedded flex sensors a soft actuator sample was repeatedly actuated at different magnitudes and durations of the input pneumatic supply.", "Fig. 8 plots the internal pressure measured against the resulting flex sensor readings, when supplied with a step pressure input of 12 Psi (82.7 kPa) for different durations.", "The plotted flex sensor readings are the result of the analog to digital conversion of the voltage received from the flex sensor, in which the 0–5v range is converted to values between 0 to 1023.", "The plotted cycle shows the readings from the flex sensor decreasing upon actuation as the internal pressure builds up, until the pneumatic supply is stopped and the SPA starts to retract back to its original shape.", "The response was observed to be fairly repeatable; with longer actuation duration causing a systematic extension to the witnessed response.", "Thus, it can be assumed that when increasing the actuation duration, the actuator will continue to bend following the same relation between the internal pressure and flex sensor readings as long as the input pressure is constant and the actuator is able to hold that pressure.", "Furthermore, the same test was repeated again, yet this time the actuation duration was fixed at 500 ms, while the SPA was actuated at pressure inputs of 10 Psi (68.9 kPa) and 12 Psi (82.7 kPa).", "Fig. 9 shows that changing the input pressure had a more significant effect on the recorded sensory response, influencing not only the final reading from the flex sensor, but also the rate of change of the response.", "This shows the need for incorporating the measured internal pressure response, if accurate models are to be derived for the estimation of the bending angle of soft actuators.", "The pressure term in this case will account for the rate of change of the flex sensory reading, allowing more generic models to be derived that are capable of estimating the bending angle at varying input pressures.", "Finally, Fig. 10 shows the relation between the measured internal pressure and the flex sensor readings at three different soft finger initial orientations of 45°, 0°, and −45°.", "A slight deviation in the response can be observed in each case, even though the input conditions were held constant.", "The deviation is more evident during the retraction of the soft finger to its initial position, since the pneumatic supply is stopped and gravity becomes the dominant force acting on the finger.", "This illustrates the significance of taking the initial orientation of soft fingers into consideration when modelling their bending response to be able to compensate for the effect of gravity and generate more accurate models [10].", "The orientation here is known for each test since the tested soft finger is fixed using the 3D printed mounts.", "Yet, in actual grasping applications the orientation can be measured in real-time using an accelerometer sensor mounted at the gripper base.", "This would be an additional sensory input that can be interfaced to the Arduino board.", "In this section, the data acquired from experimental tests is utilised for deriving an empirical model that describes the bending response of the investigated soft actuator.", "The available feedback from a typical actuation test comprises of the (1) the measured voltage from the change in resistance due to bending of the embedded flex sensor “F”, (2) internal pressure “P” measured using on-board pressure sensors, (3) and the initial orientation of the soft finger within its bending plane “ϕ” set using the tilting fixture.", "A soft actuator sample with an embedded flex sensor was tested twice at the combinations of three different initial orientations of −45°, 0°, and 45°, with step pressure inputs of 8, 10, and 12 Psi (55.2, 68.9, and 82.7 kPa).", "The resulting sensory feedback and the image frames captured for measuring the actual bending angle were recorded and synchronised as shown in Fig. 11.", "A data set of 1664 observations in total was generated at a unified sampling rate of 10 ms, with each observation being an array of four elements in the form of [F, P, ϕ, θ].", "The first three elements in the array are the input variables, while the fourth element is the target bending angle output for training and testing the derived empirical models.", "Regression analysis and neural networks are two data-driven modelling techniques that are implemented and compared here, in order to derive empirical models which will be better suited predicting and controlling the bending angle of the tested soft actuators based on real-time sensory feedback.", "A summary of the regression statistics for the derived models is given in Table 1.", "It shows how the model accuracy was improved gradually by the inclusion of the P and ϕ variables.", "It is clear that using the feedback from the flex sensor alone is not sufficient to derive a model that can accurately predict the actual bending angle under different operating conditions.", "The addition of the P term significantly improves accuracy of fit since the change in the input pressure is reflected in the model, while adding the ϕ term allows the prediction of the absolute bending angle values while accounting for the static bending under gravity.", "In order to verify the derived models, the testing dataset was fed to each model and their predicted bending angle was compared to the actual values measured using the vision system.", "The data set for testing was generated at input conditions that were not covered by the training dataset used in deriving those models.", "The results of testing the three models are summarised in Table 2, showing the mean squared error (MSE) and standard deviation (SD) of the predicted bending angles.", "It becomes evident that the inclusion of the P and ϕ parameters contribute to a more accurate empirical model that is able to accurately reproduce the bending angle values under untrained input conditions, with an MSE of only 1.36 and a SD of 1.15° (Model 3).", "Fig. 12 shows a sample of the test results comparing the predicted bending angle of each model to the target values measured using the vision system.", "It can be observed that even though models 1 and 2 result in almost the same final bending angle value, model 2 better follows the actual bending angle response since the P parameter is adjusting the output to the correct the rate of change.", "As expected, model 3 can be seen to provide the closest predictions compared to the actual target values, yet a small error of 1.54° in the final value still exists.", "The remaining deviation between the predicted and target values can be accounted to sources of non-linearity in the response that cannot be captured effectively by a linear regression model.", "Another data-driven modelling technique investigated here is the use of a feed-forward artificial neural network (ANN) that is known to cope well with handling sources of uncertainty [29], and is hence a good candidate for modelling the complex behaviour of continuum soft robots in general [30].", "The same training dataset used in the regression analysis was used again here to train and validate a feed-forward neural network with 1 hidden layer and 7 neurons using MATLAB.", "This network structure was found to reduce the MSE while avoiding overfitting.", "The inputs to the neural network are the same as model 3, which include the sensory feedback from the pressure and flex sensors, and labelled with initial orientation of the soft finger.", "The target output is again the measured bending angle of the soft finger recorded by the vision system.", "Training was conducted using the Levenberg–Marquardt algorithm [31], which would stop when the generalisation accuracy stops improving.", "The results of the training showed an excellent fit between the inputs and the target output with an R value of almost 1, as shown in Fig. 13.", "The trained ANN was tested with the same test dataset previously used for testing the regression models, and achieved a much lower MSE of only 0.37 with a SD of 0.6° (Table 3) when compared to the best performing regression model.", "This means that the trained network is able to better capture the non-linearity in the response of the soft actuator, which the linear regression model was not able to account for.", "On the other hand, the neural network is more complex in structure compared to a simple linear equation resulting from the regression analysis.", "Thus, depending on the application needs, the slightly less accurate regression model might still be favoured when deploying the model to a controller with a limited processing power, so that a faster sampling rate for the required sensory feedback can be maintained.", "This is essential especially when operating at higher input pressure, during which the actuation would typically last for less than 500 ms.", "Finally, Fig. 14 shows a sample of the test results comparing the prediction accuracy of the derived empirical model 3 and the trained neural network.", "Both techniques successfully reproduced the bending angle response at the tested finger orientation.", "It can be concluded that both techniques can be used to predict the bending angle response when given new data sets acquired at untrained operating conditions.", "Yet, the trained neural networks performed better than the derived regression model, at the expense of requiring more computational power when deployed to a controller.", "The next step after successfully deriving models that predict the bending angle of soft actuators based on the combined sensory feedback, is to utilise those models in controlling the bending angle according to a desired target value.", "A closed-loop PID controller was designed and tuned using Matlab Simulink, which (1) collects the real-time sensory feedback from the Arduino, (2) predicts the current bending angle using the derived regression model according to the sensory data, (3) calculates the duty cycle value of the PWM signal driving the valve switching, based on the difference between the current and target bending angles.", "(4) adjusts the supplied pressure to the actuator by generating a PWM signal based on the new duty cycle value and sending it to the high speed vales.", "Fig. 15 shows a schematic diagram summarising the basic operation of the MATLAB control program communicating with the Arduino controller on the pneumatic control board.", "The PID controller gains were tuned and initiated online following the Ziegler–Nichols method [32], until the settling error and oscillations were minimised.", "This involved setting the controller's integrator gain (Ki) and derivative gain (Kd) to zero while increasing the proportional gain (Kp) until the system reaches its stability boundary.", "The corresponding values of this gain and period of oscillations were recorded and used to calculate the initial values of the PID controller gains.", "Further tuning of the gain values was manually conducted online based on the monitored bending response when tested with different target values.", "The final values of the tuned PID gains were found to be: Kp = 26 (deg−1), Ki = 11 (1/deg s), and Kd = 0.2 (s/deg).", "This heuristic tuning approach is well suited for the data-driven modelling approach adopted here, since the physical model of the system is not available for typical model-based tuning and stability analysis tests to be performed.", "Instead the availability of the real-time response is utilised to tune the PID controller gains and confirm the controller stability within the required operating conditions as further demonstrated in the tests to follow.", "In order to test the accuracy and stability of the controller in meeting a target bending angle value, a series of experiments were conducted in which a step as well as sinusoidal reference signals were supplied to the controller and compared to the measured bending angle.", "Once a switch on the pneumatic controller board is toggled, the valve opens to supply a constant pressure input to the tested soft actuator with an initial duty cycle value of 100%.", "The sensory feedback from the embedded flex sensor and on-board pressure sensor is continuously fed to the derived regression model to convert it to the current bending angle value of the soft actuator.", "The difference between the target and current bending angles is fed to the PID controller as the error signal (Fig. 15), which as it decrease causes the PID controller to reduce the duty cycle value of the PWM signal accordingly.", "Reducing the duty cycle below 100% initiates the high speed valve switching, which in turn reduces the effective internal pressure supplied to the soft actuator as previously explained.", "Hence, the rate of increase in the bending of the soft actuator attenuates until settling at the target bending angle value, even though a fixed pressure input is being supplied.", "Fig. 16 shows the measured bending angle response, the measured internal pressure, and the duty cycle output from the controller, when testing an actuator at 8 Psi (55.2 kPa) pressure input and setting the target bending angle to a value of −35° (measured from the positive X-axis).", "It can be observed how the measured internal pressure response settled to a value of nearly 5.5 Psi (37.9 kPa), as the controller reduced the duty cycle value to values in the range of 60–80%.", "The fluctuations in the duty cycle value is due to the small residual oscillations in the internal pressure measurement, which is expected when using high speed valve switching to control the pressure.", "Once the actuation switch is toggled off, the input pressure is stopped causing the soft actuator to retract to its original position as shown in Fig. 16.", "Since the exhaust of the pneumatic circuit was left at atmospheric pressure, the duty cycle output from the controller settles around the value required to achieve an internal pressure that is just enough for the actuator to maintain the target bending angle.", "Alternatively, if the exhaust was instead sealed to hold the supply pressure, then the output duty cycle will continue to drop until reaching zero, which closes the valve completely to stop any further pneumatic supply.", "However, in this case the exhaust has to be manually opened for the soft actuator to retract to its original position, which means that the bending can only be controlled during the forward actuation phase.", "Moreover, further tests were conducted in which the target bending angle was supplied to the controller as stepped as well as a sinusoidal reference signal.", "For the step response experiment the reference signal increases from 30° to 400 and falls back with 5° increments, while the sinusoidal reference signal oscillated from 30° with an amplitude of 10° and a period of 3 s.", "The measured bending angle response closely followed that of both the stepped and sinusoidal reference signals with a mean error of only 0.752° and a standard deviation of 2.09° as shown in Fig. 17.", "The convergence time was around 150 ms on average, yet this largely depends on the input pressure value which for these tests was 8 Psi (55.2 kPa).", "The results of these tests confirmed that the soft actuator can be accurately controlled to follow a variable reference signal, based on the acquired real-time sensory feedback.", "Moreover, a key feature of this controller is the fact that it relies on feedback from both the embedded flex sensor as well as the internal pressure response to estimate the current bending angle.", "Thus, not only can the controller operate effectively at different pressure inputs, but also it can handle external disturbances in the form of pressure leaks.", "This was witnessed during the conducted tests, when a leak from the inlet of the supply tubes to the base of the soft actuator caused an unexpected drop in the measured internal pressure.", "Consequently, the controller automatically increased the output duty cycle value to compensate for the witnessed pressure drop until meeting the target bending angle.", "The pressure leak in this case is no different than a change in the supply pressure, which the derived model was trained to accommodate.", "Thus, the inclusion of the filtered internal pressure measurements with the feedback from the embedded flex sensor in the derived models, enhances the robustness of the controller to external disturbances.", "This also allows accurate predictions of the bending angle during both the actuation and retraction of the actuator, which is important to measure and control the fluctuations around the target bending value in both directions.", "Finally, including the initial orientation of the soft actuator in the derived models allows the user to set meaningful target bending angle values that are measured as an absolute value from a known fixed reference.", "The work presented here demonstrated an alternative approach for predicting and controlling the bending angle of a common soft pneumatic actuator using a purely data-driven approach that relies on generated datasets of sensory feedback, without the need for analytically deriving complex physical and material models.", "A resistive flex sensor was embedded within the strain limiting layer of the soft actuator, while an on-board pressure sensor measures the internal pressures response during actuation.", "The soft actuator was tested at different operating conditions using a controlled pneumatic supply.", "The resulting bending response was recorded using a high-speed camera and processed using an image processing program, to track and measure the change in bending angle during each test.", "Regression analysis and neural networks were utilised to model the measured bending angle output based on the generated sensory feedback.", "Both techniques were successful in capturing the bending response of the soft actuator, with neural networks providing more accurate predictions.", "The trained models were successfully validated using a new dataset generated at untrained operating conditions.", "Furthermore, the derived regression model was integrated as part of a closed-loop PID controller, in order to control the soft actuator bending based on real-time sensory feedback.", "The controller was tested using stepped and sinusoidal reference signals, and was able to accurately maintain the desired target angle with a mean error of only 0.752° and a standard deviation of 2.09°.", "The result of this work showed how simple empirical models and trained neural networks are able to accurately predict the bending angle of common bending SPAs at different operating conditions, using a relatively small dataset from inexpensive commercial sensors.", "Additionally, the bending angle was successfully controlled in real-time using the derived empirical model, even when the system suffered from some pressure leak.", "The main advantage of this approach lies in lifting the need for exact physical models that require prior knowledge about the geometry and material properties of the tested soft actuators.", "Instead, inexpensive commercial sensors are embedded to generate experimental data required for deriving the empirical models, which implicitly accounts for any variations that could arise during the manual fabrication of such actuators.", "Thus, the approach can be potentially adopted for other soft actuator morphologies, as long as the required sensory feedback can be generated to derive the models.", "This is part of ongoing work on developing more controllable soft gripper prototypes (as the one shown in Fig. 3), which are made of interchangeable soft fingers based on bending SPAs with embedded flexible sensors.", "The first stage of the work presented here, demonstrated how data-driven approaches can be used to model and control the free bending response of individual soft fingers using real-time sensory feedback.", "By combining those individually calibrated soft fingers to form a complete soft gripper, more controllable manipulations can be achieved.", "It is envisioned that by further training of the ANN under wider grasping conditions, contact with the grasped objects can be detected, and accurate estimations can be made about the size and position of the grasped object with respect to the gripper base.", "This will contribute towards expanding the application of soft grippers to include more complex manipulation tasks that require not only the inherited softness and compliance, but also accurate positional and force control." ]
[ "A purely data-driven approach for modelling the bending of soft pneumatic actuators.", "Evaluation of the feedback from an embedded flex sensor at different operating conditions.", "Accurate bending angle prediction at variable input pressures and even pressure leaks.", "Comparing the prediction accuracy of regression analysis and neural network methods.", "A PID controller is used to control the bending angle based on multi-sensory feedback." ]
BH3-dependent and independent activation of BAX and BAK in mitochondrial apoptosis
Mitochondria play key roles in mammalian apoptosis, a highly regulated genetic program of cell suicide. Multiple apoptotic signals culminate in mitochondrial outer membrane permeabilization (MOMP), which not only couples the mitochondria to the activation of caspases but also initiates caspase-independent mitochondrial dysfunction. The BCL-2 family proteins are central regulators of MOMP. Multidomain pro-apoptotic BAX and BAK are essential effectors responsible for MOMP, whereas anti-apoptotic BCL-2, BCL-XL, and MCL-1 preserve mitochondrial integrity. The third BCL-2 subfamily of proteins, BH3-only molecules, promotes apoptosis by either activating BAX and BAK or inactivating BCL-2, BCL-XL, and MCL-1. Through an interconnected hierarchical network of interactions, the BCL-2 family proteins integrate developmental and environmental cues to dictate the survival versus death decision of cells by regulating the integrity of the mitochondrial outer membrane. Over the past 30 years, research on the BCL-2-regulated apoptotic pathway has not only revealed its importance in both normal physiological and disease processes, but has also resulted in the first anti-cancer drug targeting protein–protein interactions.
[ "BIOC", "MEDI" ]
[]
[ "1314", "2737" ]
[ "Apoptosis is the best-studied form of programmed cell death, which is indispensable for the development and maintenance of homeostasis within multicellular organisms [1].", "Dysregulation of apoptosis incurs a wide variety of human illness ranging from neurodegenerative disorders to cancer [1].", "Apoptosis can be initiated through either intrinsic or extrinsic pathways, both of which activate caspases that are executioners of apoptosis; processing of cellular substrates by these enzymes leads to the characteristic morphological and biochemical features of apoptosis [1].", "The intrinsic pathway is activated by a wide variety of cellular stresses including DNA damage, endoplasmic reticulum (ER) stress, and deprivation of cytokines, growth factors, or nutrients, whereas the extrinsic pathway is initiated by the engagement of cell surface ‘death receptors’ such as FAS and TRAIL receptors (Figure 1).", "The intrinsic death signals culminate in MOMP, resulting in the release of apoptogenic factors including cytochrome c and SMAC [2].", "Upon binding to cytochrome c and dATP, APAF-1 oligomerizes into a heptameric complex known as the apoptosome, which recruits and activates caspases [3].", "Death receptor engagement can lead to initiator caspase-8 and subsequent effector caspase-3/7 activation in so-called ‘type I’ cells, such as T-lymphocytes.", "However, in ‘type II’ cells, such as hepatocytes, effector caspase activation requires a mitochondrial amplification loop to alleviate XIAP-mediated caspase inhibition through mitochondrial release of SMAC (Figure 1).", "The BCL-2 family proteins are central regulators of MOMP [1].", "The founding member BCL-2 was cloned from the t(14;18)(q32;q21) breakpoint, pathognomonic of human follicular lymphoma.", "The discovery that BCL-2 promotes cellular survival rather than proliferation initiated a new category of oncogenes [4].", "Over the years, at least 15 members of the BCL-2 family have been identified that either prevent or promote apoptosis.", "They are now divided into three subfamilies: first, multidomain anti-apoptotic BCL-2, BCL-XL (BCL2L1), MCL-1, BCL-W (BCL2L2), and A1 (BCL2A1); second, multidomain pro-apoptotic BAX and BAK; and third, pro-apoptotic BH3-only molecules (BH3s).", "Multidomain members share sequence homology with all four conserved BCL-2 homology domains (BH1-4), whereas BH3s only contain the BH3 domain.", "Most BCL-2 family members also harbor a C-terminal transmembrane anchor that targets these proteins to the mitochondrial outer membrane (MOM).", "Although BOK shares significant sequence homology with BAX and BAK, it neither rescues the apoptotic defects of Bax−/−Bak−/− double knockout (DKO) cells nor is regulated by other BCL-2 members, and is considered as a non-canonical BCL-2 member [5].", "Since the discovery of BAX and BAK, it was known that anti-apoptotic and pro-apoptotic BCL-2 members form heterodimers [1].", "This led to a major debate in 1990s regarding whether multidomain anti-apoptotic or pro-apoptotic BCL-2 members are downstream effectors in controlling apoptosis.", "The generation of Bax−/−Bak−/− DKO mice provided convincing evidence that BAX and BAK are the essential downstream effectors of mitochondrial apoptosis [6–8].", "However, BAX and BAK are kept inactive in viable cells and need to be activated upon death signaling to trigger MOMP.", "Hence, the next major debate was how BAX and BAK are activated and whether the ‘activator’ subgroup of BH3s are required for their activation.", "The generation of Bid−/−Bim−/−Puma−/−Noxa−/− quadruple knockout (QKO) mice deficient for all activator BH3s provided in vivo evidence supporting direct activation of BAX and BAK by BID, BIM, PUMA, and NOXA.", "It also concurrently revealed that BH3-independent autoactivation of BAX and BAK can occur when BCL-2, BCL-XL, and MCL-1 are simultaneously downregulated, but with slower kinetics compared to BH3-mediated activation [9••].", "Here, we summarize recent advances in how the BAX and BAK-dependent mitochondrion-dependent cell death program is regulated.", "BH3s are sentinels for cellular stress and function as initiator cell death signaling molecules with each BH3 coupled to a specific death signal.", "Their activity is regulated transcriptionally or by post-translational modifications.", "For example, genotoxic stress activates p53 to induce PUMA and NOXA, while cytokine/growth factor deprivation triggers nuclear translocation of FOXO1/3 to transactivate PUMA or BIM in a cell type-specific manner [1,10].", "ER stress activates BIM through CHOP-mediated transcription as well as protein phosphatase 2A-mediated dephosphorylation [11].", "In contrast, phosphorylation of BIM by the kinases ERK and RSK targets BIM for β-TRCP-mediated ubiquitination and subsequent proteasome-dependent degradation [12].", "Death receptor ligation results in caspase-8 mediated proteolytic cleavage of cytosolic BID into truncated BID (tBID), which then targets to the mitochondria to activate BAX and BAK, connecting the extrinsic pathway to the intrinsic mitochondrial apoptosis pathway [1].", "BH3s interconnect signal transduction and multidomain BCL-2 family checkpoints by either activating BAX/BAK or inactivating anti-apoptotic BCL-2 members through direct binding [13,14].", "Accordingly, BH3s have been divided into two classes, ‘activator’ and ‘inactivator’ (or ‘sensitizer’) [13,14].", "The BCL-2 family proteins regulate mitochondrial apoptosis through protein–protein interactions, all involving the same BH3 helix-in-groove structure [15] (Figure 2).", "The BH1, BH2, and BH3 domains of multidomain anti-apoptotic and pro-apoptotic members can form a hydrophobic binding groove (or canonical dimerization groove) that accommodates the amphipathic alpha-helical BH3 domain in BH3s.", "The interaction between activator BH3s and multidomain BAX or BAK has been debated for decades due to low binding affinity.", "Recent biophysical demonstration of BID, BIM or PUMA bound to BAX or BAK by NMR or crystal structures have helped resolve this controversy [16–20].", "The binding of BH3s to multidomain anti-apoptotic members is inhibitory and stable (Figure 2A), whereas the binding of activator BH3s to BAX/BAK is stimulatory and dynamic (Figure 2B).", "The major purpose of the latter interaction is to induce the exposure of the BH3 domain in BAX or BAK such that the ‘BH3-exposed’ BAX or BAK monomer can bind to the hydrophobic dimerization groove of another BAX or BAK molecule, forming symmetric homo-dimers and subsequent homo-oligomers (Figure 2C).", "Accordingly, the interaction between activator BH3 and BAX or BAK must be ‘hit-and-run’, consistent with the low binding affinity between activator BH3s and BAX or BAK.", "In contrast, BH3s bind tightly to anti-apoptotic BCL-2 members.", "By analogy, activator BH3s are death ligands, BAX/BAK are death receptors, and the anti-apoptotic BCL-2 members function like ‘decoy’ death receptors that form inert stable complexes with BH3s but are unable to assemble the homo-oligomerized death machinery.", "Notably, the BH3 domain of most BAX or BAK present in viable cells is not exposed [9••,15,16,20–23].", "Only partially activated BAX or BAK will expose the BH3 domain and as a result can bind to anti-apoptotic BCL-2 members (Figure 2D).", "Among BH3s, BID, BIM, PUMA, and NOXA are ‘activator BH3s’ that directly interact with and induce the stepwise structural reorganization of BAX and BAK [7,8,9••,13,18,24–26] (Figure 3).", "Notably, only the BH3 peptides derived from BID and BIM, but not PUMA and NOXA, can consistently recapitulate the full-length proteins in activating BAX and BAK [27–29].", "Hence, early classification of BH3s based on the activity of BH3 peptides has inherent limitations.", "In viable cells, BAX exists in the cytosol as a monomer with its α1 helix keeping the C-terminal α9 helix engaged in the dimerization groove [21,24].", "This auto-inhibited BAX monomer may be further stabilized by forming an asymmetric dimer with the α9 helix of one BAX molecule binding to the α1/α6 trigger site of the other BAX [30•].", "In contrast, BAK is constitutively inserted in the MOM via its C-terminal α9 helix and maintained as an inactive monomer by VDAC2 [31].", "Activation of BAX involves two distinct steps, mitochondrial targeting and homo-oligomerization, whereas that of BAK only involves the latter (Figure 3).", "To induce mitochondrial targeting of BAX, the activator BH3s bind to the α1 helix or the α1/α6 trigger site of BAX, resulting in the exposure of the α1 helix and secondary disengagement of the α9 helix that inserts into the MOM [24,32,33].", "Activator BH3s remain associated with the N-terminally exposed BAX through the canonical dimerization groove to drive the exposure of the BH3 domain and ensuing homo-dimerization of BAX [16,18,24].", "Binding of activator BH3s to the canonical dimerization groove of BAK also induces the exposure of the α1 helix and the BH3 domain [9••,17,19,20,22,34•].", "X-ray crystallography has shown the unfolding of BAX or BAK into an N-terminal ‘core’ (α2–α5) and a C-terminal ‘latch’ (α6–α8) upon activation by BH3 peptides [15,16,20], which may help eject activator BH3s.", "Whether this occurs in the MOM remains to be determined.", "The symmetric homo-dimers of BAX or BAK further assemble into homo-oligomers through either the α6/α6 or α3/α5 interface [35,36].", "Deciphering how the homo-oligomers of BAX or BAK permeabilize the MOM is currently an area of intense investigation.", "BAX or BAK homo-oligomers have been proposed to form either proteinaceous or lipidic pores in the MOM [37–39], and visualization of the BAX oligomers in the MOM recently became feasible through super-resolution imaging [40,41].", "Anti-apoptotic BCL-2, BCL-XL, and MCL-1 sequester activator BH3s to prevent the initiation of BAX and BAK activation [8,13], providing frontline protection (Figures 2A and 3).", "As the second line of defense, the anti-apoptotic BCL-2 members can also sequester ‘BH3-exposed’ BAX and BAK monomers to prevent the homo-oligomerization of BAX and BAK [9••,15] (Figures 2D and 3).", "The interaction between activator BH3s and anti-apoptotic BCL-2 members confers mutual inhibition because it not only prevents activator BH3s from activating BAX or BAK but also restrain the anti-apoptotics from sequestering ‘BH3-exposed’ BAX or BAK monomers.", "BID, BIM, and PUMA can prevent BCL-2, BCL-XL, and MCL-1 from sequestering BAX and BAK whereas NOXA can only inhibit MCL-1.", "This difference may contribute to the lower death-inducing activity of NOXA in comparison with BID, BIM, and PUMA.", "Consequently, Noxa deficiency only confers resistance to apoptosis in tissues or cell types that highly express NOXA, such as mouse embryonic fibroblasts (MEFs) and the small intestines [9••].", "Notably, BCL-XL is superior to BCL-2 and MCL-1 in preventing apoptosis due to its dual inhibition of BAX and BAK and higher protein stability [9••,42••].", "BCL-2 can only inhibit BAX but not BAK [9••,42••] whereas MCL-1 is prone to degradation upon death signals [43].", "The ability of BCL-2, BCL-XL, and MCL-1 to sequester tBID/BIM/PUMA is further modulated by ‘inactivator’ BH3s through high-affinity, competitive binding (Figures 3 and 4).", "Specifically, BAD, BMF, BIK, and HRK (DP5) displace sequestered BID/BIM/PUMA from BCL-2/BCL-XL and thereby activate BAX/BAK indirectly [9••,13].", "NOXA is unique among all BH3s in that it can prevent MCL-1 from sequestering BID, BIM, and PUMA due to its high binding affinity to MCL-1 [13].", "Hence, NOXA is both an activator and inactivator BH3.", "Notably, deficiency of Bid, Bim, Puma, and Noxa abrogates apoptosis triggered by overexpression of BAD, BMF, BIK or HRK [9••], supporting the BH3 hierarchy in which activator BH3s function downstream of inactivator BH3s.", "Hence, the observation that BH3 peptides of some inactivator BH3s can induce BAX-dependent or BAK-dependent liposome permeabilization is a purely in vitro phenomenon [44].", "The first unequivocal evidence of BH3-independent activation of BAX and BAK was not revealed until the generation of Bid−/−Bim−/−Puma−/−Noxa−/− QKO mice [9••].", "Autoactivation of BAX and BAK can occur in QKO cells when BCL-2, BCL-XL, and MCL-1 are simultaneously decreased upon DNA damage or silenced by siRNA, albeit with slower kinetics compared to BH3-mediated activation [9••].", "In fact, silencing both BCL-XL and MCL-1 is sufficient to trigger BAK autoactivation due to the inability of BCL-2 to bind BAK.", "Similar findings were shown in cells deficient for eight canonical BH3s created by genome editing [45••].", "The MOM appears to provide an important platform for the autoactivation of BAX and BAK, which is consistent with the ‘embedded together’ model that emphasizes the influence of the membrane milieu on BCL-2 family interactions [46].", "However, autoactivation of BAX and BAK appears less efficient in part because only a small fraction of BAX and BAK expose their BH3 domain, which is sequestered by anti-apoptotic BCL-2 members in viable cells.", "Liberation of the small fraction, ‘BH3-exposed’ BAX or BAK monomers from the anti-apoptotic BCL-2 members is sufficient to induce a ‘feed-forward’ amplification loop for the initiation of mitochondrial apoptosis.", "Therefore, activator BH3s function as catalysts for BAX and BAK activation by inducing the BH3 exposure of BAX and BAK while simultaneously restraining anti-apoptotic BCL-2 members.", "The presence of heterodimers between multidomain anti-apoptotic and pro-apoptotic members in QKO cells suggests that exposure of the BH3 domain in BAX and BAK can be generated independently of activator BH3s.", "Potential mechanisms include protein misfolding, post-translational modifications, oxidative stress, and physical stress such as heat or changes in intracellular pH [23].", "It is possible that these heterodimers also have non-apoptotic functions in the maintenance of cellular homeostasis, such as calcium homeostasis and mitochondrial fission and fusion [47,48].", "Retrotranslocation of BAX from the mitochondria to the cytosol mediated by BCL-XL appears to offer a means to reduce the BAX/BCL-XL heterodimers in the MOM [49].", "Over the years, the mouse genetic studies of BCL-2 family proteins have provided the ultimate validations for in vitro mechanistic studies.", "Consistent with the higher and broader expression of Bim than other BH3s as well as its activator BH3 activity, Bim−/− mice display the most severe phenotypes compared to other single BH3 KOs, developing lymphoid hyperplasia and fatal autoimmune diseases [50].", "Puma deficiency exacerbates the lymphoid hyperplasia and apoptotic defects of Bim KO mice [51], and mice lacking Bid, Bim, and Puma display even more severe defects and recapitulate the developmental defects of Bax−/−Bak−/− mice, including perinatal embryonic lethality, persistent interdigital webs and imperfortate vagina [6,52].", "Triple deficiency of Bid, Bim, and Puma also completely abrogates BAX/BAK-dependent apoptosis in cerebellar granule neurons [52].", "Due to the unique high expression of NOXA in MEFs, Bid−/−Bim−/−Puma−/−Noxa−/− but not Bid−/−Bim−/−Puma−/− MEFs are as resistant as Bax−/−Bak−/− MEFs to apoptosis triggered by ER stress and deprivation of growth factors or nutrients [9••].", "However, genotoxic stress can induce autoactivation of BAX/BAK through downregulation of BCL-2, BCL-XL, and MCL-1 in Bid−/−Bim−/−Puma−/−Noxa−/− MEFs [9••].", "Interestingly, DNA damage-induced downregulation of BCL-2 and BCL-XL is not observed in T-cells or the small intestine.", "Consequently, quadruple deficiency of Bid, Bim, Puma, and Noxa provides comparable protection as double deficiency of Bax and Bak against irradiation-induced apoptosis in the small intestine [9••].", "Consistent with the low expression of NOXA in lymphocytes and the absence of BAX/BAK activation detected in Bid−/−Bim−/−Puma−/− T-cells [52], Bid−/−Bim−/−Puma−/− T-cells are as resistant as Bid−/−Bim−/−Puma−/−Noxa−/− T-cells to various apoptotic signals [9••].", "However, double deficiency of Bax and Bak incurs more severe embryonic lethality than quadruple deficiency of Bid, Bim, Puma, and Noxa [6,9••], likely reflecting the presence of BAX/BAK autoactivation in certain tissues in response to developmental cues.", "Alternatively, non-apoptotic functions of BAX and BAK, such as regulation of mitochondrial fission/fusion or ER calcium homeostasis [47,48], may account for the more severe embryonic lethality of Bax−/−Bak−/− mice.", "The findings that Bim deficiency prevents the development of polycystic kidney disease and loss of melanocytes in Bcl-2−/− mice [53] support the concept that sequestration of BIM by BCL-2 prevents apoptosis.", "In addition, mice harboring the death-competent BakQ75L mutation that abrogates the BCL-XL/BAK but not MCL-1/BAK or BAK/BAK interaction display reduced T-cell and platelet survival and increased sensitivity to various apoptotic stimuli [54••], providing in vivo evidence that inhibition of BAK by BCL-XL contributes to apoptosis regulation.", "Overall, the mouse genetic studies substantiate the interconnected hierarchical regulation of apoptosis by the BCL-2 family.", "To abrogate apoptotic checkpoints, cancer cells often overexpress anti-apoptotic BCL-2 family proteins through genetic mutations such as chromosomal translocations involving BCL-2 or amplification of BCL-XL and MCL-1 [4,55].", "Counterintuitively, cancer cells also commonly express higher levels of BIM and PUMA that are transcriptionally activated by E2F1 upon malignant transformation [56] and sequestered by anti-apoptotic BCL-2 members as inert complexes.", "Hence, many cancer cells are likely ‘primed’ to undergo apoptosis upon the administration of BAD and NOXA mimetics that displace BIM/PUMA from BCL-2/BCL-XL and MCL-1, respectively, to activate the BAX/BAK apoptotic gateway (Figure 4).", "Structure-based screening efforts targeting the hydrophobic dimerization groove of BCL-XL have led to the development of the first specific small molecule inhibitor of the BCL-2 family, ABT-737 [57].", "ABT-737 and its orally bioavailable analog ABT-263 (navitoclax) function like BAD mimetics that bind and inhibit BCL-2, BCL-XL, and BCL-W, but not MCL-1 or A1 [57,58].", "Although navitoclax shows promising clinical activity, it induces a dose-dependent rapid thrombocytopenia as an on-target result of BCL-XL inhibition.", "This spurred the development of ABT-199 (venetoclax or GDC-0199), a platelet-sparing, selective BCL-2 inhibitor [59].", "Venetoclax has exhibited remarkable therapeutic efficacy for relapsed/refractory chronic lymphocytic leukemia (CLL) [60••], resulting in its approval by the FDA for the treatment of CLL patients with 17p deletion.", "Similar to inactivator BH3s, ABT-263 activates BAX/BAK indirectly through the displacement of activator BH3s from BCL-2 and BCL-XL (Figure 4).", "Hence, low expression of activator BH3s in cancers, particularly BIM, confers resistance to ABT-263 [42••].", "Another therapeutic limitation of ABT-263 is its inability to disrupt the BCL-XL/BAK interaction such that overexpression of BCL-XL confers resistance to ABT-263 [42••].", "Selective inhibitors of BCL-XL (A-1331852) or MCL-1 (S63845) with robust preclinical activity have also been generated [61,62•].", "The current progress of development of non-canonical inhibitors of anti-apoptotic BCL-2 members, along with emerging approaches to target pro-apoptotic BAX and BAK, are discussed in detail elsewhere [63].", "With BH3 mimetics entering the clinic, one major challenge is how to identify patients who will respond to a specific BCL-2 family inhibitor.", "Differential addiction of cancer cells to anti-apoptotic BCL-2, BCL-XL, or MCL-1 was reported in a panel of small cell lung cancer (SCLC) cell lines and found to correlate with the respective protein expression ratio [42••].", "If a given cell predominantly expresses a specific anti-apoptotic BCL-2 member, it will be addicted to that specific anti-apoptotic BCL-2 member for survival.", "BH3 profiling is a powerful tool to assess the baseline ‘mitochondrial priming’ or apoptotic sensitivity of cancer cells to cancer therapeutics [64], whereas dynamic BH3 profiling can be used to measure the changes in apoptotic priming in response to therapeutic agents [65•].", "However, both predict the overall apoptotic sensitivity rather than the addiction to individual anti-apoptotic BCL-2 members.", "The development of protein expression-based biomarkers to predict the differential addiction of human tumors to individual anti-apoptotic BCL-2 members will guide the future practice of precision cancer medicine targeting the BCL-2 family.", "The applications of BH3 mimetics as therapeutics extend beyond cancer treatment.", "ABT-263 was identified as a potent senolytic agent that selectively eliminates senescent cells [66•], potentially expanding its utility to treating age-related pathologies.", "Given that some BCL-2 family proteins can regulate autophagy, mitophagy, mitochondrial metabolism, mitochondrial dynamics, calcium homeostasis, and peroxisomal membrane permeability [47,48,67,68•,69], BH3 mimetics may provide useful tools for manipulating these non-apoptotic processes for future studies and therapeutic interventions.", "For example, stapled peptides modeled after the phospho-BAD BH3 helix can enhance insulin secretion and β cell survival, and improve functional β cell mass in diabetes due to activation of glucokinase by phosphorylation of the BAD BH3 domain at Ser155 [70].", "Three decades after the term ‘apoptosis or programmed cell death’ first captured the world's attention, intense research efforts encompassing molecular biology, biochemistry, structural biology, and genetically engineered mouse models have unveiled an intricately wired, interacting, regulatory network centered on the BCL-2 family proteins that adjudicate cell survival or death decisions.", "Importantly, this comprehensive knowledge has not only satiated humanity's curiosity for the unknown but also laid the foundations and provided the much needed roadmaps for the continuous development and mechanism-based application of small-molecule BH3 mimetics in cancer therapy.", "In the next 10 years, we envision that many long unresolved questions will be answered with new pharmacological tools and research technologies, yet novel opportunities will arise to challenge existing and inspire new cell death researchers.", "The authors declare no conflict of interest.", "Papers of particular interest, published within the period of review, have been highlighted as:", "• of special interest", "•• of outstanding interest" ]
[ "BH3s initiate apoptosis by activating BAX/BAK or inactivating anti-death BCL-2s.", "BID, BIM, PUMA, and NOXA directly induce stepwise bimodal activation of BAX and BAK.", "BH3-independent autoactivation of BAX/BAK occurs when anti-death BCL-2s are decreased.", "Anti-death BCL-2 members sequester BH3s or BH3-exposed BAX/BAK to inhibit apoptosis.", "BCL-2 family interactions dictate the efficacy of BH3 mimetics in cancer therapy." ]
Intelligence in youth and mental health at age 50
Background Few cognitive epidemiology studies on mental health have focused on the links between pre-morbid intelligence and self-reports of common mental disorders, such as depression, sleep difficulties, and mental health status. The current study examines these associations in 50-year-old adults. Methods The study uses data from the 5793 participants in the National Longitudinal Survey of Youth 1979 cohort (NLSY-79) who responded to questions on mental health at age 50 and had IQ measured with the Armed Forces Qualification Test (AFQT) when they were aged between 15 and 23 years in 1980. Mental health outcomes were: life-time diagnosis of depression; the mental component score of the 12-item short-form Health Survey (SF-12); the 7-item Center for Epidemiological Studies Depression Scale (CES-D); and a summary measure of sleep difficulty. Results & conclusion Higher intelligence in youth is associated with a reduced risk of self-reported mental health problems at age 50, with age-at-first-interview and sex adjusted Bs as follows: CES-depression (B = − 0.16, C.I. − 0.19 to − 0.12, p < 0.001), sleep difficulties (B = − 0.11, C.I. − 0.13 to − 0.08, p < 0.001), and SF-12 mental health status (OR = 0.78, C.I. 0.72 to 0.85, p < 0.001; r = − 0.03 p = 0.075). Conversely, intelligence in youth is linked with an increased risk of receiving a diagnosis of depression by the age of 50 (OR 1.11, C.I. 1.01 to 1.22, p = 0.024; r = 0.03, p = 0.109). No sex differences were observed in the associations. Adjusting for adult SES accounted for most of the association between IQ and the mental health outcomes, except for having reported a diagnosis of depression, in which case adjusting for adult SES led to an increase in the size of the positive association (OR = 1.32, C.I. 1.16 to 1.51, p < 0.001).
[ "ARTS", "PSYC" ]
[ "AFQT", "Age 50", "Cognitive epidemiology", "Depression", "Intelligence", "Mental health", "NLSY-79", "SES", "Sleep difficulties" ]
[ "1201", "3204", "3205" ]
[ "Approximately 25% of American adults are affected by mental health problems, with anxiety disorders and depression being the most common problems (American Psychiatric Association, 2015).", "In 2012, both anxiety and depression were in the top 5 leading causes of disability globally (Mathers, Fat, & Boerma, 2008; Whiteford et al., 2013).", "In view of this, it is of public health interest to discover the factors that increase the risk of developing these disorders across the life course.", "There is evidence in the literature of a link between intelligence test scores in youth and the risk of mental health difficulties in adulthood.", "For example, a lower IQ in childhood is associated with increased risk of schizophrenia (David, Malmberg, Brandt, Allebeck, & Lewis, 1997; Osler, Lawlor, & Nordentoft, 2007; Gunnell, Harrison, Rasmussen, Fouskakis, & Tynelius, 2002; Zammit et al., 2004; Dickson, Laurens, Cullen, & Hodgins, 2012), post-traumatic stress disorder (PTSD) (Koenen, Moffitt, Poulton, Martin, & Caspi, 2007; Kremen et al., 2007; Gale, Deary et al., 2008; Gale, Batty, Tynelius, Deary, & Rasmussen, 2010) and depression (Zammit et al., 2004; Gale, Deary et al., 2008).", "Previous research has found that a SD increase of intelligence in youth tends to be associated with a 13% to 43% reduced risk of the above-mentioned mental health difficulties in adulthood (Gale, Deary et al., 2008; Gale et al., 2010; Koenen et al., 2007; Osler et al., 2007).", "When these studies adjusted for indicators of socio-economic status (i.e. parental occupation or education, or own education or income) the effect sizes in many cases were somewhat reduced; however, the majority of effects remained significant.", "Much of this previous work was based on records of hospital admissions for mental illnesses.", "However, around 40% of adults in the U.S with a mental illness may not receive treatment (American Psychiatric Association, 2015).", "Therefore, it is important to test if this relationship between intelligence and mental disorders holds for the common, less severe cases of mental illness.", "Some work in the cognitive epidemiology of mental health has focused on sub-clinical mental health problems based on self-reported measures.", "In two British cohorts, the 1958 National Child Development Survey and the 1970 British Cohort Study, IQ in childhood was inversely associated with risk of psychological distress in individuals in their early 30's.", "In these two cohorts, Childhood IQ and scores on a the Malaise depression scale correlated − 0.11 and − 0.18, respectively (Gale, Hatch, Batty & Deary, 2008).", "The current study aims to expand these findings by testing for links between intelligence in youth and self-reports of mental health in an American cohort of adults around the age of 50.", "It is important to focus on middle-aged adults, because much of the previous work has looked at mental health problems in younger adults under the age of 40.", "Moreover, it is not uncommon for someone aged 45–54 to have a mental health problem (Kessler et al., 2009); for example, approximately 14% of British men and 23% of women in this age group had a common mental disorder in 2007, with similar rates for those aged 16–44 (McManus, Meltzer, Brugha, Bebbington, & Jenkins, 2009).", "There is some evidence that the relationship between intelligence in youth and mental health outcomes in adulthood might be different for men and women (Hatch et al., 2007).", "As the current study is based on a cohort that is made up of both men and women, a sex-by-IQ interaction will be included in the analysis to test for differences in the association between intelligence and mental health outcomes in men and women.", "The present study follows up on previous analyses of the NLSY-79 cohort at the age of 40 years (Der, Batty, & Deary, 2009).", "In that study, higher IQ in youth was significantly linked with self-reports of better overall mental health and lower levels of depression at age 40.", "There were also fewer self-reports of sleeping difficulties and lifetime cases of depression by the age of 40 in people with higher IQ at entry to the study.", "A standard deviation higher score in IQ was associated with a 23% to 12% reduced risk of having sleeping difficulties and depression, respectively, at age 40; it was also associated with approximately a fifth of a standard deviation lower depression score as well as a marginally better global mental health status score.", "There are a number of ways in which the study by Der, Batty and Deary (2009) could be improved.", "For example, it would be useful to test how intelligence relates to both mental health status at age 50 as well as the lifetime prevalence of depression diagnosis up to age 50.", "In addition to this, both childhood (parental) and adult SES are associated with mental health inequalities (Wilkinson & Marmot, 2003) and intelligence (Lubinski, 2009; McLoyd, 1998) but only childhood SES was adjusted for in the models reported by Der, Batty and Deary (2009).", "It is important to adjust for adulthood SES, in addition to childhood SES, as this could help to highlight a possible mediation pathway along which pre-morbid intelligence affects mental health outcomes.", "With regard to lower socio-economic status, it is important to mention that poverty and mental illness very likely interact dynamically, in a cycle: i.e., poverty could increase the risk of a mental illness, as explained by the social causation theory (Lund, 2012; Saraceno, Levav, & Kohn, 2005; Stansfeld, Clark, Rodgers, Caldwell, & Power, 2011).", "Mental illness might, in turn, increase the risk of poverty, as explained by the social drift theory (Dembling, Rovnyak, Mackey, & Blank, 2002; Rodgers & Mann, 1993; Lund, 2012; Saraceno et al., 2005; Stansfeld et al., 2011; Kessler, 2004).", "Whereas SES is often represented by a compound index, as it is in the present study, it is also potentially informative to examine individually the impact of its components – namely, education, income, and occupational status – as possible mediators of the relationship between childhood intelligence and later health.", "We also note the value of having a measure of intelligence that was taken in youth, because this increases the likelihood that the measure is truly pre-morbid; it helps to avoid the possibility that poor mental health in youth confounds the association between lower IQ and poor mental health later in adulthood.", "The present study also tests for associations between intelligence in youth and sleep difficulties at age 50.", "Although sleep difficulties are not often included in studies on mental health, they were included in the present study primarily because sleep difficulties often co-occur with psychiatric disorders such as anxiety or depression (Bixler, Vgontzas, Lin, Vela-Bueno, & Kales, 2002; Szelenberger & Soldatos, 2005; Ford & Kamerow, 1989; Kales, Caldwell, Soldatos, Bixler, & Kales, 1983).", "Der et al. (2009) found that a SD increment in intelligence in youth was associated with a 23% reduction in the likelihood of reporting sleep difficulties at age 40.", "The current study will test this association at the age of 50 years.", "The present study is particularly interested in testing how intelligence in youth is associated with different self-reported mental health outcomes (mental health status, sleep difficulties, and levels of depression) at age 50, and with self-reports of a lifetime diagnosis of depression by the age of 50.", "The current study also tests the role played by childhood and adult SES, and the latter's three components (education, income, and occupation status), in the above-mentioned relationships.", "It is hypothesized that higher IQ in youth will be associated with better mental health across all outcomes.", "It is hypothesized that adult SES will have a greater attenuating effect on the relationship between intelligence and mental health outcomes than childhood SES because adult SES is thought to mediate some of the association between intelligence in youth and mental health outcomes in adulthood.", "Finally, the present study will also test for sex-based differences in intelligence-mental health associations.", "This study was based on data from the National Longitudinal Survey of Youth 1979 (NLSY-79).", "The initial sample was representative of non-institutionalized young people who lived in the United States.", "It was a random household sample and consisted of 12,686 individuals aged 14–21 years on 31st of December 1978.", "There were 6283 males (50%) and 6403 females (50%); 16% were Hispanic/Latino, 25% were Black, and 59% were non-Black & non-Hispanic; ethnic minorities were intentionally over-represented in order to obtain a large-enough sample size of these groups.", "The NLSY-79 survey collected information on a variety of topics such as health, education, achievement tests, employment, and attitudes.", "The initial interview for NLSY-79 took place in 1979 and respondents were re-interviewed annually until 1994 and biennially thereafter.", "The most recent data available derive from the 2012 wave.", "It had a 73.3% retention rate from the initial sample to the 2012 wave, which consisted of 7301 individuals (48% males).", "The respondents were between 47 and 56 years of age (Bureau of Labor Statistics, 2015).", "The 50 + health module was used in the present study.", "The data in this module were collected over three waves in 2008, 2010, and 2012.", "Respondents completed this module when they were approximately 50 years old (range 49–55).", "In total, 6893 (46%) of the initial NLSY-79 sample completed the 50 + health module (48% males).", "The three following measures appeared in both the 40 + health module and the 50 + health module: the 7-item Center for Epidemiological Studies Depression Scale (CES-D), the 12-item Short-Form Health Survey—mental health status (SF-12), and a question about sleep difficulty.", "The other question included in this study, which asked about a lifetime diagnosis of depression, was novel to the 50 + health module (NLSY-79).", "The data were downloaded from the National Longitudinal Study (NLS) Web Investigator site on 15/11/2014 (NLSY-79).", "The measure of intelligence used in the NLSY-79 was the Armed Forces Qualification Test (AFQT), 1989 re-normed version.", "The participants in the NLSY-79 completed the AFQT in 1980 when they were aged between 15 and 23 years.", "This score is derived from four of the 10 subtests in the Armed Services Vocational Aptitude Battery (ASVAB).", "The subtests assessed the following: arithmetic reasoning (AR), mathematics knowledge (MK), word knowledge (WK), and paragraph comprehension (PC).", "The ASVAB has been found to be a reliable and valid measure of intelligence (Welsh, Kucinkas, & Curran, 1990).", "It has been found to be a predictor of academic and job performance as well as a predictor of first-term attrition rates from a military job and self-paced school completion time (Palmer, Hartke, Ree, Welsh & Valentine, 1988; Welsh et al., 1990).", "To be consistent with the study done on the 40 + health module (Der et al., 2009), the AFQT variable used in the present study was downloaded from The Bell Curve Page (Herrnstein & Murray, 1994).", "This variable was scored as a percentile, and was then z-scored.", "The current study examines four mental health outcomes from the 50 + health module, as follows.", "The first outcome was a self-report of ever having had a diagnosis of depression.", "This was elicited by the question, “Has a doctor ever told you that you have depression” and it could receive a Yes/No response.", "The three further mental health outcomes were summary measures for current mental health status, levels of depression, and sleep difficulties, as follows.", "The measure of mental health status was taken from the 12-item short-form Health Survey (SF-12) (Ware, Kosinski, & Keller, 1996), which has strong validity and reliability (Resnick & Parker, 2001).", "This is a summary measure of the 6 questions in the SF-12 that evaluate the overall levels of mental health status at the time of testing.", "Scores can have a minimum value of 11.33 and a maximum value of 70.33; a higher score on this variable indicates better mental health (Ware et al., 1996).", "This variable was included as a dichotomous outcome because, as a continuous measure, this variable had a non-normal distribution that could not be effectively transformed to a normal distribution.", "This item was dichotomized at 47.74, which was the twentieth percentile of values.", "A score that fell below 47.74 was categorized as poor mental health and was given a value of 1.", "A score above this point was categorized as good mental health and received a value of 0.", "In a sensitivity analysis the SF-12 was used as a continuous measure.", "For this, it was reversed scored to have a minimum score of 1 and a maximum score of 60, with a higher score indicating poorer mental health status.", "This variable was reversed scored so that its interpretation was consistent with the other outcomes and so that a higher score indicated poorer mental health status.", "This variable also received a square root transformation to help reduce the affect of the non-normal distribution, and was z-transformed to zero mean and unit SD.", "The summary measure for depression was the 7-item Center for Epidemiological Studies Depression Scale (CES-D) (Radloff, 1977; Levine, 2013; Ross & Mirowsky, 1989).", "Each item is scored 0 to 3, and the total summed scores, therefore, potentially range from 0 to 21, with a higher score indicating more depression.", "The CES-D scale has strong reliability and validity (Levine, 2013; Radloff, 1977).", "The sleep difficulty summary measure was made up of four questions that were taken from the Sleep Habits Question that was used in the Sleep Heart Health Study (SHHS) (Redline et al., 1998).", "This scale was adapted from a scale used in the Tuscan Epidemiologic Study of Obstructive Airway Disease (Klink & Quan, 1987; Quan et al., 1997; Bixler & Kales, 1979).", "Each question asked about frequency of different sleep problems.", "For example, one question asked the following, “How often do you…have trouble falling asleep?”.", "Each item is given a score between 1 and 4, with a higher score indicating less sleep difficulty.", "Responses took the form “Almost always (4 + times per week)” = score 1; “Often (2–3 times per week)” = score 2; etc.", "This variable was reversed scored and the scale went from 1 to 13; this was done so that a higher score indicated more sleep problems.", "Potential confounding and mediating variables were as follows: age at initial interview when recruited to NLSY-79, adult age when the health at 50 module was completed, sex, childhood SES, and adult SES (along with its three constituent parts of education, income, and occupational status).", "We now describe how these variables were created.", "Childhood SES was a z-transformed composite variable of parental income, education, and occupation status, which was derived by Herrnstein and Murray (1994).", "Higher scores on the childhood SES variable indicate a more advantaged socio-economic position (Herrnstein & Murray, 1994).", "The adult SES variable was also a derived variable.", "The method used to make this variable was similar to that used by Herrnstein and Murray (1994) to derive childhood-SES.", "In other words, adult SES is an average of z-scored adult educational attainment, income, and occupation status.", "For adult educational attainment the variable ‘Highest Grade Completed’, as of 2012 was used and was z-transformed.", "The variable for income was ‘Total Net Family Income In Past Year’.", "Income received a square root transformation prior to a z-transformation.", "The third component of the adult SES variable was occupation status.", "This was coded according to the US 3-digit, 2000 census code; this is explained in NLSY-79 Attachment 1: Census Industrial & Occupational Classification (Bureau of Labor Statistics, 2015).", "This was then used to derive an Occupational status hierarchy.", "Herrnstein and Murray (1994) used the 1960 Duncan socio-economic index (SEI) scale but, because many changes had been made to the census occupation classification system between 1980 and 1990 (Frederick, 2010), an updated version of the 1960 Duncan SEI scale was used here.", "This scale was developed by Hauser and Warren (1996) and was constructed in a similar way to the Duncan SEI (Frederick, 2010).", "Occupation status received a log transformation and was then z-scored.", "Higher scores on the composite adult SES variable and on each of its constituent components indicate more advantaged socio-economic position.", "Two sets of analyses were conducted.", "The primary set of analyses was performed using a complete case analysis.", "The other set of analyses was a multiple imputations analysis.", "Each set of analyses comprised a series of hierarchical regression analyses.", "Logistic regressions were conducted for diagnosed depression and the dichotomized SF-12 mental health status score.", "Generalized linear models were applied for sleep difficulty and CES-Depression, because these outcomes were both heavily skewed to the right.", "Both models assumed a gamma distribution and used a log link.", "For each mental health outcome, six separate models were analyzed.", "The baseline model adjusted for age at NLSY-79 baseline (when AFQT was tested) and sex.", "Model 2 additionally adjusted for childhood SES.", "Model 3 added composite adult SES to the variables included in Model 2.", "Models 4, 5, and 6 were the same as Model 3 but each replaced the composite adult SES with one of its constituents: income, education, or occupational status.", "A sex by IQ interaction was tested in all models.", "All analyses were conducted in STATA 13.0.", "A second set of analyses was performed using multiple imputations to the data.", "The imputed analysis was selected due to the relatively high proportion of missing data for the adult SES variable.", "Twenty-eight imputations were created using the multivariate normal regression method for arbitrary patterns of missing data.", "The variables imputed were income, education, and occupation status, because these had the highest rates of missing data, with 21%, 6% and 14% missing, respectively.", "Twenty-eight imputations were selected, because 28% of the composite-Adult SES values were missing.", "Imputations were conducted on only those who were present for the 50 + health module.", "As an additional check of the effect of intelligence in youth on sleep difficulty and CES-Depression, logistic sensitivity models were run on these outcomes.", "To run a logistic model, both scales had to be dichotomized.", "For CES-Depression, the accepted cut-off point for depression of > 8 was used to create a dichotomous variable (Levine, 2013).", "As the sleep difficulty scale used in the present study was a subset of a larger scale and no standard cutoff point was known, the top of the first quartile was used as a cut-off point, and a score < 11 was used to indicate that a sleep problem was present.", "A linear regression was also conducted with the continuous SF-12 mental (health) measure as part of the sensitivity analysis.", "An additional set of complete case analyses was conducted which was weighted for ethnicity to correct for the over representation of ethnic minorities in the NLSY-79.", "The non-weighted complete case analyses are the primary focus of the results section.", "The results from the imputed analysis are briefly covered at the end of the results, and are covered in more detail as Supplementary material.", "The present analysis was based upon a sample of 5793 participants who responded to the 50 + health module.", "There were 1619 missing values for adult SES, 1239 were missing for income, 818 for occupational status, and 339 for education.", "For IQ, 293 were missing.", "The complete cases analysis was conducted on those 4015 respondents who had complete data for IQ, age, sex, educational attainment, occupational status, and adult income.", "The numbers used for each analysis vary slightly from this due to small numbers (< 3%) of missing data for the health outcomes.", "Table 1 shows differences in selected characteristics between those who did and did not complete the 50 + health module.", "Those who completed it were significantly more likely to be female, had slightly lower IQ scores (− 0.04 of a SD) and were significantly older (by a mean of 1.29 years) than those who did not complete the 50 + health module.", "There were no significant differences between the two groups in income, education, occupation status, or adult SES.", "Among the 4015 participants who completed the 50 + health module included in the complete case analysis, there was a significant and positive association between IQ and childhood SES (r =.56, p <.001), adult SES (r =.64, p <.001), and the sub-components of Adult SES, which includes income (r =.49, p <.001), education (r =.60, p <.001), and occupational status (r =.48, p <.001) (Table A.1 Appendix A).", "Pairwise correlations between AFQT and CES-depression and SF-12 mental health status were run to estimate the strength of the true relationship, with adjustment for the reliability of these measures.", "The parallel forms reliability coefficients of AFQT was 0.92 (Welsh et al., 1990), and the internal consistency alpha coefficient of SF-12 mental health status and CES-depression was 0.80 (Resnick & Parker, 2001) and 0.82 (Levine, 2013), respectively.", "The correlations between AFQT and SF-12 mental health status increased from −.028 to −.033 and the correlation between AFQT and CES-depression increased from −.139 to −.160 (Table A.5).", "Table 2 displays the descriptive statistics for the four mental health outcome.", "variables.", "The mean for CES-depression, SF-12 mental (health) status, and sleep difficulties was significantly higher for women than for men.", "The standard deviation was greater in all three outcomes for women than it was for men.", "A greater percentage of women (22%) had a lifetime diagnosis of depression than men (10%).", "Table 3 displays the result of the generalized linear regression analysis of CES-depression and sleep difficulty regressed on IQ.", "When age at first interview and sex were adjusted for in the initial model, higher IQ was significantly associated with lower scores for depression and sleep difficulties.", "For CES-Depression a one standard deviation higher score in IQ was associated with B1 = − 0.16 (95% C.I. − 0.19 to − 0.12, p <.001).", "For sleep difficulty a one standard deviation higher score in IQ was associated with B = − 0.11 (95% C.I. − 0.13 to − 0.08), p <.001.", "When childhood SES was included there was only slight attenuation in the effect size for the relationship between IQ and sleep difficulty and CES-Depression, and both associations remained significant.", "Including adult age and adult SES in Model 3 had a substantial attenuating effect on associations between IQ and CES-Depression and Sleep Difficulty, which became non-significant.", "The sex by IQ interaction was not significant for any of these continuous mental health outcomes.", "For example, the sex by IQ interaction in the initial model for sleep difficulty and CES-depression had B = 0.01 (p =.68), and B = 0.03 (p =.30), respectively.", "Table 3 also displays the result of the logistic regression for the relationship between IQ and self-reported diagnosis of depression and the dichotomized SF-12 mental health status variable.", "In the baseline model, IQ was significantly and negatively associated with SF-12 mental health status and was nominally significantly and positively associated with a doctor diagnosis of depression.", "A one standard deviation higher score in IQ was associated with reduced odds of having poor mental health status, with an odds ratio of 0.78 (95% C.I. 0.72 to 0.85, r = −.03).", "A one standard deviation higher score in IQ was associated with increased odds of lifetime diagnosis of depression, with an odds ratio of 1.11 (95% C.I. 1.01 to 1.22, r =.03).", "Including childhood SES led to the attenuation of the effect size for a doctor diagnosis of depression to non-significant levels, but it did not attenuate the effect size for SF-12.", "When adult SES was included, the association between IQ and SF-12 mental health status was almost wholly attenuated and was no longer significant.", "Including adult SES led to an increase in effect size for doctor diagnosis of depression and resulted in an odds ratio of 1.32 (95% C.I. 1.16 to 1.51).", "The sex by IQ interaction was not significant for either of these dichotomized mental health outcomes.", "The sex by IQ interaction in the initial model for SF-12 and diagnosis of depression was OR 1.07 (p =.36) and OR 1.10 (p =.29), respectively.", "The results of the regression analysis that adjusted for the three sub-components of adult SES (income, education, and occupation status) for all of the mental health outcomes in the complete case analyses are presented in Table A.2 in the Appendix.", "This table shows how IQ in youth is associated with the four different mental health outcomes when income, education, and occupation status are adjusted for separately.", "Adjusting for the composite adult SES variable led to greater attenuation of the associations between IQ and CES-depression, sleep difficulty, and SF-12 mental health status than adjusting for income, education, and occupation status individually.", "Of the three adult SES components, adjusting for income led to more attenuation of the associations between IQ and CES-depression, sleep difficulty, and SF-12 mental health status than did education and occupation status.", "Looking at the self-report diagnosis of depression, adjusting for the composite adult SES led to more amplification of the positive association with IQ than income, education, or occupation status did individually.", "Much of the amplification effect of adjusting for adult SES was also found when adjusting for income.", "Fig. 1 displays the results of the four mental health outcomes by IQ quintile for the two models that adjust for age at first interview and sex, and additionally for adult SES, in the complete case analyses.", "Table 4 shows descriptive statistics of AFQT score broken into quintiles.", "The complete case analyses were repeated, weighted for ethnicity (Table A.6) and overall the results were similar to the original complete case analysis.", "The most notable deviation in results was that the association between intelligence in youth and a lifetime diagnosis of depression was not significant in the weighted analysis, in the baseline model.", "However, in the other five models that analyzed the association between intelligence in youth and a lifetime diagnosis of depression, the effect sizes and significance levels in the weighted analysis were similar to those in the complete case analyses.", "One other notably different result was that the strength of the association between intelligence and sleep difficulties increased and became significant after adjusting for adult SES.", "Weighting for race and ethnicity, at most, only marginally influenced all of the other results across the four mental health outcomes.", "The results for the complete cases analysis were also compared to the results from the multiply imputed analysis (Table A.3 on the appendix).", "The overall patterns were similar between the imputed and complete case analysis across the four mental health outcomes.", "A detailed description of these results can be found in the supplementary materials.", "The results of the logistic and linear sensitivity analyses of the relationship between intelligence in youth and CSE-depression, sleep difficulty, and SF-12 mental health status were similar to those found in the complete case analysis.", "These results are described in the supplementary materials.", "The present study examined the relationship between IQ in youth and four mental health outcomes taken at age 50.", "It also examined how associations changed after adjusting for childhood and adult SES.", "The results suggest that pre-morbid intelligence was significantly associated with mid-life mental health outcomes.", "In the baseline model that adjusted for age at IQ test and sex, a higher pre-morbid IQ was associated with less CES-depression, less sleep difficulty, and better SF-12 mental health status at age 50.", "In contrast, a higher pre-morbid intelligence was associated with higher rates of a lifetime diagnosis of depression.", "Adjusting for childhood SES had little attenuating effect.", "Adjusting for adult SES led to substantial attenuation of IQ's association with CES-depression, sleep difficulty, and SF-12 mental health, but it amplified the effect size of the association with a diagnosis of depression.", "No significant sex differences were found in the relationship between IQ in youth and any of the mental health outcomes, which is consistent with other studies (Gale, Batty, Cooper and Deary, 2009; Mikkelsen, Flensborg-Madsen, Eliasen, & Mortensen, 2014; Johnson, Corley, Starr, & Deary, 2011).", "The present study provides a novel contribution to our understanding of the association between intelligence in youth and adult mental-health.", "It is one of the few studies to report associations between intelligence and both a history of depression diagnosis and current self-reported mental health problems in both men and women in mid-life, around age 50 years.", "Higher pre-morbid intelligence was significantly associated with less depression, less sleep difficulty, and a better overall mental health status at age 50.", "These results were similar to those found at age 40 (Der et al., 2009) and they suggest that higher intelligence in youth, in both men and women, may have a protective effect on mental health into middle age.", "An intriguing aspect of the results is the contrast in the results between CES-depression scores and a lifetime diagnosis of depression.", "The results suggest that higher intelligence is linked with lower rates of depression at age 50 but with a higher lifetime diagnosis of depression.", "This suggests that those with higher intelligence in youth are at an increased risk of being diagnosed with depression by the age of 50.", "Two other studies reported a positive relationship between cognitive ability and lifetime cases of depression (Luciano et al., 2015; Cullen et al., 2015).", "A small number of studies have also found some evidence that suggests there may be a positive correlation between IQ and increased risk of bipolar disorder (Gale et al., 2013; Higier et al., 2014; Smith et al., 2015) and mania in adulthood (Koenen et al., 2009).", "However, the current findings on depression diagnosis do deviate from the general trend in the literature which tends to find higher intelligence is associated with less mental illness (Der et al., 2009; Gale, Hatch, et al., 2008; Martin et al., 2007; Weeks et al., 2014).", "One possible explanation for the positive association between intelligence and lifetime diagnosis of depression in the current study could be health literacy.", "Evidence suggests health literacy is strongly associated with general intelligence (Reeve & Basalik, 2014; Gottfredson, 2004; Mottus et al., 2014; Murray, Johnson, Wolf, & Deary, 2011; Wolf et al., 2012).", "Following from this, people with higher intelligence may also have higher mental health literacy.", "Those with higher intelligence might be more able to identify their symptoms of depression, which could motivate them to consult a doctor for diagnosis and advice; they might also be likely to have accurate reporting of such diagnoses in the health module (Beier & Ackerman, 2003; Gottfredson, 2004).", "Conversely, people with a lower IQ might not be aware of symptoms of depression and may therefore be less likely to become diagnosed with depression and therefore less likely to report a diagnosis of depression than people with higher levels of intelligence.", "Another possible explanation for the positive association between IQ and a reported diagnosis of depression, as observed in the current study, could relate to incentives for more intelligent people to seek out a diagnosis of depression in the United States.", "It is possible that more intelligent Americans are motivated to strategically get a diagnosis of depression if they experience symptoms of psychological distress, even if they are not actually depressed, because it would allow them to get insurance coverage for treatment, which they might think they would benefit from (APAPO, 2002).", "As intelligence is strongly correlated with social class, evidence for this may be found by referring to socio-economic class differences in mental health treatment.", "In the US the higher social classes are disproportionally represented among those receiving privately-funded mental health treatment (Hollingshead & Redlich, 2007).", "Higher social class individuals are also more likely to receive psychotherapy as their treatment, whereas lower class individuals are more likely to receive biological treatment (i.e. drugs or electro-shock therapy) for the same condition (Hollingshead & Redlich, 2007).", "This might highlight a deterrent for people from a lower social class to seek out a diagnosis of depression, as their treatment is likely to be more invasive.", "The less invasive psychotherapy treatment, on the other hand, might act as an incentive for people from a higher social class to seek out a diagnosis.", "This hypothesis could be used to guide future studies that seek to analyze the interplay between intelligence, social class, and lifetime diagnosis of depression.", "Childhood SES was found to explain at most a small part of the relationship between intelligence in youth and each of the mental health outcomes, which is similar to the results found in other studies (Der et al., 2009; Koenen et al., 2007; Gale, Hatch, et al., 2008; Martin et al., 2007; Weeks et al., 2014; Wrulich, Brunner, Schalke, Keller & Martin, 2014).", "This suggests that the relationship between intelligence in youth and mental health outcomes is not confounded by childhood SES.", "Adult SES accounted for much of the relationship between pre-morbid intelligence and CES-depression, sleep difficulty, and SF-12 mental health status at age 50.", "After adjusting for adult SES the betas for CES-depression and sleep difficulty were both reduced.", "The odds ratio for SF-12 mental health status was also reduced.", "None of these effects remained significant.", "The composite adult SES variable had a greater attenuating effect than income, education, and occupation status did on the association between IQ in youth and all three of these outcomes.", "Income had a greater attenuating effect than education and occupation status and therefore explained much, but not all of, the attenuation by adult SES.", "Other studies had similar findings.", "Wrulich et al. (2013) found similar results in their study.", "They found that the combination of adult SES and education partially mediated the effect of intelligence in youth on functional health and overall subjective health (as measured by a rating of their overall health status, their health status compared to peers, and their overall satisfaction with their health) in adulthood, reducing the standardized regression coefficients from beta = 0.18 and beta = 0.11 to beta = 0.07 and beta = 0.06, respectively.", "One possible explanation for this could be that stress is a well-established determinant of mental health problems.", "Individuals with a higher IQ are more likely to be in a higher social class and make more money, and therefore tend to experience less stress (Marmot, 2004; Sapolsky, 2005), as IQ and these indicators of SES are strongly correlated.", "Adjusting for adult SES led to an increase in effect size between pre-morbid intelligence and a lifetime diagnosis of depression.", "The odds ratios went from 1.11 in the age and sex adjusted model to 1.32.", "The composite adult SES variable had a greater strengthening effect than income, education, and occupation status did.", "Income had a greater strengthening effect than either education or occupation status; therefore, much of the impact of adult SES is explained by income.", "This suggests that income in adulthood may facilitate the impact of intelligence in youth on the risk of ever having a lifetime diagnosis of depression.", "This might indicate that people with high intelligence, but a low income, might be less likely to receive a diagnosis of depression.", "This may also be explained by the US health care system and the need for health insurance, whereby higher income might afford a greater likelihood of presenting to private health care and obtaining a diagnosis of depression.", "The current research has some strengths.", "The first is that this study utilises a measure of intelligence that is taken in youth, and has an almost 30-year gap between that and the mental health measures.", "Youth is an optimal time for cognitive measurement, as it is towards the end of most fluid cognitive maturation and prior to any expected age-associated cognitive decline.", "It reduces the likelihood of reverse causation, whereby mental health might affect IQ test performance.", "By adolescence, shared environmental influences on intelligence have almost disappeared and much of the observed differences in intelligence are due to genetic factors (Haworth et al., 2010).", "A second strength of this study is that it is based on a large and representative sample of adult Americans aged 50 years old.", "Middle age adults are underrepresented in mental health literature, including the mental health literature that looks at intelligence as a risk factor.", "Much of the work in this area has focused on mental health in youth and adults under the age of 40.", "This is important because mental health problems are known to be an issue at middle age (McManus et al., 2009).", "Another strength is that unlike other studies in the field that studied IQ's associations with mental health, the present study was based on a sample that includes women.", "A further strength of this study is that it included a question on lifetime diagnoses of depression.", "Regular self-report questions tend to have poorer agreement with medical records when the condition of interest has vaguely-defined diagnostic criteria than when it has clearly defined diagnostic criteria (Haapanen, Miilunpalo, Pasanen, Oja, & Vuori, 1997).", "Explicitly asking whether or not respondents have ever received a clinical diagnosis of depression, instead of just relying on retrospective self-report, reduces the risk of inaccurate recall and recall bias (Coughlin, 1990; Newman & Bland, 1998).", "This study also has some limitations.", "First, there are a high number of missing data for the adult SES variable.", "Because of this, the number of observations used in the complete cases analysis was reduced, though still large.", "The mental health measures in this study are self-reported.", "Such measures tend to have lower validity than clinically diagnosed conditions in medical records.", "However, the scales used in the study do have high reliability and validity (Resnick & Parker, 2001; Levine, 2013; Radloff, 1977; Bixler & Kale, 1979).", "An additional limitation is that this study does not include a measure of mental health that was taken at the same time as the measure of intelligence.", "This is not available in the current data set, but would have been useful to have as a covariate because it could have adjusted for the potential effect of poorer mental health earlier in life possibly influencing IQ test performance, and also leading to lower social status in adulthood.", "The current study also did not account for potential intervening variables that might mediate the relationship between intelligence in youth and mental health outcomes at age 50.", "Future research in this area may benefit from looking at how possible intervening variables, such as stressful life events (parental divorce, death in the family, or hospitalization etc.) or physical illness might influence the relationships observed in the present study.", "There is also the possibility of reporting bias due to differences in mental health literacy (Beier & Ackerman, 2003; Gottfredson, 2004), whereby those with higher intelligence might have better symptom recognition, better recall of conditions, and more accurate reporting of these in the health module.", "The net effect of such reporting bias would be to bias some results towards the null, i.e. our estimates of effect size here might be under-estimates.", "The findings of this study demonstrate that there is a relationship between intelligence in youth and mental health in middle age.", "The results suggest that higher intelligence in youth is linked with lower rates of self-reported mental health problems at age 50.", "Adjusting for adult SES seems to substantially attenuate the association between IQ and depression and sleep problems at age 50.", "The current study also found that higher intelligence is linked with higher rates of receiving a diagnosis of depression by 50 years of age.", "These findings may be due to an artifact and may be explained by those with lower intelligence not receiving a diagnosis of depression when they are depressed and/or not seeking a diagnosis due to fear of stigma.", "If this is the case, then this study provides evidence that there is a need to tailor mental health interventions for those with lower intelligence to help increase symptom recognition of depression and to help them overcome stigma surrounding depression.", "The current findings contribute to our understanding of how intelligence is related to mental health.", "Higher intelligence seems to help reduce the risk of mental health problems.", "These findings can be used to help inform mental health policy as well as mental health interventions that target mental illness in different sub-groups of the population." ]
[ "Links between IQ in youth and four mental health outcomes at age 50 were examined.", "Higher IQ was associated with a greater risk of a depression diagnosis (OR 1.11).", "Adjusting for adult SES amplified the diagnosis of depression coefficient (OR 1.32)", "Higher IQ was associated with a lower risk of the other three mental health outcomes." ]
Use of constrained focused waves to measure extreme loading of a taut moored floating wave energy converter
This paper concerns experimental measurements of the interaction of a taut moored floating body, representing a point absorbing wave energy converter in survivability mode, with extreme waves. The extreme waves are modelled in four ways. NewWave theory is first used to generate focused wave groups of varying steepness. Steepness is shown to have negligible effect on peak mooring loads, but causes significant differences in the resulting motion. The NewWave group is then constrained into both regular and irregular background wave trains so that the floating body has a load history caused by previous waves when interacting with the focused wave group. It is shown that an independent focused wave group is insufficient to properly model the extreme response of the floating body. However differences between the target and measured constrained time series due to non-linear wave-wave interaction limited the potential benefits of this approach. Finally the results from these tests are compared with measurements taken using irregular waves without any deterministic focused wave groups present. This comparison found cases where the floats response was greater than during any of the constrained NewWave tests, indicating that the assumption made that NewWave will generate the largest response was incorrect in this case.
[ "ENGI", "ENVI" ]
[ "Constrained NewWave", "Floating bodies", "Moorings", "NewWave", "Survivability", "Wave impact" ]
[ "2212", "2305" ]
[ "Floating wave energy converters must be designed to withstand the largest waves experienced during storms of magnitude equal to their design condition.", "This forms part of a wave energy device's survivability criterion, part of the dual requirements of any marine energy device: the ability to extract energy in small to moderate seas, while surviving more extreme conditions (Barstow et al., 2008 p. 52).", "Achieving an understanding of the response to these extreme waves is important.", "Large degrees of uncertainty in the expected loads often lead to the use of conservative assumptions which can negatively influence the commercial viability of a device (O'Neill et al., 2006).", "Most floating offshore wave energy converters are being designed to deploy in arrays.", "Spacing between individual devices within an array depends on many factors, including maximising power generation, provision of maintenance access and achieving an acceptable collision risk.", "For this last point it is important to be able to also predict the maximum expected displacement of devices during extreme events.", "Both experimental and numerical techniques are used to model a device's response to extreme waves.", "In both cases a deterministic focused wave group based on NewWave theory is often used to generate a time series of an extreme wave.", "NewWave theory, as described by Tromans et al. (1991), models the statistically most probable surface elevation shape associated with the occurrence of an extreme wave crest with a specified exceedance probability (Pinna and Cassidy, 2004).", "NewWave theory has the advantage that it generates an extreme event within a relatively short time series when compared to relying on randomly occurring extreme events in an irregular time series of the sea state in question.", "The short time series means, in a correctly designed experiment, that all important wave-structure interactions occur before any significant influence from wave reflections from basin walls occur.", "The wave group generated by NewWave theory propagates into calm water.", "This and again the relatively short time series involved, means that NewWave focused wave groups are well-suited for proving validation data for computationally expensive computational fluid dynamic (CFD) models.", "These advantages of using NewWave focused wave groups to measure extreme wave interactions have led to the approach being used in a wide number of applications.", "In the offshore environment extreme wave impacts on fixed cylinders relevant to a wide range of structures have been assessed both experimental and numerically using NewWave theory (Walker and Eatock Taylor, 2005; Ransley et al., 2013; Zang et al., 2010), while Stallard et al. (2009a) measured the forces on a vertical cylinder moving through a stationary fluid with a motion devised from NewWave theory.", "Rozario et al. (1993) successfully compared the loads predicted by NewWave on a North Sea oil platform with simulations of random seas.", "Westphalen et al. (2014) conducted Volume of Fluid and SPH CFD simulations of NewWave wave groups interacting with the Manchester Bobber wave energy device, comparing results to those from 70th scale experimental measurements (Stallard et al., 2009b; Weller et al., 2013).", "More recently NewWave theory has been used to investigate the impact of extreme waves in the coastal environment.", "Borthwick et al. (2006) measured wave kinematics of NewWaves impacting on a 1:20 beach plane, while Whittaker et al. (2017) measured wave runup of a plane beach.", "Focused wave groups such as those generated by NewWave theory are used to represent the design load case for fixed structures (Stallard et al., 2009b).", "Their use as design cases for dynamic structures and devices is questionable however.", "When dynamic response to waves is expected, the response to a specific extreme wave will depend not only on the load induced by the wave, but the load history caused by the previous wave train (Pinna and Cassidy, 2004).", "This dependency is not investigated when testing with a single focused wave group.", "The device or structure in question has a stationary initial state and only interacts with the deterministic wave crests and troughs that made up the initial part of the focused wave group before interacting with the extreme central crest.", "Constraining (or embedding) the NewWave within a random background sea state is an approach which allows the effect that a device or structures load history has on that device or structures response to a deterministic extreme event to be investigated.", "Introduced by Taylor et al. (1997), a Constrained NewWave (CNW) consists of a NewWave group which is constrained into an irregular background sea state with the same characteristic spectrum, such that the resulting time series is statistically indistinguishable from a randomly occurring wave train.", "The impact of the device's load history variation and the resulting distribution of the responses to the extreme wave can then be investigated by conducting multiple simulations or experiments with the focused wave group constrained into different random background time series.", "Various numerical studies have concluded that using the CNW technique is a viable alternative to conducting simulations with random irregular wave time series.", "For example Cassidy (1999) and Cassidy et al. (2001) demonstrated the use of CNW to determine the short-term extreme response statistics of a Jack-up structure, using 5 NewWaves each constrained into 200 random backgrounds.", "Their results were found to be comparable to those found from 100 3 h simulations of random seas.", "Pinna and Cassidy (2004) conducted similar simulations on a fixed monopod platform using 100 CNW cases while Enderami et al. (2010) modelled the extreme response of a Jacket offshore platform using 200 CNW, both with ABAQUS.", "Both compared results with simulations of 3 h long irregular sea states and found that the maximum response were of a similar magnitude.", "Bennett et al. (2012) report what they believed to be the first experimental use of CNW.", "They compared the use of an independent NewWave, CNW and an ‘optimised sea state’ to model the rigid body motions of a travelling ship in abnormal waves, where the optimised sea state requires an iterative adjustment of the phases in a random sea state so that a target extreme wave occurs.", "The three different approaches to generating ‘abnormal’ sea conditions were tested for three JONSWAP sea states with increasing significant wave height.", "Discrepancies between the target and generated wave were reported for the CNW, which was considered to be potentially due to the combination of two wave spectra.", "Göteman et al. (2015) constrained NewWave into regular waves when measuring the wave load on a point-absorbing wave energy device.", "The focused wave was constrained into different phase locations within regular waves with four different periods, resulting in 32 cases in total.", "A correlation between wave height and measured mooring force is reported.", "This paper presents a systematic study into the experimental use of independent NewWave and CNW in the investigation of a moored floating body's response to an extreme wave.", "Results from four sets of experiments are reported and compared.", "In the first series the interaction of the moored floating body with an independent NewWave focused wave group was measured, along with the interaction with three other focused groups of increasing steepness.", "In the second series 24 constrained NewWave cases were tested within two regular background waves.", "In the third series the NewWave was constrained into 180 unique irregular wave backgrounds.", "The final experimental series consisted of two 3 h long irregular wave time series, without any NewWave group present.", "A single sea state, representing a 100 year return period at Wave Hub, a wave energy test site of the North Cornwall Coast (UK) was used throughout.", "The differences between the maximum mooring loads and surge measured during the four set of experiments are specific to the float geometry, mooring arrangement and wave conditions tested here.", "However by comparing the results from the four different sets of experiments conclusions are drawn and recommendations made about the application of independent and constrained NewWave to the experimental determination of the extreme response of a moored floating body.", "The floating body tested was the same as used in Hann et al. (2015), but with an altered mooring arrangement (Fig. 1).", "It is a 0.5 m diameter floating body, consisting of a hemisphere and 0.25 m high cylinder, with a dry mass of 43.2 kg and made from steel with a concrete ballast.", "The floats centre of mass was about the central axis of the float, 0.319 m from the top of the cylinder.", "Moments of inertia were Ixx = Iyy = 1.61 kg m2 and Izz = 1.25 kg m2.", "A single taut mooring was used consisting of 1.38 m of 3 mm diameter Dyneema® rope (spring constant, k ≈ 35 N/mm) in series with a 12.5 mm diameter linear spring (k = 0.066 N/mm), which provided the mooring's extension.", "The initial and maximum rated length of the spring was 0.63 m and 1.145 m respectively.", "In still water the spring was extended by 0.31 m. End stops, consisting of 4 Dyneema® ropes around the spring, were installed to prevent the spring from being overextended.", "Throughout these experiments the spring was never extended to its maximum length, and as such these end stops were never reached.", "Mooring loads were measured using a 500N load cell (40 mm high, 44 mm diameter) attached in line with the mooring to the tank floor via a 74 mm long, 22 mm diameter universal joint (Fig. 1).", "Three 0.02 m shackles connected the spring to the load cell, the spring to the mooring line and the mooring line to the float.", "Mooring loads are reported as the difference between the dynamic mooring load and the moorings static, equilibrium load (i.e. the additional load).", "An optical tracking system was used to record the motion of the model in 6 degrees of freedom about a global coordinate system.", "The reported motions are of the centre of mass of the model.", "The averaged measured residual in the optical tracking system was ±1.15 mm.", "Decay tests conducted on the moored float identified the system to have resonance frequencies of 0.93 Hz in heave, 0.75 Hz in pitch and 0.07 Hz in surge.", "Although the floating body and mooring are not representative of a specific full scale wave energy device, the experiments are considered to be conducted at 50th scale based on the scaling of the wave conditions (section 3.1).", "Measurements were conducted in a 35 m × 15.5 m ocean basin at Plymouth University's COAST laboratory.", "The variable floor depth was set at 2.8 m.", "The facility has 24 2.0 m deep hinged wave paddles across one side of the tank.", "Wave reflections are reduced using a parabolic beach and the wave paddle's active absorption.", "The model was moored, in its resting position, along the centre line of the basin, 18.8 m from the front of the wave paddles.", "A series of 9 resistance type wave gauges were positioned along a line 1.46 m away from the tanks centre line.", "The location of these gauges relative to wave paddles and the model are given in Table 1.", "Wave gauge 6 was positioned so that it was approximately aligned with the front face of the model.", "All waves generated in this sequence of measurements were long crested and normally-incident.", "It is assumed that the waves impacting the model are identical to those recorded 1.46 m to the side of the model.", "Two further wave gauges were positioned on the tank's centre line in line with wave gauge 2 and 3 to confirm this assumption.", "The first series of tests measured the response of the floating body to four independent focused wave groups.", "The first was a NewWave group generated using a Pierson-Moskowitz (PM) spectrum with a peak period and wave height based on 50th scale hindcast data (Halcrow, 2006 p. 19) for a 100-year return period storm at Wave Hub, a wave energy test facility off the north Cornwall coast in the south west of the UK (Tz = 1.99 s, Hs = 0.288 m).", "The theoretical focus location of each wave group is wave gauge 6, the approximate location of the front of the model when stationary.", "Fernández et al. (2014) discuss various reasons why focused waves do not generally come to focus at the theoretical focus location and time, as defined by linear theory.", "This includes non-linear wave-wave interactions within the wave packet (as investigated by Baldock et al. (1996)) creating components that do not satisfy the linear dispersion relation.", "As used by Baldock et al. (1996) and Ning et al. (2009), a trial and error process was used to adjust the theoretical focus location of the wave group to achieve focus at the required location (±0.05 m).", "The wave group was judged to be in focus when the surface elevation time history was symmetrical i.e. the troughs on either side of the main crest had the same magnitude.", "Fig. 2 plots the difference between the adjusted theoretical focus location and the target/actual focus location against the theoretical crest steepness.", "An approximate linear relationship can be observed, indicating the increasing influence of non-linear effects as wave steepness increases.", "The CNW approach potentially provides a means to investigate the effect that a device's load history, as generated by previous waves, has on the response of the float to a specific extreme wave.", "The NewWave previously tested as an individual focused wave (S1) was constrained into 24 regular wave and 180 irregular wave time series.", "The methodology used to generate the CNW time sequence was based on that used by Cassidy (1999) and Bennett et al. (2012).", "Fig. 3 presents an example of this embedding procedure for one of the irregular cases, showing the initial random wave sequence, the initial independent NewWave and the resulting CNW.", "Varying the phase location at which the NewWave was constrained into the 2 regular waves systematically alters the floating body's motion at the point the focused wave interacts with the model.", "The phase location was varied from −150° to 180° in 30° intervals.", "Fig. 4 shows the NewWave constrained so the central crest occurs at a phase of 0°, 90° and 180° within the regular wave.", "Each of the 180 different irregular background sea states into which the NewWave was constrained were 120 s long.", "The NewWave's were constrained such that the main crest occurred at 60 s.", "The background sea states were formed by generating two 3 h long irregular wave time series and splitting each of these into 90, 120 s sections.", "The two 3 h long time series were generated using a Pierson-Moskowitz spectrum and the same Tz and Hs values as used to generate the NewWave.", "Each of the 180 constrained NewWave time series were used as an input into the basin's wave generation software, with the theoretical focus location set to be the location of the front face of the model in its static location.", "Unlike with the four individual focused wave groups, it was not practical to use a trial and error approach to adjust the theoretical focus location of the 24 regular and 180 irregular constrained cases to achieve an improved focus.", "The repeatability of the wave generation was assessed by repeating 1 of the 180 irregular constrained cases six times.", "Fig. 5 shows the measured wave at WG6 of these 6 repeats around the CNW.", "The average RMSE between the 1st wave and the following 5 repeats wave, excluding the first 20 s of the time series, was 0.012 m, indicating a good level of repeatability in wave generation.", "A similar level of repeatability was observed in the mooring loads, with an average RMSE value between the first measurement and the following 5 repeats of 0.79 N, compare to an average peak load of 18.42 N.", "Fig. 7 shows the measured time history of the 4 independent focused wave groups.", "The measured main crest amplitude is presented in Table 2.", "The crest amplitude progressively increases as steepness increases, with the percentage difference between measured and target amplitude increasing from 2.2% to 19.1%.", "The wave troughs immediately preceding and following the main crest are, assuming linear wave theory, also expected to have the same elevation across the four wave groups.", "Fig. 7 shows that as wave steepness increased the measured group troughs were actually progressively less deep than the target waves.", "This observed behaviour of the main crest and troughs is in agreement with that investigated by Ning et al. (2009), who concluded that this is due to non-linear wave-wave interactions (particularly 3rd order).", "Fig. 8 presents the mooring loads measured during the individual focused wave tests.", "Mooring load has been normalised with respect to the static mooring load.", "Overall peak load increased with wave steepness.", "However this load increase was only 1.3% between S1 and S4, compared to a 16.5% increase in crest elevation and a 74.9% increase in wave steepness.", "There was also no appreciable difference in the peak load generated by S2 and S3, with the generated loads lying between that generated by S1 and S4.", "This trend matches that observed in Hann et al. (2015) where snatch loading, generated when a taut mooring with reduced capacity compared to the one used in these tests reached its end stop, was only observed to increase by 4.7% as steepness increased between the same four wave groups.", "The difference in the mooring load peak subsequent to the largest peak, at around 2.6 s, increases to 8.8% between S1 and S4.", "This increase was monotonic in nature, with the load progressively increasing with wave steepness.", "The corresponding motions of the float are presented in Fig. 9.", "No relationship was observed between focused wave group steepness and the peak in heave generated by the central wave crest, with the largest peak generated by S2 and the smallest by S4.", "However larger differences and monotonic increases with respect to wave group steepness are observed in surge and pitch.", "A 10.4% increase between S1 and S4 is observed in the peak in surge directly caused by the central wave crest of the focused wave, increasing to a 172% difference in the following peak.", "The magnitude of pitch for the first peak that followed interaction with the central wave crest demonstrated a 111% increase between S1 and S4.", "These results indicate that the steepness of the focused wave crest does not significantly affect the mooring load directly generated by this crest.", "However the deviation in the float's surge and pitch response with changing wave steepness would result in an increased deviation in the mooring load measured between the 4 cases if there was to be an identical second wave later in the time series.", "This indicates the need to consider the effect of previous waves when modelling the effect of an extreme wave as even small changes in the previous wave history could result in the float being in a significantly different location and motion state when the extreme wave arrives.", "Theoretically the CNW should have the same crest amplitude regardless of the background sea state it is embedded into (Fig. 4).", "However this approach is based on linear theory and does not take into account higher order wave-wave interactions that occur as waves propagate from the paddles to the focus location.", "Fig. 10 shows how the measured central crest amplitude changes relative to the phase location in the regular wave at which it is constrained.", "A clear relationship between the phase position and the generated crest amplitude can be observed.", "As can be observed in Fig. 4, the trough preceding the central crest of the CNW changes slightly in both magnitude and time depending on the phase position in the regular wave into which it is constrained.", "This affects the steepness of the CNW.", "Fig. 11 shows how both the target and measured central crest steepness changes with phase position.", "The difference between the target and measured steepness changes with both the embedded phase position and the frequency of the background regular wave.", "Tests were conducted with regular CNW as a means to systematically alter the position and motion of the model at the time where it interacted with the NewWave group.", "However the observed differences between the target and measured central crest amplitude and steepness make it difficult to separate these effects from those of changing the models position and motion on its response to the NewWave group.", "Fig. 12 presents how the peak mooring load generated by the regular CNW changes with the phase location.", "In Fig. 13 the same load is compared to the measured amplitude of the central crest of the CNW.", "A strong correlation can be observed between amplitude and load for f2, however a much weaker correlation is observed for f1 (the R2 value of a line of best fit is 0.54 for f1 as compared to 0.87 for f2).", "This suggests that either the previous motions generated by regular wave f1 have a greater influence than for f2 on the load generated by the CNW, or that the larger range over which the CNW amplitude varies in f1 has resulted in a non-linear relation between amplitude and load.", "Fig. 13 provides evidence that the former is true, with different phase locations that have approximately the same amplitude generating differing loads.", "Without testing with other regular wave frequencies it is not possible to conclude whether the f1 background had a larger influence than f2 due to differences in the regular wave steepness or due to its relation to the model's resonance frequencies.", "There is a less obvious correlation between the steepness of the CNW and the load (Fig. 14), which is in agreement with the very small effect of crest steepness observed in the first experimental series.", "Fig. 15 plots the change in maximum surge with phase position.", "Unlike with peak mooring load, for the majority of phase locations the maximum surge generated by the CNW was less than that generated by the individual NewWave.", "There is also a strong dependence on the background regular wave frequency, with the maximum surge generated in the f2 cases covering a larger range.", "Comparing Fig. 15 with Fig. 10 it can be observed that there is a correlation between the CNW amplitude and the peak surge for f2, although not for f1 (fitting a line of best fit to amplitude against maximum surge data results in an R2 value of 0.89 for f2 compared to 0.17 for f1).", "This is consistent with the peak load results.", "As with the regular cases, the main crest of the CNW is reproduced less well than the underlying irregular sea state.", "In Fig. 16 the distribution of the measured embedded crest amplitudes are presented, normalised with respect to the NewWave theoretical crest amplitude.", "The distribution of crest amplitudes is approximately normal, with a standard deviation of 0.155 and a mean of 0.969 of the target crest amplitude.", "In 97 of the 180 cases measured crest amplitude was within ±10% of the target theoretical amplitude.", "A similar comparison of the wave steepness of the embedded focused wave crest with the steepness of the theoretical independent NewWave is shown in Fig. 17.", "The mean of this distribution is 1.25, with a standard deviation of 0.42.", "This distribution has a positive skew of 0.995.", "Unlike crest amplitude, the focused wave constraining process does not conserve the steepness of the focussed wave groups central crest.", "The distribution of the target wave steepness had a mean of 1.08, standard deviation of 0.330 and a positive skew of 0.958.", "It can be observed that, on average, the embedded focused wave was steeper than the initial target.", "The distribution of the peak mooring loads resulting from the 180 irregular CNW are presented in Fig. 18.", "Loads have been normalised with respect to the peak load produced by the individual NewWave.", "The distribution has a mean of 1.11, standard deviation of 0.14 and a skew of 0.22.", "Comparing Fig. 18 to the distribution of crest amplitude (Fig. 16) and crest steepness (Fig. 17) it cannot be concluded that the observed distribution in mooring load is due to the distribution of the CNW properties.", "In Figs. 19 and 20 the measured loads are plotted against the normalised crest amplitudes and crest steepnesses respectively.", "The lack of strong correlation in both figures suggest that the variation in properties of the generated CNW are not the only causes of the distribution in loads.", "This suggests that the influence of the previous wave field is a significant factor, demonstrating the necessity to consider the influence of this when investigating the interaction between an extreme wave and a moored floating body.", "The distribution of the measured maximum surge generated by the CNW is plotted in Fig. 21.", "As with the other properties this is approximately normal, with a mean of 1.12 of the surge generated by the individual NW, a standard deviation of 0.33 and a skew of −0.076.", "This larger standard deviation, as compared to the distributions in crest amplitudes and mooring loads, is considered to be an indication that the horizontal excursion of the model is less well predicted by an individual focused wave than the maximum mooring load.", "It also again emphasises the need to include the influence of the previous wave field when predicting response to an extreme wave.", "A comparison of the results from the two irregular wave experiments with the irregular CNW results identified four cases where the peak mooring load exceeded the largest measured in the CNW cases.", "These were 1.47, 1.52, 1.53 and 1.60 times the load measured using the individual NewWave, where the largest load measured during the irregular CNW case was 1.465.", "The sections of the irregular wave time history that generated these four largest loads are presented in Fig. 22 and are compared with the measured independent NewWave.", "The time series have been shifted so that the wave crests that generated the large loads occur at t = 0.", "The properties of these four waves have a wide range, with crest amplitude ranging from 0.781 to 1.09 of the target amplitude of the NW and a crest steepness ranging from 1.01 to 2.08 times the steepness of the NW.", "This further demonstrates the dependence of the mooring load on the previously generated model motions.", "The variation between the irregular and CNW cases was less for the measured surge than it was for the mooring loads.", "The largest measured value of surge was 2.31 times the maximum surge generated by the individual NewWave for the irregular case and 2.23 times from the irregular CNW cases.", "Results are reported from four series of experiments designed to measure the response of a taut moored floating body, representing a point absorbing wave energy converter, to an extreme wave.", "Tests with individual focused waves based on NewWave theory demonstrated that the steepness of the main crest of the focused wave had negligible effect on the response of the floating body.", "However the floats motion once the central crest has passed the model showed significant differences resulting from the differences in steepness.", "This demonstrates the non-linear response of the model, with initially very small changes induced by changes in wave steepness resulting in significantly increased changes in motion once the wave has passed.", "This is significant when trying to develop methods to predict dynamic model responses to extreme waves as it indicates that even small changes in previous wave conditions could have a significant effect on the position and motion of a model when interacting with the extreme wave.", "To explore the effect of a device's load history (as generated by the preceding wave field) on its response to an extreme wave, a NewWave constrained into a series of regular and irregular waves were used.", "In both cases differences between the target and generated constrained time series have limited the potential benefit of this approach.", "Bennett et al. (2012) report a similar finding when generating constrained NewWave experimentally.", "It is potentially feasible that an iterative approach could be used to adjust the wave generator input signal to improve the agreement between the target and generated constrained NewWave.", "However this would be too time consuming when conducting enough irregular constrained NewWave experiments to draw meaningful conclusions from the results.", "The impact of this approach on the background sea state is also unknown.", "Regardless of the differences between the target and generated time series it is still concluded from the CNW experiments that the use of an independent focused wave is insufficient to properly model the response of a moored floating body such as wave energy converter to an extreme wave.", "Instead the body's load history must also be modelled.", "Comparisons between the irregular and regular CNW cases indicate that it is also insufficient to model this load history using regular waves.", "Although the use of regular waves allows for a more systematic study, potentially reducing the number of test cases, result here found significantly higher mooring loads and surge levels from the comparable irregular CNW cases.", "The regular CNW results did demonstrate a dependence on the frequency of the background wave, so it is possible that other frequencies would have generated loads and surges more comparable to the irregular cases.", "However this seems unlikely considering the range of responses seen for the two frequencies tested here.", "Ideally the background wave properties associated with the largest responses to the irregular CNW could be identified and used to limit the number of CNW tests that need to be conducted.", "Attempts to do this were inconclusive however.", "As results from the individual focused waves showed, even small changes in a specific wave can result in significant difference later on.", "Any conclusion that could have been drawn would likely have been device geometry and mooring specific as well, therefore making the use of such an approach limited.", "Finally results from the two 3 h long irregular wave runs found 4 times where generated mooring loads were larger than any measured in the CNW cases.", "With the CNW approach the assumption is made that the NewWave will generate the largest response, with only the changes in the previous sea state being significant.", "It can be concluded from the irregular wave cases that this is not necessarily the case, and that for a particular sea state the traditional experimental approach of generating a random time series may be the better method of finding the maximum expected load and surge as this assumption is avoided.", "The random nature of this approach however also requires multiple cases of the full length sea state to be run (as opposed to the shorter time series involved with the CNW approach) to gain greater certainty in what the largest expected responses are.", "This is prohibitively expensive to do experimentally and indicates that numerical approaches, validated by representative experimental results, might be a better approach." ]
[ "Measurements of the interaction of a taut moored floating body representing a WEC, with extreme waves are reported.", "Focused wave group steepness has no significant impact on directly generated mooring load but influences later motion.", "The effect of load history on extreme response is explored by constraining focused wave groups into background sea states.", "Using regular constrained focused wave groups appears inadequate to assess extreme loads experienced by a taut moored WEC.", "Model response to constrained groups indicate that the largest waves do not necessarily result in the largest response." ]
Thermal performance analysis of a concentrated solar power system (CSP) integrated with natural gas combined cycle (NGCC) power plant
In this study, a concentrated solar thermal power plant (CSP) with parabolic trough collectors integrated into a reference triple-pressure NGCC power plant as an Integrated Solar Combined Cycle (ISCC). The aim is to study and assessment the overall performance of different hybrid schemes of ISCC plants. Thus, A detailed thermodynamic model of the proposed ISCC plants has been developed to investigate the thermodynamic impact of solar heat rate injected in the high and intermediate pressure sections (HP, IP) of the NGCC plant at different seasons of the year. The cycles have been characterized and simulated under southern Egypt climate. An increase in the NGCC output power of 52.315 MWe with peak net thermal efficiency of 64.86% and solar to electric conservation efficiency of 46.95% are obtained from the integration process in the HP section. The increase in power output reaches a limit of 43.38 MWe with a net efficiency of 63.82% and solar to electric efficiency of 38.85% for integration in the IP section. The results of the analysis have been indicated that the higher the solar energy injected the higher is the solar electricity production and the higher is the overall thermal efficiency.
[ "CENG", "ENGI" ]
[ "Concentrated solar power integrated solar combined cycle solar energy", "Solar thermal energy performance" ]
[ "1507", "2201" ]
[ "The use of fossil fuels is largely responsible for increasing pollution and resulting climate change and if continue to be depleted at the present rate, they will be exhausted soon.", "Thus, renewable energy sources, particularly solar energy can help countries meet their sustainable development goals through provision of access to clean, secure and affordable energy.", "Solar energy is clean, environmentally friendly and freely available over the planet earth, as well as it can be used for producing thermal and electrical energy.", "Recently years, there has been rapid development in solar thermal electricity technologies and photovoltaic (PV) materials.", "This development brought cost-effectiveness to solar devices [1].", "Solar thermal power plants (STP), also known as concentrated solar power (CSP) are one of the main renewable energy alternative technologies for the production of heat or electricity generation using mirrors that captures and concentrates the solar energy to heat a liquid, solid or gas at temperatures between 400 and 1000 °C for producing of steam required to drive a heat engine for electricity generation, rather than using fossil fuels or nuclear reactions [2].", "Unlike photovoltaic installations, STP does not appear to be a logical choice in terms of distributed generation, but at the level of a large-scale system.", "In particular, they can be integrated with thermal storage or in a hybrid process, providing power with fixed capacity and can be customized.", "This allows STP to fill the gaps in intermittent resources of CO2-free energy, like wind energy.", "Solar thermal electricity can meet the maximum and minimum demand level together.", "CSP plants are suitable for areas exposed to large amounts of solar radiation, such as southern Europe, North Africa, the Middle East, South Africa, some parts of India, China, the southern United States, and Australia.", "Today, STP plants range from a few megawatts to more than 400 MW but can be increased.", "Currently, four CSP technologies are considered: linear Fresnel reflector system (LF), parabolic trough collector system (PTC), power tower or central receiver system (CRS), and parabolic dish/engine system (DE).", "The forms of mirrors, solar tracking methods, and energy saving methods vary, but all follow the same principle: stimulating a heat engine often a steam turbine to generate electricity.", "The most advanced and commercially widely proven is the PTC plants when compared with all other types of CSP plants [3].", "They have the ability to concentrate the solar radiation with a concentration ratio ranges from 70 to 80 suns and operating temperature in the range of 290–550 °C [4].", "Several decades ago, NGCC power plants are the most efficient thermal-to mechanical energy conversion systems.", "They are only power plants in the world that can release electricity to consumers with high thermal efficiency up to 55–60% when operating in condensation mode.", "Therefore, they have been commercially installed at high power rate levels (several hundreds of MW) and widely used [5].", "The integration of solar thermal technologies such as CSP technology with the NGCC power plants is a novel power generation plants known as integrated solar combined cycles (ISCC).", "ISCC may become an interesting choice for power generation because they are exhibit several advantages, including (1) higher conservation efficiency of solar energy to electric energy; (2) hybridization can provide an efficient reduction in fuel consumption and CO2 emissions as compared to NGCC power plants; (3) integration arrangements has lower investment costs when compared to solar only and conventional power plants.", "A large number of ISCC power plants now are in operation all around the world (North Africa, Iran, Italy, Spain, and the USA), several projects are planned and under construction (North Africa, Mexico, China, and the USA) as reviewed in Ref. [6].", "The majority of these plants based on PTC technology.", "In recent years, a number of studies have been thermodynamically analyzed the ISCC plants for identification the integration points and limits of solar energy injected into existing NGCC power plants to maximize cycles performance.", "Therefore, the objective of this study is the comparison in terms of daily, yearly electricity production and solar to electric conservation efficiency of two different hybrid schemes of ISCC using PTC solar field under design point and off-design operations.", "A detailed new thermodynamic model of the two ISCC plants has been built in the Mathcad environment to investigate the thermodynamic impact of solar heat integration into a triple-pressure NGCC plant at higher and intermediate pressure sections.", "The model allows the designers to change the input data according to the different design and operating conditions.", "In this study, both two ISCC plants are characterized and simulated in location: Aswan, Egypt.", "The configuration of ISCC basically consists of a PTC solar field coupled to the bottoming steam cycle of a NGCC power plant.", "Two different operation strategies for PTCs in ISCC plants are considered: direct steam generation technology when the working fluid runs in the absorber pipes of PTC solar field is water/steam in which the feed water is heated and then transferred directly to the steam cycle.", "Another scheme consists of heat transfer fluid (HTF) in which synthetic thermal oil is used to transfer heat.", "Saturated/Superheated steam at high pressure through a heat exchanger can be produced by the hot synthetic thermal oil passing through a heat exchanger and fed into a steam turbine for electricity generation.", "Most currently parabolic trough thermal plants use HTF technology [7,8], which is assumed in our present study.", "The flowsheets of the considered configurations of ISCC plants are given in Figs. 1 and 2.", "The system is composed of a triple-pressure NGCC coupled with a PTC solar field and a solar steam generator (SSG) modeled in the Mathcad® environment.", "The NGCC plant is composed by a topping gas turbine unit (GT) type M701F4 (Mitsubishi heavy industry) and a bottoming steam Rankine cycle.", "The bottoming steam cycle consists of a steam turbine (ST) unit divided into three stages: the high-pressure turbine stage (HPT), the intermediate-pressure stage (IPT) and the low-pressure turbine stage (LPT).", "The steam turbine connected to a triple-pressure heat recovery steam generator (HRSG) with one reheat at the intermediate pressure stage.", "The output power of the NGCC is 503 MWe with a net thermal efficiency of 58.75% at nominal conditions.", "All technical and design parameters for the NGCC and HRSG of the steam cycle shown in Table 1.", "The working principle of the ISCC plant is derived from the NGCC power plant, where the exhaust gases with high temperature from the gas turbine pass through the HRSG to generate superheated/saturated steam that used to drive the steam turbine.", "The solar energy collected by the solar field is used to generate an additional amount of steam through the SSG to supplement the steam being generated in the HRSG.", "The steam generated in SSG and fed into the HRSG of the NGCC plant allowing for two different integration strategies modes: power boosting and fuel saving mode.", "The former is considered when the additional solar steam increasing the NGCC power output while maintaining constant fuel input, the later when solar steam is used to reducing natural gas consumption while keeping power output constant [9].", "In the present study power boosting mode is considered for two cases of integration between PTC solar field and NGCC power plant at IP stage (ISCC–I, Fig. 1) and HP stage (ISSC-II, Fig. 2).", "During periods of solar activity, apart the feed water from the HRSG is sent to SSG, in which water is heated and transferred into saturated/superheated steam.", "The resulting steam is returned to HRSG, where it overheats to the parameters required for supply to the steam turbine cycle, see Figs. 1 and 2 [10,11].", "These configuration circuits provide an increase in the flow rate of the generated steam, and, as a consequence, the increase in power realized in the NGCC power plant.", "During periods of relatively low solar activity due to cloudiness and at night, the ISCC unit operates as a conventional combined-cycle plant.", "According to the flow diagram of ISCC-I shown in Fig. 1, the PTC solar field heats thermal oil “TherminolVP-1” to a temperature limited to a level of about 400 °C, which is then sent through a series of shell-and-tube heat exchangers (SSG).", "The SSG receives part of the preheated feed water (steam) from the intermediate pressure economizer (IPE) in the HRSG and returns superheated steam at 378 °C, which is mixed with the IP steam coming out of the IP superheater (IPSH) circuit.", "The mixture is reheated to the final conditions of the IP steam turbine to approximately 540 °C in the reheater.", "The flow diagram of ISCC-II shown in Fig. 2 represents the integration of solar steam in the HP circuit of HRSG, in this case a fraction of the generated steam in the HP economizer I (HPE_I) is sent to SSG for superheating to approximately 378 °C outside the HRSG, while the maintaining part is evaporated in the HRSG.", "The superheated steam from SSG returns back to the HRSG, where it is mixed with HP steam coming out of the HP evaporator circuit.", "The mixture of two streams is then farther superheated in the HP superheater to approximately 560 °C before entering the HP turbine.", "Egypt enjoys favorable solar radiation intensity.", "It has between 2900 and 3200 h of sunshine annually with annual Direct Normal Irradiance (DNI) of 1970–3200 kWh/m2 from the north to the south of Egypt, see Fig. 3.", "An average sunshine duration of 12 h was observed by NASA Surface Meteorology and Solar Energy in Egypt all over the year with average daily maximum temperature ranges from 18 °C to 38 °C and average daily minimum temperature ranges from 9 °C to 22 °C.", "due to the available solar resource in the country with the number of sunny days per year, the longer sunshine duration and the high solar radiation intensity, Egypt has a huge potential of solar energy that Makes it one of the most appropriate regions for the design of solar energy conversion system both for electricity generation and thermal heating applications.", "In several previous research, the researchers have been used the DNI for determining the performance and the nature of the PTC system.", "Therefore, a Mathcad Internet mathematical model was developed to estimate the direct component of solar irradiation and the amount of solar energy collected by PTCs based on the selected site and available climate data (see Appendix A) [12].", "The initial input data to the solar field model are the location (latitude and longitude); time (day of the year, hour); geometric (dimensions of the solar field) and optic characteristics (coefficients of the absorption and the transmission of collector components).", "The geometrical and optical characteristics of PTCs shown in Table 2.", "Two thermodynamic models of the ISCC plants have been developed in detail in Mathcad environment based on the thermodynamic laws, relation, and energy balance.", "The models include solar radiation, solar field modeling, and modeling of the power conversion cycles.", "All cycle's calculations are designed using Mathcad Internet and available online in Refs. [13,14].", "The models are validated with experimental and numerical results obtained from Refs. [8–11].", "Models start with analysis and determining the performance of the solar field which has the main effect on the ISCC performance.", "In this model, the solar field works in variable flow strategy, therefore the inlet and outlet temperature of HTF are set constant at 291 °C and 393 °C respectively.", "The variation of solar energy absorbed by solar field and the HTF mass flow rate determined by using the model in three selected days, 22nd June, 23rd March, and 21st December on hourly basis shown in Fig. 4.", "From the figure, we can conclude that the solar energy collected by the solar field might reach up to 110.8 MW in summer.", "This value can be lower than 32 MW in winter.", "This due to the higher the solar radiation results in an increase in the thermal energy gain by the solar field in summer compared to lower solar radiation in winter.", "The solar energy gained can be reached to more than 85 MW in spring and autumn.", "Also, it is obvious that the higher solar energy the higher is the HTF mass flow rate.", "Where it might reach to 444 kg/s on 22nd June and 134.5 kg/s on 21st December.", "Fig. 5 shows the effect of the solar heat input to SSG on the steam mass flow rate generated in both cycles.", "It is clear that the flow rate of solar steam generated gradually increases in the HRSG with increasing the solar thermal heat input which leads to a decrease in the exhaust stack temperature and hence increasing the HRSG efficiency.", "For low solar heat integration, the steam flow rate generated is relatively low for both cycles (ms = 5 kg/s at Qsolar = 10 MW).", "At Qsolar = 100 MW the mass of steam generated reaches to 53 kg/s in ISCC-II compared to 46.57 kg/s in ISCC-I this due to the difference in the temperature of steam input to SSG.", "The proposed ISCC power plants in this study are working in power boosting mode, this means the higher the solar energy input the higher the electricity production by the power plant.", "The reason behind that was mentioned above, when the solar heat input increases, the steam mass flow in the HRSG also increases, which will lead to the generation of more steam in Rankine steam bottoming cycle up to 55 kg/s and consequently more power output will be produced by ISCC plant [15].", "The overall performance of the two ISCC during a year has been analyzed, the total power output produced at the three selected days shown in Fig. 6.", "The output power varies during a day with solar radiation intensity, it can reach to the peak value of 546.43 MWe with an increase of 43.38 MWe from NGCC for ISCC-I and 555.36 MWe for ISCC-II with an increase of 52.315 MWe in summer.", "The difference is due to the amount of solar steam entered to the HRSG.", "Minimum values of 515.8 MWe and 517.6 MWe for both cycles are observed during the low radiation periods (winter).", "Solar thermal electricity (Nsolar) represent the power produced by solar energy injected to the cycle and equal to the difference between the power output of steam bottoming cycle in case of NGCC and in ISCC power plant [16].", "Fig. 7 shows the impact of the amount of solar heat injected to the HRSG on the solar thermal electricity produced.", "In fact, solar thermal electricity is largely depending on the amount of solar thermal input.", "At Qsolar = 0 MW the two ISCC plants are working as a conventional NGCC thus, the solar power output is Nsolar = 0 MWe.", "When the solar energy increases the solar power increases (Nsolar), it could reach to 40 MWe in ISCC-I and 47.3 MWe in ISCC-II at Qsolar = 100 MW.", "Similarly, the effect of solar energy injected on the net thermal efficiency of the two ISCC plants shown in Fig. 8.", "It is obvious that the overall plant efficiency is a strong function of the solar heat input.", "It can reach the limit of 65% in case of high-pressure integration (ISCC-II) in summer days, this limit decrees to 60.5% in winter days.", "For IP integration process represented in ISCC-I, the overall efficiency can reach a maximum of 63.8%.", "From the above, we can deduce that the increase in the power output and thermal efficiency of the plant for the integration in HP section (ISCC-II) is higher than that one in IP section (ISCC–I) at high solar heat input, but the two cycles give approximately the results at relatively low solar heat input.", "Here, in order to obtain a complete overview and assess the ISCC system's real benefits, a simulation of the annual performance of the proposed ISCC plants are carried out and shown in Table 3.", "Since the cycles operate on power boosting mode, the gas turbine works all the time at full load and thus, the annual fuel consumption is constant.", "The most important keys of annual performance assessment are: annual solar thermal share (defined as the ratio of annual solar electricity production (Nsolar) to the total ISCC system net power output, (NISCC)), annual solar thermal into electrical efficiency (represent the ratio of Nsolar and the net electricity generation by ISCC plant) [16] and the annual overall thermal efficiency.", "This study proposes and compares the performance of two configurations of ISCC power plants using PTC solar field technology with a reference conventional triple-pressure NGCC power plant.", "In Both configurations, the solar field generates a part of the steam of the high-pressure level (ISCC-II) and intermediate-pressure level (ISCC–I) of the heat recovery steam generator in parallel to the steam generated in the corresponding evaporators.", "Thus, two thermodynamic simulation models were developed in Mathcad environment to simulate the components of the proposed ISCC plants.", "Both models are used to analyze and evaluate the thermodynamic performance of the cycles at any operating condition.", "The effect of solar heat rate input on the main parameters of the systems was investigated such as the mass flow rate of HTF in the solar field, the mass of generated steam in the solar steam generator (SSG), the net output power and the net thermal efficiency of ISCC.", "The comparisons between the two plants are made also in terms of annual electrical energy production and overall thermal efficiency.", "The simulations for the two ISCC system show that:", "The integration process into the HP stage (ISCC-II) provides more generation of steam flow rate in HRSG than the integration into the IP stage (ISCC–I), hence more net electrical power out for ISCC-II than ISCC-I.", "For ISCC-II, the net power output can be reached as high as 555.36 MWe in summer compared to 546.43 MWe for ISCC-I, which is higher than that in the reference NGCC system by about 52.315, 43.38 MWe respectively.", "Also, the overall efficiency increased from 58.75% to 65% for both cycles.", "The values decrease in winter days due to less solar radiation available.", "An annual reduction of CO2 of 73586.42 (ton/yr.)", "could be achieved by ISCC-II with annual fossil fuel saving ratio (SRf) of 11.28% and 62885 (ton/yr.)", "of CO2 reduced by ISCC-I with annual SRf = 9.6%.", "The study is limited for using PTCs solar filed with NGCC power plant, therefore using other types of CSP technologies with NGCC, comparing between them and using thermal storage systems are suggested improvements and future works in this line of research." ]
[ "The thermal performance for two configurations of integrated solar combined cycles ISCC power plants using PTC solar field technology with a reference conventional triple-pressure NGCC power plant are analyzed.", "An increase in the net power output of NGCC power plant by about 52.315 MWe at high-pressure integration and 43.38 MWe at the intermediate pressure section, which improved the overall efficiency from 58.75% to 65%." ]
Differentiation of Vascular Characteristics Using Contrast-Enhanced Ultrasound Imaging
Ultrasound contrast imaging has been used to assess tumour growth and regression by assessing the flow through the macro- and micro-vasculature. Our aim was to differentiate the blood kinetics of vessels such as veins, arteries and microvasculature within the limits of the spatial resolution of contrast-enhanced ultrasound imaging. The highly vascularised ovine ovary was used as a biological model. Perfusion of the ovary with SonoVue was recorded with a Philips iU22 scanner in contrast imaging mode. One ewe was treated with prostaglandin to induce vascular regression. Time-intensity curves (TIC) for different regions of interest were obtained, a lognormal model was fitted and flow parameters calculated. Parametric maps of the whole imaging plane were generated for 2 × 2 pixel regions of interest. Further analysis of TICs from selected locations helped specify parameters associated with differentiation into four categories of vessels (arteries, veins, medium-sized vessels and micro-vessels). Time-dependent parameters were associated with large veins, whereas intensity-dependent parameters were associated with large arteries. Further development may enable automation of the technique as an efficient way of monitoring vessel distributions in a clinical setting using currently available scanners.
[ "BIOC", "HEAL", "PHYS" ]
[ "Capillary", "Contrast agent", "Microbubble", "Ultrasound", "Vessel imaging" ]
[ "1304", "3102", "3614" ]
[ "Contrast-enhanced ultrasound (CEUS) imaging is an expanding field with a wide range of current and potential applications.", "One area yet to fully benefit from the potential of ultrasound contrast agents is the imaging of the microcirculation.", "Very narrow and evolving blood vessels are found in tumour vasculature (Folkman 2003) and angiogenesis within atherosclerosis (Moreno et al. 2006), with their structure, development and appearance being useful diagnostic markers.", "Positron emission tomography scanning currently can distinguish between the malignant and non-malignant tissue by enabling quantification of macroscopic flow (Dijkmans et al. 2006; El-Galaly et al. 2018).", "However, the high cost and associated radiation hazard render it largely impractical for significant clinical use, particularly for repetitive monitoring of treatment.", "Contrast-enhanced magnetic resonance imaging (MRI) also has the capability of providing flow information on micro-vessels within tumours (Lagaru and Gambhir 2013; Simoncic et al. 2017).", "However, MRI contrast agents do not just remain within the vasculature; a proportion of the agent distributes to the extracellular spaces, thus enabling the evaluation of vascular permeability instead of providing vascular flow assessment (Mori et al. 2017).", "Ultrasound contrast agents are blood pool agents and unable to pass into the extracellular spaces making them ideal markers for assessing blood flow through micro-vessels (Mori et al. 2017).", "In general, a very large number of diseases have altered blood flow, which directly links to the pathophysiology and the progression of the disease.", "Dynamic CEUS has been shown to have potential in the assessment of perfusion of microvascular structures such as tumours.", "Time-intensity curve (TIC) analysis appears promising in assessing tumour therapy using anti-angiogenic agents (Lassau et al. 2014; Lassau et al. 2012) where the area under the curve (AUC) was shown to be the most significant indicator of tumour stability when the whole tumour was selected as the region of interest (ROI) (Lassau et al. 2014).", "However, to date the research in conventional CEUS approaches has not produced quantitative vascular indices that are clinically robust.", "The recent advances in super-resolution imaging (SRI) provide maps of the structure and dynamics of the vascular bed with resolution that potentially can exceed 10-fold that of CEUS.", "Initial work on in vivo animal models (Christensen-Jeffries et al. 2015; Errico et al. 2015) has been followed by the potential identification of tumour characteristics (Opacic et al. 2018).", "The method deploys microbubble (MB) localization similar to that utilised in region localisation in fluorescent optical microscopy or astronomy.", "Given that this method works through single microbubble localisation, and there are large numbers of vessels in view (e.g., in a 2-D CEUS image plane), all vessels need to be crossed several times by single microbubbles.", "This is challenging because only a few isolated microbubbles can fit in an image.", "The vascular network imposes another restriction.", "Vessels of different diameters, from micrometers to centimeters, provide a large variation in vascular volume and consequently orders of magnitude variations in microbubble density.", "In practice, achieving a few single microbubbles in the microvasculature may require a high-density microbubble population from the millimetre-sized vessels that supply these.", "This means that millimetre-sized vessels would not have distinguishable single microbubbles for super-resolution processing.", "On the other hand, the reduction of microbubble concentration results in a significant increase in the required image data required for recovering the vascular structure in view, which translates into clinically impractically long examination times.", "Further, because larger vessels contain larger MB concentrations, the likelihood of multiple or overlapping MB echoes increases and the MB localisation and tracking may be compromised.", "In addition, it is difficult at this stage to envisage single emission (Errico et al. 2015) protocols useful for imaging entire organs.", "This is because the acoustic pressure varies significantly across the image and this introduces a variable MB density (Sboros et al. 2003).", "As a consequence, the link between MB count and vascular volume is variable.", "Focused transmission may ensure a less variable acoustic pressure field across an image, higher depth penetration and thus the potential, through SRI, to provide good representation of the vasculature of entire organs.", "The microbubble transit time physically imposes the minimum duration for the vascular bed to be mapped.", "Bolus injections of microbubbles take a few minutes to partially washout; therefore, to maintain an examination time of a few minutes, it is likely that a larger concentration is needed at least for the larger vessels in view.", "The tracking of microbubbles may not be the only method that provides improved resolution.", "Recently methods deploying pixel dynamics have shown significant promise in capturing vascular structure in high resolution (van Sloun et al. 2018; van Sloun et al. 2019), although not as much as SRI.", "Vessels that are usually above the resolution limit may significantly benefit from such methods.", "The conventional method of investigating these is through parametric mapping.", "The first parametric maps were provided by the visualisation of vascular structure using maximum intensity projection.", "The maximum intensity of each pixel over a number of consecutive frames is displayed in a meta-image, enabling the highlighting of the contrast agent path and thus the structure of the vessels (Forsberg et al. 2011).", "Generally, in CEUS, parametric mapping refers to images where each pixel provides the value of a parameter as fitted to the TIC of the particular pixel (Greis 2011).", "The method appears to provide more quantitative results on tissue disease state (Dizeux et al. 2017).", "This approach has evolved to mapping parameters not directly derived from the TIC, but the result of a more complex calculation of a physical parameter, a candidate biomarker, associated with the vascular expression of pathology.", "Such are the diffusion and dispersion parameters that provide biomarkers for tumours and have shown to be superior to TIC-derived parameters (Kuenen et al. 2011; Kuenen et al. 2013).", "A significant advance, similar to the concept of microbubble tracking is a direct translation of diffusion tensor tractography in MRI into 3-D vascular imaging by ultrasound tractography (van Sloun et al. 2018).", "The fractal dimension representing wash in rate and peak intensity (Ip) determined from dynamic CEUS was used to differentiate between two prostate cancer models comprising different microvascular structures (Saidov et al. 2016).", "All these studies demonstrate significant potential in revealing abnormal vascular dynamics using CEUS.", "Importantly, they demonstrate that the pixel statistics may be important to improving resolution and image sensitivity.", "The underlying reasons for making some of these methods work better than others are not fully clarified, partly because it is difficult to have good quality ground truth in in vivo investigations.", "Most of ground truth currently relies in histologic or other non-live evaluation.", "Although all these methods enable the visualisation of vascular-related parameters, there is still little knowledge available on the type or approximate size of the vessels that this information originates from.", "To date there is no study that investigates the ability of conventional CEUS to detect different sizes of vessels.", "This is the subject of the investigation in this article.", "The distinction between large vessels and the rest of the vasculature using frame sequences from ultrasound contrast imaging of the ovine corpus luteum (CL) (Perperidis et al. 2014) demonstrated the ability to differentiate between large vessels and the rest of the vasculature using frame sequences from ultrasound contrast imaging of the ovine CL.", "This article further investigates the differentiation of different sized vessels.", "The ovine CL provides a suitable in vivo model as different vessel sizes can be found at different locations in the image plane.", "The CL is a transient vascular structure on the ovary with a complex microvascular bed responsible for progesterone secretion.", "In maturity, it is an approximately spherical structure of 0.5–2 cm in diameter, comparable in size to a small tumour, and is suitable for developing CEUS methodology (Sboros et al. 2011; Strouthos et al. 2010).", "It is also a model to study vascularisation because its generation and regression are predictable and can be synchronised (Fraser and Duncan 2009).", "The ovary contains a network of larger vessels that branch out of the ovarian artery and cover the periphery of the CL.", "Within the CL itself, there is a high-density microvascular network such that each luteal cell is in direct contact with an endothelial cell (Fraser and Duncan 2009).", "Thus, the centre of the CL is nearly entirely composed of capillaries and venules, providing a large ROI in the ultrasound imaging plane that is dominated by the microvascular bed, representing a hi-fidelity in vivo biological model of microvascular flow.", "The CL microvasculature then feeds into the venous system which is spatially located alongside the arterial feeds.", "This means that the CL containing ovary is particularly suitable for imaging ROIs containing different sized vessels and for the implementation of parametric mapping methodology to enable vessel differentiation.", "Our aim here was to investigate this distinction to allow more specific identification of veins, small and large arteries and capillaries, using the CL within the ovary as an in vivo biological model of vascular flow.", "In a comprehensive study of all TIC parameters, new criteria for differentiating vessel types within the resolution of CEUS were developed.", "Finally, vascular regression within the CL was imaged, in a proof-of-principle attempt to demonstrate the ability to map changes in microvasculature.", "The results have implications in understanding the uncertainties associated with CEUS analysis and may work synergistically to recent SRI methodologies to boost CEUS resolution.", "As mentioned above, the CL-containing ovine ovary was used as a model for a highly vascularised system, where large and small blood vessels could be imaged together in one imaging plane.", "The methods have been previously described (Sboros et al. 2011).", "In short, ewes (n = 11) were synchronised to the mid luteal phase using standard animal husbandry methods using progesterone sponge administration and prostaglandin, prostaglandin F2 alpha (PGF2α) administration.", "About 10 d after sponge withdrawal ewes were anaesthetised and their ovaries exposed, with a fully maintained blood supply, by laparotomy and covered in scanning gel to allow fixed plane scanning.", "This method provided a vascular source where optimal data from the contrast agent could be recorded without loss of information due to sheep anatomy or inability to achieve a good image plane.", "To assess proof of concept one ewe had an injection of PGF2α (0.5 mL prostaglandin estrumate; Schering Plough Animal Health, Welwyn Garden City, UK) 24 h before scanning.", "This causes a predictable and marked reduction in microvascular density (Baird and McNeilly 1981; Sboros 2014; Vonnahme et al. 2006).", "For the study presented here it provided a data set with a regressed vasculature to show how differences and changes in vasculature may potentially be assessed.", "Using 2.4 mL bolus injections of Sonovue (Bracco, Geneva, Switzerland) followed by 5-mL saline, videos were acquired of the contrast agent through wash-in, Ip and wash out phases.", "Each bolus injection contained between 2.4 and 12 × 108 microbubbles.", "A Philips iU22 ultrasound scanner (Philips Medical Systems, Bothell, WA, USA) with a L9-3 linear array probe was used for all imaging with mechanical index 0.05.", "Recording commenced at the same time the saline flush was administered.", "Dual image b-mode and contrast mode videos were acquired.", "Multiple videos for each ovary were recorded, and Doppler scans provided additional information on the larger blood vessels.", "In total, 23 contrast videos of individual bolus injections, from 11 different ovaries were available for the study presented here.", "The videos were saved and exported from the scanner in DICOM format.", "After scanning, each sheep was euthanised and the ovaries removed and fixed for histologic analysis.", "All surgical work was conducted under UK Home Office approval (Project Licence McNeilly PPL 60/3906).", "Ovaries were fixed in Bouin's solution for 8 h and embedded in paraffin wax.", "Sections (5 µm thick) were cut, de-waxed, rehydrated and antigen retrieval was achieved for 20 min in a pressure cooker using citrate buffer 0.01 M. Endogenous peroxidase was inactivated by immersion in a solution of 3% H2 O2 in methanol for 20 min followed by avidin and biotin blocking (Vector Labs, UK).", "Biotinylated Lectin BS-1 (Sigma, UK), used to specifically bind to endothelial cells, was diluted 1/80 in normal goat serum (NGS)/Tris-buffered saline (TBS)/5% bovine serum albumin (BSA) and applied overnight to tissue sections at 4°C in a humidified chamber.", "Detection was performed for 30 min at room temperature using streptavidin-HRP (Vector Labs) diluted 1/1000 in NGS/TBS/5% BSA.", "Development of signal was made using Impact diaminobenzidine (Vector Labs) to stain the endothelial cells brown and identify vessels.", "Sections were washed, dehydrated and mounted using Pertex.", "The section that was a close to the ultrasound image plane as possible was chosen the represent the ROI and was analysed using a light microscope (Olympus BH2) coupled to a digital camera for image capture.", "Vessels across the whole plane were measured and counted in order to determine the average vessel content of the cross section.", "Vessels were grouped into those less than 30 µm diameter, those which were 30–200 µm diameter and those which were greater than 200 µm diameter.", "Initial processing of large ROIs was performed in QLab (Philips Medical Systems) with Matlab (Mathworks, Natick, MA, USA) being used for further automated pixel processing (each pixel measuring 140 × 140 µm).", "In general, the parametric maps derived were of lower resolution compared to the original data (Kuenen et al. 2013).", "We tested 1 × 1 to 4 × 4 pixel ROIs and concluded that a 2 × 2 pixel ROI being the best compromise between resolution and good signal-to-noise ratio in the TIC curves, which in turn resulted in reproducible parametric data.", "For the videos, rigid movements of the ovary due to respiration had to be removed.", "To correct this, each frame in the acquired video was aligned to a reference frame, chosen at the end of a respiration cycle.", "To achieve the alignment, a Nelder and Mead's Simplex optimisation method (Lewis 1995; Nelder and Mead 1965) was used for derivation of the rigid transformation which maximised the normalized cross-correlation between the registered images (current frame to reference frame).", "Time-intensity curves are commonly used for analysing the flow of contrast agent in CEUS imaging (Dietrich et al. 2012).", "For example, Figure 1a shows example TICs obtained for a small region of interest (ROI A) within the CL, which was dominated by micro-vessels (arterioles, capillaries and venules), as well as for a larger ROI B which covers the whole ovary and included the larger feeding vessels as well as the artery and vein that branch out into the respective ovarian vessels.", "For each TIC, a smoothing spline fit was used to remove high levels of noise.", "The resulting smoothed TIC was used to extract the flow- and volume-related parameters by fitting a Log-normal model.", "The following parameters were then calculated from the TICs: (i) Ip, (ii) time for the peak intensity (tp) to occur, (iii) mean transit time (MTT) of the contrast agent, (iv) wash in time (WIT) (time between 5% and 95% of Ip), (v) rate of increase during wash in (WIR) (5%–95% of Ip), (vi) wash out time (WOT) (time between 95% and 45% of Ip, a 50% drop), (vii) rate of decrease during wash out (WOR), (viii) area under the lognormal curve and (ix) the regression coefficient of the data to the model (R2) (Strouthos et al. 2010).", "Following this, the TICs for 2 × 2 pixel ROIs over the entire scan plane were calculated.", "The TICs were then used to enable parametric mapping of the entire image plane.", "These maps provided localised information on individual curve parameters as generated by the lognormal model that was fitted to the local TIC.", "It was observed that regions of very-low-density contrast flow produced noisy data resulting in TICs from which some flow parameter values were overestimated due to the broad shape of the fitted TIC.", "This often created an erroneous outlying saturated parametric signal.", "To account for this, the parametric maps and TICs were inspected and ROIs with erroneous parameter estimation were assigned the lowest detectable intensity values for each parameter, respectively.", "These regions of very low flow were not used for the later location specific parametric analysis.", "For 11 different ROIs (four different ovaries), videos comprising two or three consecutive ultrasound contrast scans were selected.", "TICs for 2 × 2 pixel ROIs were chosen to be in regions of good contrast echo.", "The same ROI by pixel number was chosen in each set of consecutive videos.", "Parameters associated with the TIC were calculated for each curve.", "Mean, standard deviation and standard error were calculated for each group.", "The parameter maps of the ovary indicated where the large veins and arteries and smaller vessel networks could be identified.", "Aided by the histologic analysis as well as prior knowledge of the vascular structure of the ovary and the parameter maps, TICs for 2 × 2 pixel ROIs from manually selected regions of specific vessel types were recalculated.", "These locations were chosen using (i) parametric maps of each ovary, (ii) histology, (iii) prior knowledge of the structure of the CL and ovary and (iv) user observations of the videos.", "The identifiable vessel types were arteries, veins, medium-sized vessels and micro-vessels.", "All the vessels greater than 200 µm in diameter were identified as “large,” whereas micro-vessels were below 30 µm.", "All the vessels in between belonged to the “medium”-sized vessels grouping.", "These sizes were used in the histologic evaluation and do not directly refer to the interpretation of the ultrasound images.", "Given that the size of each pixel was 140 × 140 µm2, the 2 × 2 pixel region was 280 × 280 µm2.", "Thus, it was implied that the regions were dominated by the respective vessel type.", "For example, micro-vessel regions were those thought to be dominated by capillaries and may include larger vessels but was unlikely to include “large” vessels.", "Similarly, medium vessel regions were dominated by vessels between 30 and 200 µm, which may include capillaries, but did not include large vessels.", "In addition, arteries were identified as the regions to show the first inflow of contrast agent.", "Veins were similar in size and provided delayed contrast, which typically would appear at the same time or later compared to the inner microvascular CL and remained bright toward the end of the contrast perfusion.", "Medium-sized vessels were classified as filling and clearing in between the times of the large arteries and veins.", "Time-intensity curves and all associated flow parameters were derived for the new 2 × 2 pixel ROIs.", "In total 54 ROIs associated with arteries, 35 ROIs associated with veins, 41 ROIs associated with medium-sized vessels and 67 ROIs associated with micro-vessels were selected.", "The same parameters were calculated and normalised to the intensity of the largest artery signal.", "From these, mean and standard deviation for each parameter associated with each vessel type were calculated because the distributions of these parameters were Gaussian.", "One-way analysis of variance (ANOVA) with a post hoc Tukey test (SPSS Statistics 25, IBM, Portsmouth, Hampshire, UK) were performed to determine if differentiation of the vessel types was achievable from flow characteristics of individual small ROIs.", "Figure 1a shows the TICs for the large overlapping ROIs.", "ROI A is within the CL and contains the smallest vessels in the ovary, whereas ROI B is the whole ovary which includes the entire CL structure with its vascular supply.", "ROI B (red) has the broadest TIC due to the largest veins and arteries that branch out from the ovarian artery and vein, and the main vessels which connect to the CL.", "In ROI A (yellow), the peak is lower than that of the ROI B because the region contains a higher proportion of smaller vessels and the bubble density is lower on average.", "Furthermore, ROI A has a narrower, later-to-peak curve as the smaller vessels fill after the feeding arteries and empty before the veins.", "On the other hand, ROI B produces a curve which is earlier to peak as the signal is dominated by the large arteries.", "These arteries have high concentrations of contrast and feed the vessels in the CL.", "Figure 1b shows the relative proportions of vessels in different size ranges within two ROIs of the CL.", "The data presented are the mean of six ovaries from six different sheep.", "ROI A within the CL is primarily (95%) composed of vessels less than 30 µm diameter.", "For ROI B, approximately 2% of the total vessel content are vessels which are larger than 200 µm diameter.", "These larger vessels are not located within ROI A, and the actual proportion of large vessels will be higher for ROI B when the larger vessels, removed during excising and processing, are included.", "Note, that although vessels larger than 200 μm are only 2% of the total vessel number, it is their volume that matters as the area they occupy in the ultrasound image and the blood volume within is largest, and thus, the contrast intensity is highest.", "This results in a much larger intensity for ROI B compared to ROI A in Figure 1a.", "Figure 1c shows the ratio of the MTT (ROI B)/MTT (ROI A) along with the same ratio for WIT and their correlation with the ratio for AUC (Averkiou et al. 2010).", "Both the MTT and WIT ratios have positive correlation coefficients with the AUC ratio (0.69 and 0.83, respectively).", "Based on this information it was concluded that the proportion of different vessels within a ROI impacts on the flow kinetic parameters derived from the fitted flow model.", "The larger ROI B provides larger intensity (AUC) and this appears to be associated with longer transit time (WIT and MTT).", "In general, transit time varies between different vessel sizes, as well as the starting time of the TIC of each vessel (e.g., a vein TIC starts later than an artery TIC).", "Because vessels of different type and size appear in the same image, it is expected that the TIC of any given ROI is a sum of the TICs of the different vessels that appear in the ROI.", "In the CL, ROI A is dominated by microvascular flow, whereas ROI B surrounding ROI A is dominated by medium to large vessel flow, thus providing a good initial knowledge on the locations of different vessel types.", "This leads to the hypothesis that conventional CEUS is able to provide information on flow behaviour of specific vessel types.", "This was further investigated using multiple, small regions of interest as follows.", "Typical examples of TICs for 2 × 2 pixel ROIs are shown in (Fig. 2a–c).", "The best fit was seen in regions with the strongest signal (i.e., the highest contrast concentration such as the supply arteries [Fig. 2a]); the artery ROI was chosen based on the first large vessel to fill in the ultrasound video.", "The worst lognormal fit was for micro-vessels less than 30 µm (Fig. 2c, 2d); the associated ROI was chosen from the highly microvascular central area of the CL.", "For low signal-to-noise TICs, as in Figure 2d, where parameters such as MTT are overestimated due to the broad, low shape of the fitted curve, the parametric images were adjusted accordingly.", "In particular, the region of overestimation was assumed to contain very small vessels where parameters were set to the minimum measurable value in the data set (e.g., those which would be calculated from the curve type of Figure 2c).", "The TICs for small ROIs within regions determined to be arteries and veins peak at different times as expected due to the flow dynamics through the ovary.", "The difference in mean MTT for 35 vein ROIs and 53 artery ROIs (from 13 different videos) was 3.1 ± 1.8 s. Similarly, for Tp, the difference between vein and artery ROIs was 3.7 ± 1.5 s.", "There was good reproducibly between parameters calculated from TICs in consecutive videos of the same ovary.", "Table 1 shows the minimum and maximum mean values for each parameter, over all videos assessed, with the corresponding standard error.", "Example parametric maps are displayed in Figure 3.", "The larger veins (blue arrow) and arteries (red arrow) are clearly seen toward the bottom of each image; the artery and vein were confirmed by surveying the video sequence.", "The area comprising smaller vessels is known to be the upper region and central region of the image plane.", "In Figure 3, the R2 parameter map is consistently bright for the all vessel types apart from the smallest capillaries.", "It is also seen that the Ip, AUC, WIR and WOR values are higher in the large arteries (red arrow in Ip map), thus making the main arteries and other arterioles (yellow arrow) the dominant vessels in each respective map.", "On the other hand, for the MTT, WIT and WOT of the vein produce the dominant signal (blue arrow).", "Capillaries generally provide the lowest parameter values throughout apart from the MTT, where they fall within similar values to all other arteries.", "Figure 4 illustrates the mean value for each parameter with error bar of one standard deviation across different vessel types.", "Significance for each parameter was determined by one-way ANOVA with a post hoc Tukey test used to identify the vessel groups between which there was significant difference.", "The vessel groups within each parameter which were significantly different (p < 0.05) are indicated.", "As with Figure 3 the bar chart highlights, with statistical significance, that the intensity-dependent parameters have artery dominant behaviour (Ip, AUC, WIR, WOR) and time-dependent parameters have vein dominant behaviour (MTT, WIT, WOT).", "The capillaries are also confirmed to have the lowest values in all parameters apart from the MTT.", "From the data it was determined that there are values and relative values of parameters which can be attributed to a specific vessel type.", "Figure 5 shows the output from analysis for a further three different sheep ovaries including the one that has undergone vascular regression (CL4) including (a) the colour Doppler images, (b) the image from the peak contrast frame throughout the video, (c) the parameter map for Ip and (d) the tiled microscopy image (histologic evaluation) confirming the high vascular density within the region of the CL.", "The histology slices are in general agreement with the overall vascular structure shown in the ultrasound images.", "For each CL, the central area is densely populated with micro-vessels and larger vessels are found toward the outer region.", "Yet, it is well understood that a quantitative comparison between histologic slices and medical imaging data is not easy to achieve.", "However, there are several arrowed features (orange arrow) that confirm a strong similarity between the parametric maps and the histologic slices, mainly for the medium to large vessels.", "Although the resolution of the original CEUS is not fundamentally different from the parameter map, the latter provides some structure detail that is not distinguishable in the contrast video.", "On the other hand, visual inspection of the colour Doppler data, the current method for locating large blood vessels in vivo, provides cruder information on the location of the largest vessels, overestimating the actual size of the large vessels.", "Figure 5 CL4 shows proof-of-principle data from an ovary which had been treated with prostaglandin to induce vascular regression of the CL.", "Of particular importance is the capillary dense area.", "In the normal CLs (Fig. 5 CL2 and CL3), vascular flow is depicted by the presence of a network of arteriolar flow (medium-sized vessels).", "From histology we know that only vessels less than 200 μm are located within the central region of the CL.", "The histology also shows that the vascular density is significantly decreased in the centre of the CL.", "Although small submillimetre cysts may appear in normal mature CLs, the size of the vacant space exceeds 3 mm.", "In the regressed CL this network is absent, thus making the inner CL area appear dark in the parameter map, indicating the absence of vessels in that region.", "This study of time-intensity kinetics of ultrasound contrast demonstrates that vessel differentiation is possible within the standard resolution of CEUS.", "Our study confirms the work of others that makes use of TICs from single pixels to extract parameters which can be used to identify the nature of vascular structure (Kuenen et al. 2011; Kuenen et al. 2013; Pitre-Champagnat et al. 2015).", "We have shown that large ROI analysis using CEUS is generally burdened by significant uncertainties and these should be replaced by parametric map analysis.", "Studies utilising TICs to assess perfusion and blood volume use large ROIs which span multiple sized vessels in an organ (Averkiou et al. 2010; Lassau et al. 2010).", "Given that an ultrasound image covers large areas, including both microvasculature and larger vessels, ROI analysis, as used in Figure 1, results in erroneous estimation of most parameters compared to the values that can be found in pixel analysis.", "The structure of the ovine ovary enabled the deduction of vessel types in different areas of the parametric maps.", "The CL is a transient gland that needs to grow fast and reach maturity within 9–12 d; it has a dense central microvasculature and most importantly the larger vessels are located in the periphery of the tissue (Fig. 1b), which is clearly visible in the parametric maps.", "The findings here suggest that the inclusion of all vessel types in ROI analysis increases the likelihood of systematic errors, particularly for transit time–related parameters as supported by calculations of the mean difference in MTT (3.1 ± 1.8 s) and Tp (3.7 ± 1.5 s) between veins and arteries.", "Veins fill with contrast several seconds later, and have longer contrast transit, compared to arteries.", "This explains the discrepancy between ROI A and ROI B in Figure 1 and the volume-dependent calculation of transit-related parameters shown in Figure 1c.", "In normal tissue, millimetre-sized arteries and veins often are spatially paired, as is the case for the vessels that support the CL.", "Thus, the overall transit measured in a large ROI is augmented by the presence of both arterial and venous flow.", "The high reproducibility in WIT within the CL in repeated injections and identical scan planes was previously established to below 10% and as low as 3% (Sboros et al. 2011).", "The results here suggest that such low uncertainties are plausible in all tissue examinations if the kinetics of different vessels are investigated using higher resolution parametric maps.", "In addition to rectifying larger vessel induced uncertainties, parametric maps could be further deployed to assess tumour perfusion kinetics.", "Dynamic CEUS imaging is a tool for assessing tumour perfusion (Dietrich et al. 2012).", "Parametric imaging has provided additional information on tumour blood volume changes, microvascular density and the assessment of vascular changes in response to treatment (Panfilova et al. 2019; Pitre-Champagnat et al. 2015; Rognin et al. 2010; Williams et al. 2011).", "However, to date, it has not been possible “to distinguish the different categories of vessel diameter or their maturity” (Pitre-Champagnat et al. 2015).", "In contrary to the CL, for most tissue types larger vessels are structurally more mixed with the microvascular bed, and it is difficult to have a clear anatomic separation between vessel types.", "In particular, tumours present vascular heterogeneity which makes the distinction of different vessel sizes difficult (Panfilova et al. 2019; Pitre-Champagnat et al. 2015).", "The work here shows that the combination of high-contrast and temporal resolution enable this delineation.", "By utilising all the available information from the parametric maps (Fig. 4) it is possible to effectively differentiate vessel types.", "The results here show that the microvascular bed has the lowest values in most calculated flow parameters apart from the MTT.", "Large arteries produce large values for parameters such as Ip, AUC, WIR and WOR, whereas veins dominate time-dependent parameter maps such as MTT, WIT and WOT (Fig. 4).", "These are expected results.", "The main feeding arteries have the shortest time from injection to Ip and veins have the longest.", "Flow kinetics after injection produce a lower contrast agent density with time; therefore, lower echo intensities and higher transit times are found further downstream (Hudson et al. 2015).", "This information coupled with prior knowledge may form the basis for vessel classification in the future, even at micro-vessel level.", "Vessels that are larger than the resolution limit appear to be depicted more accurately compared to colour Doppler (Fig. 5), but further work is required to establish the accuracy of sizing these vessels.", "The 2 × 2 pixel ROIs may contain more than one vessel type.", "Even in the case of larger, millimetre-sized, arteries and veins, it is likely that signal from other overlapping vessels is captured in the same pixels.", "In light of this, the hypothesis for differentiating vessel types through parametric maps refers to ROIs where the dominant signal originates from one type of vessel and likely the largest vessel in the ROI.", "In the example, parametric maps regions of venous flow can be differentiated from arterial flow irrespective of its direction of flow (blue and red arrows in Fig. 3).", "When the size of the vessels is much smaller than the resolution of the image, their depiction will not provide an accurate measurement of their size.", "Each pixel is 140 × 140 µm (therefore the parametric map has pixel size 280 × 280 µm), and the ultrasound image plane has a thickness of at least 1 mm.", "The classification of the flow kinetics (Fig. 2) matches the broad vessel groupings in the histology, but cannot provide clarity on the sizes of the different vessel types.", "Figure 2c shows a strong signal and is likely to include signals from arterioles up to several tens of micrometres.", "Generally, micro-vessels do not provide a good fit to the lognormal model (Strouthos et al. 2010) due to their small contrast volumes that produce a noisy signal (Fig. 2c, 2d).", "However, the different intensity and temporal characteristics of the different vessel types should be further explored in the future to enable reproducible vessel differentiation.", "The recent development of SRI (Christensen-Jeffries et al. 2015; Errico et al. 2016; O'Reilly et al. 2014; Opacic et al. 2018) may be coupled with the vessel classification proposed here.", "SRI promises to provide significant resolution gains in vascular mapping that may reach above 10-fold improvement (van Sloun et al. 2018).", "As explained in the introduction, a clinically applicable SRI method may require larger concentrations of microbubbles that ensure the image is well populated by scatter resulting in the larger vessels having very dense contrast.", "Therefore, hybrid methodologies may be developed that also deploy information from high-density MB distributions, such as the one presented here.", "Such an advance may reduce acquisition time, and thus patient examination time, by making optimal use of the available data at larger MB concentrations and the spatiotemporal information they contain.", "For vessels with large concentrations, a solution may be available through a largely heuristic approach proposed by Perperidis et al. by creating binary masks which highlight (as verified by associated histology slices) larger vessels (mostly around the perimetry of the CL).", "Yet, this approach needs to be treated with caution as it tends to overestimate the regions occupied by the medium-sized vessels.", "One way to look at this is as a sparsity problem that may require a highly supervised approach.", "The low frame rate translates into undersampling of larger vessels with high velocities (i.e., 10–15 Hz is inadequate to sample fragments of bent vessels that have blood speeds above 1 cm/s) also the microbubble transit is finite (at least 2 min).", "Thus, the improvement of resolution requires the deployment of prior knowledge on the vascular flow and structural properties that have to compensate the sampling problem facing SRI.", "Currently, the literature relies on localisation and tracking approaches with little input from prior knowledge.", "Deep learning has also been attempted to provide SRI and has shown promise as there was also a significant resolution gain (van Sloun et al. 2018).", "Knowledge on the nature of the vessels may help by providing associations between microbubble density, velocity and vessel size.", "In addition, traditional machine learning methodologies, adopting a classifier (such as Support Vector Machines) on a handcrafted 8-dimensional feature space (incorporating all parametric maps) (Boser et al. 1992; Pratondo et al. 2017) or custom convolutional neural networks directly on the 2-D + time data sets are anticipated to improve the classification between larger vessels and microvasculature.", "Binary masks and future iterations may provide hybrid methodologies that enable the identification of disease state or response to therapy, by means of automated vascular structure classification.", "An important limitation in this study is the comparison with a gold standard of histology.", "A known consequence of tissue processing for microscopic evaluation is tissue shrinkage and shape distortion (Kerns et al. 2008).", "Histology evaluates slices with a thickness of a few micrometres that are physically impossible to match exactly with the ultrasound data.", "Ultrasound images are routinely compared with histology to evaluate prostate lesion findings.", "In these studies, the comparison is performed in 3-D reconstructed volumes.", "This process results in substantial spatial errors of a few millimetres (Wildeboer et al. 2018).", "Consequently, no direct quantitative comparison between the vascular structure of the CL and the ultrasound video data was performed here.", "Histology was used as a guide, in conjunction with well-understood overall structure of the ovary and CL in the deduction of the vessel types.", "Finally, the parameter map (CL4 Fig. 5) shows regions where vascular regression, induced by prostaglandin, has occurred.", "This could be used to monitor tumour regression or to locate tumour necrotic regions.", "The compromised vascular bed is clearly seen as a large region devoid of vascular structure.", "Specifically, and as noted in the results section, it is the lack of medium vessels in the central region which indicates the absence of vascular structure.", "It is known that better knowledge of the flow characteristics of ultrasound contrast agent through a vascular network can aid in diagnosis (Eisenbrey et al. 2013).", "TIC analysis has been previously shown to be a good method by which to assess vascular regression of tumours in a range of solid tumour types (Lassau et al. 2014; Lassau et al. 2012).", "AUC has been shown to be the most significant parameter for indication of tumour stability when the whole tumour was selected as the ROI (Lassau et al. 2014).", "The use of all available pixel kinetics information may lead to improved characterisation of therapeutic vascular regression.", "This study demonstrates the ability of parametric maps, across a regular grid of TICs, derived from CEUS frame sequences, to assist in the visual differentiation of different vessel types, including arteries, veins and capillaries.", "Parameters associated with the differentiation of specific structures have been identified.", "Furthermore, parametric mapping has demonstrated the potential to indicate regions where vascular regression has occurred and can potentially be used to monitor tumour regression.", "Classification algorithms can potentially use this information to generate binary masks differentiating microvasculature from larger vessels, thus potentially enriching current super-resolution methodologies with much needed prior knowledge." ]
[]
Mutations in the TaPIN1 peptidyl prolyl isomerase gene in Theileria annulata parasites isolated in Sudan
The tick-borne parasite Theileria annulata is the causative agent of tropical theileriosis or Mediterranean theileriosis. Infection of bovine leukocytes by the obligate intracellular parasites induces proliferative and invasive phenotypes associated with activated signaling pathways. The transformed phenotypes of infected cells are reversible by treatment with the theilericidal drug buparvaquone. Recent reports of resistance to buparvaquone in Africa and Asia highlight the need to investigate the mechanisms and prevalence of drug resistance. We screened 67 T. annulata isolates from Sudan to investigate mutations in the T. annulata prolyl isomerase I gene (TaPIN1). The secreted TaPin1 interacts with host proteins to induce pathways driving oncogenic transformation and metabolic reprogramming. We found an Alanine-to-Proline mutation at position 53 (A53P) in the catalytic loop that was previously found in Tunisian drug-resistant samples. This is the first study reporting independent confirmation of the A53P mutation in geographically isolated samples. We found several additional mutations in the predicted N-terminal signal peptide that might affect TaPin1 processing or targeting. We found that many parasites also share mutations in both the TaPIN1 and the cytochrome b genes, suggesting that these two genes represent important biomarkers to follow the spread of resistance in Africa, the Middle East and Asia.
[ "IMMU", "MEDI" ]
[ "Drug-resistance", "Markers", "Point mutations", "Prolyl isomerization", "Theileria", "Theileriosis" ]
[ "2405", "2725", "2736" ]
[ "Bovine Tropical Theileriosis (BTT) is a lymphoproliferative, tick-borne disease of cattle caused by the protozoan parasite Theileria annulata.", "The intracellular Theileria parasites transform mammalian host cells by inducing cancer-like phenotypes, such as immortalization, dissemination and hyper-proliferation of the infected leukocytes (Morrison, 2009).", "During the pathogenesis of the disease, the infected host cells spread throughout the animal, causing enlargement of lymph nodes and eventual lethality.", "Theileriosis has been reported in southern Europe, North Africa, India, China and across Asia with substantial socio-economic impact on farming and veterinary health.", "The molecular mechanisms underlying host cell transformation by Theileria parasites likely involve changes in signaling pathways that affect the host epigenome and cause changes in host cell gene/protein expression and activation (Cheeseman and Weitzman, 2015; Lüder et al., 2009; Morrison, 2009).", "For example, Theileria infection of bovine leukocytes modulates oncogenic signaling pathways such as JNK and IKK (Heussler et al., 2002; Lizundia et al., 2007) and activation of host transcription factors such as c-Myc, AP-1 and HIF-1α (Chaussepied et al., 1998; Dessauge et al., 2005; Marsolier et al., 2013; Medjkane et al., 2014; Metheni et al., 2015).", "Furthermore, Theileria parasites interact with host microtubules and recruit end-binding protein 1 to the parasite surface (von Schubert et al., 2010; Woods et al., 2013).", "We recently identified the parasite-encoded Peptidyl Prolyl Isomerase Pin1 (designated TaPin1) in T. annulata as a secreted protein (Marsolier et al., 2015).", "The parasite TaPin1 protein can interact with host cell proteins and modulates oncogenic signaling pathways For example, the TaPin1 prolyl isomerase interacts with the host ubiquitin ligase Fbw7, leading to its degradation and subsequent stabilization of c-Jun which promotes host cell transformation (Marsolier et al., 2015).", "TaPin1 can also stabilize the host pyruvate kinase isoform M2 (PKM2) leading to HIF-1α-dependent metabolic reprogramming (Marsolier et al., 2019).", "Parasite-induced transformation of host cells is reversible with the anti-Theilerial drug buparvaquone (McHardy et al., 1985).", "Buparvaquone administration results in significant damage of both schizont and piroplasm stages of T. annulata causing reduction of these parasitic stages to low or undetectable levels within 7 days (Mhadhbi et al., 2010).", "At least one mechanism of buparvaquone action may be via inhibition of TaPin1 isomerase activity (Marsolier et al., 2019, 2015).", "The socio-economic impact of Theileriosis has been exacerbated by the emergency of resistance to buparvaquone, the frontline drug in the treatment of BTT.", "Recently, cases of buparvaquone-resistant T. annulata isolates were reported in Sudan, Tunisia, Iran and Turkey (Chatanga et al., 2019; Mhadhbi et al., 2015, 2010; Sharifiyazdi et al., 2012).", "Drug-resistance poses a serious threat to the dairy industry in the endemic countries where the disease continues to cause significant losses due to deaths, as well as operational costs to mitigate the impact of the disease.", "Several of these cases have been linked to mutations in the parasite mitochondrion cytochrome B gene (Chatanga et al., 2019; Mhadhbi et al., 2015; Sharifiyazdi et al., 2012).", "We also reported mutations in the TaPIN1 gene in buparvaquone-resistant parasite isolated in Tunisia (Marsolier et al., 2015).", "Specifically, an alanine to proline mutation at residue 53 (A53P) in the catalytic loop of TaPin1 caused loss of sensitivity to buparvaquone in recombinant TaPin1 protein (Marsolier et al., 2015).", "Both in vitro and in vivo experiments demonstrated that mutant TaPIN1 was not inhibited by PIN1 inhibitors, such as anti-Theilerial drug buparvaquone (Marsolier et al., 2015).", "Here, we report studies on T. annulata resistance to buparvaquone in isolates from Sudan.", "We sequenced the parasite TaPIN1 gene to determine the extent of mutations in the resistant isolates.", "Our results show that mutations in TaPIN1 may be widespread in drug-resistant parasites.", "We examined sixty-seven DNA samples from blood from cattle clinically diagnosed, and confirmed by both Giemsa blood smears and polymerase chain reaction (PCR), to be infected with T. annulata in Sudan.", "This was a parallel study of our initial study (Chatanga et al., 2019) which was approved by the University of Khartoum, Sudan (Approval no. 2017BS), and Sudan University of Science and Technology, Khartoum, Sudan (Approval no.28–46), and informed consent was sought from animal owners.", "Thus, no separate approval was required.", "The samples were collected from Omdurman (15.38 °N, 32.28 °E) (n = 17) and Khartoum North (15.63 °N, 32.56 °E) (n = 31) and Khartoum (15.55 °N, 32.53 °E) (n = 19) in December 2016 to March 2018.", "These samples were collected from farms where buparvaquone is actively used in the treatment of bovine tropical theileriosis in Khartoum state, Sudan.", "These samples were collected from cattle clinically diagnosed and confirmed by Giemsa blood smears to be infected with T. annulata.", "These samples came from farm owners who specifically reported that these animal were not responding to buparvaquone treatment at the normal dose.", "DNA was extracted using DNAzol BD Reagent (Invitrogen, MA, USA) or QIAmp DNA Blood Mini kit protocol (Qiagen, Tokyo, Japan) and stored at −20 °C until analysis.", "PCR was performed using the primers TaPIN1 forward 5′-GTCTGTCAAATAGGTAGAAATC-3′ and reverse 5′-GAGAGGAAGTTGAATCAAACAT-3′ to amplify the 526 bp encompassing the full length of the gene.", "The reaction was conducted in a 25 μl reaction mixture containing 12.5 μl of 2xGflex PCR Buffer (Mg2+, dNTP plus), 0.5 μl of Tks Gflex DNA Polymerase 1.25 units/μl (TaKaRa Bio Inc., Shiga, Japan), 200 nM of each primer, 1.0 μl of template DNA and molecular-grade water.", "The cycling conditions were initial denaturation at 94 °C for 1 min followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 57 °C for 15 s, extension at 68 °C for 45 s and final extension at 68 °C for 5 min.", "The amplicons were electrophoresed in 1.5% agarose gel stained with Gel-Red (Biotium, Hayward, CA, USA) and visualized under UV light.", "The amplicons were purified using the NucleoSpin Gel and PCR Clean Up Kit (TaKaRa Bio Inc.) and sequenced using the Big Dye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) utilizing an ABI Prism 3130 x genetic analyser (Applied Biosystems) according to the manufacturers' instructions.", "Two sequence fragments were generated for each sample and ATGC software version 9.1 (GENETYX Corporation, Tokyo, Japan) was used for manual editing to correct possible base calling errors and remove primer annealing sites.", "The regions of unambiguously aligned sequences were eventually joined to reconstruct sequences of 482bp of T. annulata TaPIN1 gene which were retained for final analysis.", "The 482 bp nucleotide sequences corresponding to the PPlase TaPIN1 were successfully sequenced for 67 T. annulata samples from Sudan and the sequences obtained in the present study were submitted to the Mendeley Data Repository.", "These consensus sequences were translated into 143 amino acids and the protein multiple alignment was generated by Molecular Evolutionary Genetics Analysis (MEGA) and viewed with Jalview softwares respectively (Waterhouse et al., 2009) using TaPIN1 (PiroplasmaDB: TA18945) as reference sequence.", "We aligned the amino acid sequences corresponding to the TaPin1 protein from the 67 Sudanese isolates and compared with the annotated, reference sequence for T. annulata PIN1 PPlase (TA18945).", "We discovered 8 positions in the isolates from Sudan where the sequences differed from the reference TA18945 sequences (Fig. 1, Table 1).", "Interestingly, nearly two thirds of the isolate sequences (40/67) had a sequence that differed from the annotated TaPin1 sequence.", "This is even higher than the number of mutations detected (18 sequences) in the T. annulata cytochrome b gene in our previous study (Chatanga et al., 2019).", "And nine of the isolates presented two mutations in the TaPIN1 gene (Fig. 1, Table 1).", "Sixteen of the isolate sequences had a M1L mutation and two of the sequences had a STOP143Y mutation.", "These mutations would likely result in the loss of TaPin1 protein, so we cannot speculate on their functional relevance.", "A comparison of the TaPIN1 results with analysis of the cytochrome b mutations revealed that 11 (16%) of the isolates presented mutations in both the parasite genes (Fig. 2).", "In a previous study of buparvaquone-resistant parasites isolated in Tunisia, we reported the identification of a mutation (A53P) in the catalytic loop of TaPin1 which could account for drug resistance (Marsolier et al., 2015).", "TaPin1 protein carrying this mutation maintained prolyl isomerase activity, measured by an in vitro assay for catalytic isomerization, but was no longer inhibited by buparvaquone treatment (Marsolier et al., 2015).", "Interestingly, one of the Sudanese samples (isolate B241) contained the same A53P mutation that predicts a functional isomerase which is not functionally inhibited by buparvaquone (Fig. 1, Table 1).", "This is the first independent confirmation of this mutation in two geographically distinct isolates.", "Our initial analysis of the Tunisian isolates had identified additional mutations that we did not pursue at the time as they fell within the sequence that we predicted as a signal peptide that could be cleaved upon TaPin1 secretion (Marsolier et al., 2015).", "These included, T22I and A26L.", "Intriguingly, we found a number of mutations in the Sudanese isolates that also affected residues in the proposed signal peptide.", "These included T22A, I23N, I24V, A26L and A26P (Fig. 2, Table 1).", "Thus, three of the positions mutated in the Tunisian samples (residues 22, 26 and 53) are also mutated in the Sudanese samples (accounting for 25 of the isolated samples).", "The emergence of resistance to anti-parasite drugs should drive our search for drug targets and the mechanisms of resistance.", "BTT is a case in point, as resistance to buparvaquone, the leading drug treatment for Theileriosis, has been observed across North Africa.", "Here, we report the discovery of mutations in the TaPIN1 gene in isolates from Sudan.", "Indeed, 40 of the 67 Sudanese sequences (60%) have a mutation that differs from the TaPIN1 reference sequence.", "Notably, we report the first independent confirmation of the A53P mutation previously characterized in drug-resistant T. annulata parasites from Tunisia (Marsolier et al., 2015).", "This result strengthens our initial claim that TaPin1 could be a direct target for buparvaquone as the same functional mutation has now been described in two isolates from distant geographical locations.", "The mutation to Proline in the TaPin1 catalytic loop is likely to induce the conformational alterations affecting access of buparvaquone to the PPlase active site.", "We also observed several other mutations, particularly at positions 22, 23, 24 and 26.", "Interestingly, two of these (T22I and A26L) were also found in the Tunisian samples (Marsolier et al., 2015).", "These positions are unlikely to affect the catalytic activity directly, but they could affect the secretion, processing or targeting of the TaPin1 protein.", "As noted previously, the signal peptide sequence is not found in non-transforming Theileria species, but is conserved in T. parva.", "We do not currently have any experimental evidence that the predicted signal peptide is actually cleaved.", "It is possible that this N-terminal sequence is critical for targeting to a particular organelle or for proteins interactions, and this merits future investigation.", "In addition, two sample sequences had a *143Y mutation, representing a change in the STOP codon; it is unclear at the moment what the functional consequences of a longer protein could be.", "Another important finding of our study is the overlap with mutations in the T. annulata cytochrome b gene.", "Several reports have shown mutations in this gene in drug-resistant isolates from around the world (Chatanga et al., 2019; Mhadhbi et al., 2015, 2010).", "But this is the first time that both genes have been investigated in the same samples and the high degree of overlap suggests that perhaps both genes contribute together to drug resistance.", "Futhermore, while 40 out of the 67 samples have a mutation in either of these two genes, we currently have no indication of what could account for the bupavaquone-resistance in the other 27 samples, suggesting that additional genes remain to be discovered.", "Our results highlight the need to develop molecular marker(s) and phenotypic tests to detect the occurrence of resistance and determine its prevalence in the population (Mhadhbi et al., 2015).", "TaPIN1 and cytochrome b should both be used as markers to screen resistant strains of T. annulata to buparvaquone in field isolates.", "In conclusion, this study of field isolates from Sudan extends our understanding of buparvaquone resistance in T. annulata parasites.", "We confirm the importance of the A53P mutation in the catalytic loop and identify additional N-terminal mutations that could affect TaPin1 processing or targeting.", "Finally, the high frequency of mutations in the TaPIN1 and cytochrome b genes suggests that both genes should be used as molecular markers to detect the spread of resistance in the field across endemic areas in Africa and the Middle East.", "All data generated or analyzed during this study is publicly available on the Mendeley Data Repository." ]
[]
Glutamatergic correlates of gamma-band oscillatory activity during cognition: A concurrent ER-MRS and EEG study
Frequency specific synchronisation of neuronal firing within the gamma-band (30-70. Hz) appears to be a fundamental correlate of both basic sensory and higher cognitive processing. In-vitro studies suggest that the neurochemical basis of gamma-band oscillatory activity is based on interactions between excitatory (i.e. glutamate) and inhibitory (i.e. GABA) neurotransmitter concentrations. However, the nature of the relationship between excitatory neurotransmitter concentration and changes in gamma band activity in humans remains undetermined. Here, we examine the links between dynamic glutamate concentration and the formation of functional gamma-band oscillatory networks. Using concurrently acquired event-related magnetic resonance spectroscopy and electroencephalography, during a repetition-priming paradigm, we demonstrate an interaction between stimulus type (object vs. abstract pictures) and repetition in evoked gamma-band oscillatory activity, and find that glutamate levels within the lateral occipital cortex, differ in response to these distinct stimulus categories. Importantly, we show that dynamic glutamate levels are related to the amplitude of stimulus evoked gamma-band (but not to beta, alpha or theta or ERP) activity. These results highlight the specific connection between excitatory neurotransmitter concentration and amplitude of oscillatory response, providing a novel insight into the relationship between the neurochemical and neurophysiological processes underlying cognition. © 2013 Elsevier Inc.
[ "NEUR" ]
[ "Evoked gamma-band oscillatory activity", "Functional MRS", "Glutamate", "Repetition suppression", "Repetition-enhancement", "Repetition-priming" ]
[ "2805", "2808" ]
[ "Converging evidence from multiple neuroscience disciplines indicates that frequency-specific temporal synchronisation of neuronal firing is critical for the co-ordination of neuronal network assemblies underlying both basic sensory and motor processing (Singer, 1999; Womelsdorf et al., 2006) as well as a variety of cognitive functions (for a recent review see for instance:Herrmann et al., 2010; Jensen et al., 2007).", "In both humans and animals, synchronisation in the gamma-band frequency (30–70 Hz) is considered to play an important role in local cortical information processing (Fries, 2009).", "Gamma-band oscillatory activity can typically be observed as an early evoked and a later induced response (Tallon-Baudry and Bertrand, 1999).", "Evoked oscillatory activity is tightly time-locked to stimulus-onset and typically occurs less than 250 ms after stimulus onset.", "Evoked gamma-band activity is modulated by physical stimulus properties and there is increasing evidence showing evoked gamma-band oscillatory modulation also by higher cognitive processes, like memory, attention and context processing (Busch et al., 2008; Debener et al., 2003; Fründ et al., 2008; Haenschel et al., 2000; Herrmann et al., 2004; Oppermann et al., 2012; Roye et al., 2010).", "The induced gamma-band response occurs after a variable time lag across trials, and reflects the subsequent processing stages within cortical networks (Hassler et al., 2011; Martinovic and Busch, 2011).", "Both evoked and induced gamma band activity have been linked to in-vitro measured local field potential gamma-band oscillatory activity (Haenschel et al., 2000; Hall et al., 2005; Ronnqvist et al., 2013).", "As the importance of gamma-band oscillatory activity has become increasingly appreciated (e.g. Fries, 2009) a considerable effort has been made to reveal the neural mechanisms for its generation.", "In-vitro studies of changes in neuronal assembly activity in response to specific pharmacological agents indicate that gamma-band activity can be generated by local networks of chemically and electrically coupled inhibitory gamma-aminobutyric acid (GABA)-ergic interneurons, and controlled by excitatory glutamatergic receptor activation (Fuchs et al., 2007; Whittington et al., 1995).", "More specifically, gamma oscillations can be elicited by tonic activation of ionotropic glutamate kainate, as well as cholinergic receptors (Buhl et al., 1998; Cunningham et al., 2003; Rodriguez et al., 2004; Wespatat et al., 2004), and transiently by activation of ionotropic (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AMPA, N-methyl-d-aspartate; NMDA) or metabotropic glutamate receptors.", "Combining both in-vitro and in-vivo techniques, research in animal models confirms that the balance between excitation and inhibition modulates the gamma-band oscillatory frequency (Atallah and Scanziani, 2009).", "The links between gamma-band activity and changes in neurotransmitter concentrations within humans remain largely unknown.", "Fortunately, proton magnetic resonance spectroscopy (1H-MRS) allows in-vivo measurement of neurotransmitter concentrations (such as GABA and glutamate) in humans, and can be combined with neurophysiological measures of oscillatory activity.", "A recent study measured resting-state GABA concentration in the visual cortex across individuals and correlated these levels with subsequent measures of peak induced gamma-band oscillatory frequency during a simple visual task measured with magnetoencephalography (Muthukumaraswamy et al., 2009).", "However, static measures of neurotransmitter concentration during a resting-state period may ignore critical changes in concentrations that occur during task performance.", "Furthermore, a direct relationship between glutamate levels and gamma-band activity, in either peak frequency or power, has not been demonstrated.", "1H-MRS is well established to provide reliable static resting-state measures of glutamate concentration, with several optimal acquisition schemes being suggested (Hancu, 2009; Henry et al., 2011; Jensen et al., 2009; Mayer and Spielman, 2005; Mullins et al., 2008; Schubert et al., 2004).", "Recently, functional changes in glutamate levels due to experimental manipulation have also been reported (Mangia et al., 2007; Mullins et al., 2005).", "Nonetheless, the abovementioned 1H-MRS experiments used long blocks of repetitive stimulation (5 + minutes), potentially causing glutamatergic response attenuation through stimulus adaptation.", "Surmounting this limitation, Gussew et al. (2010) instead acquired time-locked spectral measures of glutamate levels post-stimulus onset (in response to pain).", "Here, we wish to develop this method further by acquiring event-related 1H-MRS (ER-MRS) measures of glutamate levels during a cognitive task, in a similar vein to event-related fMRI designs, while also simultaneously collecting EEG.", "Glutamate, the primary excitatory neurotransmitter, is released by approximately 80% of synapses (Magistretti et al., 1999) and is considered to play a fundamental role in learning and memory.", "Presynaptic glutamate release activates three different glutamate gated ion channels on postsynaptic membranes: NMDA, AMPA and kainate receptors.", "Both AMPA and NMDA receptor activity have been shown to jointly modulate learning and memory, whereas the role of kainate receptors in synaptic plasticity is less well understood.", "AMPA receptors are more common and mediate fast excitation, whereas NMDA receptors generate a much slower and longer-lasting current, and, in addition are important for Ca2 +-dependent plasticity.", "The classical view is that AMPA receptors affect short-term changes in synaptic strength; whereas NMDA receptors regulate genes that are required for the long-term maintenance of these changes (Myme et al., 2003; Rao and Finkbeiner, 2007).", "Indeed, long-term potentiation, the proposed mechanism of learning and memory, can be abolished by blocking glutamatergic NMDA receptors (Bliss and Collingridge, 1993), with consequent impairment in learning and memory (Morris, 1989).", "However, more recent evidence suggests that both AMPA and NMDA receptors have a role in long-term plasticity (Rao and Finkbeiner, 2007).", "NMDA and AMPA receptors are scaled proportionally so that the ratio of currents through these channels is relatively fixed, which may help to preserve the information content of synaptic transmission or may play an important role in coupling synaptic activity to long-term modification via gene expression (Myme et al., 2003; Rao and Finkbeiner, 2007).", "Hence, glutamate is likely to play an important role in the formation of learning related networks.", "To assess the temporal relationship between event-related modulations of glutamate concentration and gamma-band activity, we simultaneously recorded stimulus-elicited changes in both 1H-MRS and electroencephalography (EEG) during a repetition-priming task.", "We repeatedly presented pictures of familiar objects and unfamiliar abstract stimuli, respectively.", "The repetition of familiar stimuli usually results in a decrease in neural activity (repetition suppression), whereas the repetition of unfamiliar stimuli is typically accompanied by an increase in neural activity (repetition enhancement; see e.g. Henson et al., 2000; Martens and Gruber, 2012).", "Thus, repetition-priming paradigms are especially suited to examine learning related changes within cortical networks.", "We focused our analysis primarily on the early-evoked gamma responses.", "The reason for this approach was threefold:", "1H-MRS can only be measured from one voxel, hence we had to choose an area (2 cm3) to measure changes in glutamate concentration during repetition priming.", "We focused on an early visual area, specifically the lateral occipital cortex (LOC), a region known to be object selective (Grill-Spector et al., 1999; Grill-Spector et al., 2001) and also localized as a source of evoked gamma-band oscillatory activity (Gruber et al., 2006).", "There is increasing evidence showing that evoked gamma band activity is sensitive to mnemonic functioning (see above).", "Specifically, evoked gamma band activity has been shown to be larger for objects compared to abstract stimuli during speeded responses (Fründ et al., 2008; Herrmann et al., 2004) and to exhibit repetition suppression in some participants with a strong behavioural repetition-priming effect again when instructed to respond as quickly as possible (Busch et al., 2008).", "The prompt appearance of evoked gamma-band activity highlights that it is an especially good index of rapid mental processes, such as the fast processing of incoming information (Busch et al., 2008; Fründ et al., 2008; Oppermann et al., 2012).", "The quantification of induced, but not evoked, gamma-band oscillatory activity using EEG remains controversial due to the potentially confounding signal interference caused by miniature eye movements (Yuval-Greenberg et al., 2008).", "Even though there are published algorithms that can deal with such artefacts (Hassler et al., 2011), it remains an open question how to deal with these artefacts when recoding EEG data inside the MRI scanner.", "This is because the removal of miniature saccade-related and MRI-related artefacts is both based on independent component analysis (ICA).", "If ICA is applied twice and an artefact-related component is pruned in the first run of the ICA, the prerequisites for the second run of ICA (namely independence) cannot thereafter be guaranteed.", "For these reasons, we chose to instruct participants to respond as quickly and accurately as possible and to focus our analysis primarily on the early-evoked responses (especially in the gamma-band, but we also included evoked theta, alpha and beta oscillatory activity and ERPs as control analyses for the specificity of our results).", "In summary, the aim of the present study was to investigate if there was a relationship between event-related glutamate concentrations in the LOC and concurrently acquired task-specific evoked gamma-band oscillatory activity during early mnemonic processing.", "Fourteen healthy right-handed participants (8 males; M = 23.79 years, SD = 3.9) were recruited via advertisements on the Bangor University Psychology research participation forum.", "Participants reported no history of neurological disorder, had normal or corrected-to-normal vision and were financially remunerated for their time.", "Informed consent was obtained from all participants and the study was approved by the ethics committee of the School of Psychology at Bangor University, United Kingdom.", "The behavioural task has been described in detail elsewhere (Gruber and Müller, 2005).", "In brief, participants categorised sequentially presented line drawings as either object or abstract stimuli (scrambled versions of unpresented objects; Fig. 1A).", "The stimulus set comprised of 256 unique randomly selected stimuli, 128 familiar objects and 128 abstract forms.", "Each stimulus was repeated three times with between one-to-four (randomised) intervening items, thus engaging processes of visual repetition priming.", "Categorisation was performed via (counterbalanced) button press.", "Participants were instructed to respond as quickly and accurately as possible.", "Each trial consisted of a 1850–2050 ms baseline period during which a fixation cross was shown, followed by presentation of a stimulus picture for 700 ms. The stimulus was then replaced by the fixation cross which remained onscreen for 250–450 ms, such that each individual trial was 3 s in length.", "There were 768 trials in total, which were spread across three same-session runs.", "The task was presented using E-Prime 2.0 software (Psychology Software Tools, Pittsburgh, PA).", "EEG was recorded continuously at 5 kHz using a compatible EEG BrainAmp MR system (BrainProducts, Munich, Germany) and an MRI compatible BrainCap electrode cap (Falk Minow Services, Herrsching-Breitbrunn, Germany) containing 64 sintered Ag/AgCl electrodes.", "Each subject's heartbeat was measured using two bipolar electrocardiogram (ECG) electrodes placed approximately on the mid-clavicle line around the third intercostal space of the chest and the fifth intercostal space of the back.", "One additional electrode was placed below the eye to record eye-movements.", "The impedance of each electrode was maintained below 20 kΩ.", "MRI acquisition was synced to the BrainAmp MR system using a SyncBox (Brain Products).", "For the analysis, a low pass filter was set to 250 Hz and the electrodes were referenced to Cz.", "An ICA based on the Infomax (Bell and Sejnowski, 1995) optimization-learning algorithm was applied to correct for MRI specific artefacts (e.g. cardioballistic interference).", "The ICA aggregated variance associated with the ECG electrodes in a rolling averaged template, which updated (every 21 intervals of 600 ms) as it proceeded throughout the data.", "Subsequently, EEG data were visually inspected so that heartbeat and other artefacts (muscle movement and eye blinks) could be removed.", "Thereafter, data were segmented into epochs from 200 ms before, to 600 ms after, stimulus onset using Brain Vision Analyser 2 (Brain Products, Munich, Germany).", "Evoked gamma-band activity was analysed by Morlet wavelet analysis, as proposed by Bertrand and Pantev (1994), using custom Matlab (version 7.7; Mathworks, Natick, MA, USA) scripts, from 19 electrodes around central, parietal and occipital electrode positions (mounted at or around the following standard electrode positions: Cz-C2-CP2, CPz, Cz-C1-CP1, CP2-CP4, P2, Pz, P1, CP1-CP3, P6-CP6, P4-PO4, POz-PO4, POz-PO3, P3-PO3, P5-CP5, P8-PO8, O2, Oz, O1, P7-PO7).", "These electrodes were chosen based on our a priori hypothesis that evoked oscillatory gamma-band activity, our focus here, would be primarily localized to posterior regions, as they were with previous use of this task (Gruber and Müller, 2005) and because of our aim to correlate evoked gamma activity with glutamate concentration measured at LOC.", "This Morlet wavelet analysis technique permits the examination of frequency band variations across the time domain.", "Specifically, the evoked response was calculated per condition (stimulus type × presentation) for each subject by averaging the EEG signal across all trials.", "Next, the averaged signal was transformed into the time-frequency domain via convolution with the Morlet wavelet, the absolute value of this transformation was quantified and the post-stimulus-onset period was baseline corrected by the averaged 200 ms period of activity prior to stimulus-onset.", "Data from the different electrode channels were averaged following wavelet decomposition.", "In order to achieve good time and frequency resolution in the gamma frequency range the wavelet family used was defined by a constant of 7, with an analysis frequency ranging from 0.97 to 97.66 Hz in 0.49 Hz steps; statistical analysis was performed between 30 and 50 Hz.", "In addition, we analysed evoked theta (4–7Hz), alpha (8–12 Hz), and beta (12–30 Hz) band activity in order to examine if there was a relationship between these frequency bands and stimulus repetitions, as well as with glutamate concentration.", "Only correct trials were included in the analysis following artefact reduction.", "Because gamma-band activity was mainly distributed between 30 and 50 Hz, we determined the peak frequency by identifying the highest amplitude across task conditions for each participant within our time-frame and frequency range of interest.", "One participant was excluded from the oscillatory analysis and subsequent correlations because of electrical interference.", "ERP data were analysed using the Brain Vision Analyser 2 software (Brain Products, Munich, Germany).", "A 30 Hz low-pass filter (0.01 Hz high pass filter, with a 12 dB/oct slope) was applied to the data before the event related potential (ERP) analysis, prior to segmenting into epochs 200 ms before to 600 ms after stimulus onset.", "Any epoch containing residual scanner, cardioballistic, movement or eye blink artefacts, identified through visual inspection was removed.", "Baseline correction was applied using the pre-stimulus interval prior to averaging and re-referencing to a global average reference.", "Only correct trials were included in the averages.", "Four main ERP components, P1, N1, L1 and L2, were identified based on the topography, deflection and latency characteristics of the respective grand average ERPs time-locked to stimulus presentation.", "Epochs of interest for each component were defined on the basis of deflection extreme in the mean global field power (MGFP; Picton et al., 2000).", "Peak detection was time-locked to the electrode of maximal amplitude (nearest 10–20 equivalent site given) for each component; P1 (90–150 ms) maximal at Oz, N1 (151–190 ms) maximal at PO8, L1 (210–350 ms) maximal at P8 and L2 (360–520 ms) maximal at P8.", "Peak and mean amplitudes and latencies were analysed using stimulus type (object, abstract) × repetition (1st, 2nd, 3rd) × laterality (right, left) × electrode.", "A cluster of three electrodes were taken around the maximal site, with a corresponding cluster in the opposite hemisphere.", "Greenhouse-Geisser corrections were applied to all analyses of ERP data, unless otherwise stated only significant main effects and interactions where corrected p < .05 are reported.", "MR images and single voxel 1H-MRS were acquired on a Philips 3 T Achieva MRI System (Eindhoven, Netherlands) using an 8-channel SENSE head coil.", "For each participant we obtained a high resolution 3D T1 weighted image covering the whole brain (1 × 1 × 1 mm voxel resolution, field of view = 240 mm, number of excitations = 1, resolution = 240 × 240, 150 slices, TR (repetition time) = 8.40 ms, TE (echo time) = 3.80 ms, flip angle = 8°) to allow anatomical localisation for MRS voxel placement.", "Time-locked spectra, between 950 and 1150 ms after each stimulus presentation, were acquired in all subjects using a single voxel PRESS acquisition (20 × 20 × 20 mm, TR = 3000 ms, TE = 40 ms) in the lateral occipital cortex (LOC), an area of the visual cortex shown to preferentially respond to objects compared to other stimuli (Grill-Spector et al., 2001) and also a localized region of evoked gamma-band oscillatory activity with this task (Gruber et al., 2006).", "The LOC was located using anatomical landmarks and confirmed with a blood-oxygen-level-dependent functional magnetic resonance imaging localizer (TR = 2000 ms, TE = 40 ms) in one subject using a block design (Fig. 4A).", "A PRESS 1H-MRS sequence with a TE of 40 ms was chosen as this combination has previously been shown to provide reliable measures of glutamate at 3 T by reducing the overlapping signals from glutamine that previously hampered separation of these two metabolites (Hancu, 2009; Hancu and Port, 2011; Mullins et al., 2008).", "Shim settings were manually optimized for each participant to achieve full-width half maximum < 12 Hz.", "Higher order shims were used for all cases.", "As the use of an ER-MRS acquisition has only been reported twice before (Gussew et al., 2010; Nishitani, 2003), further explanation of 1H-MRS acquisition in an event related paradigm with particular consideration of temporal specificity is warranted.", "For each experimental trial (N = 768) of the repetition priming paradigm, one 1H-MRS measurement occurred.", "Each individual 1H-MRS acquisition involved excitation by a train of radiofrequency pulses centered on the main resonance frequency of tissue water in the voxel of interest, followed by the collection of signal from the tissue 40 ms after excitation.", "This signal is a decaying waveform (the free induction decay; FID) composed of frequencies corresponding to the resonance frequencies of the neurometabolites present within the voxel.", "A Fourier transform of this decaying waveform allows the spectrum of resonance frequencies corresponding to these metabolites to be visualized, with peak height, or signal strength being determined by chemical concentration amongst other factors (for a good summary of these factors see (Alger, 2010)).", "Here, the FID was acquired over 1024 ms, after Fourier transformation the spectra produced is a weighted average measure of the chemical levels across this time period, with the first points of the FID providing the majority of signal power.", "The 1H-MRS acquisition window of 950–1150 ms post-stimulus onset in this experiment corresponds to the time window in which 1H-MRS data acquisition began, and was chosen to provide a clear EEG signal without the overlapping magnetic gradient artefacts.", "Here, stimulus presentation was triggered from the previous 1H-MRS data acquisition (synchronized with the first excitation phase) with the individual 1H-MRS spectra always separated by a repetition time of 3000 ms. To prevent possible entrainment and anticipation, the stimulus presentation was jittered, leading to the individual spectral collection starting between 950 and 1150 ms after stimulus onset.", "This timing means that here we are sampling the metabolic concentrations at a time point ~ 1 s after stimulus onset.", "This time point was based purely on practical concerns, aiming to provide the best chance of clean EEG data with minimal 1H-MRS gradient artefact.", "However, very few studies have examined glutamate related activity changes in this fashion, and it is unclear how they may relate to EEG measures.", "In addition, as there are no data from 1H-MRS studies describing the time course of the glutamatergic response, the precise temporal profile of the glutamate response is not known.", "Thus, it is undetermined when the 1H-MRS measured glutamatergic signal may peak and how long the signal may remain elevated following a single event.", "Future studies sampling at a wider spread of time points may help to address this issue.", "However, this technique provides a specific snapshot of a particular time point and not an average across a period of stimulus presentation, which is one advantage of the time-locked ER-MRS acquisition strategy.", "Spectra from incorrect trials were removed, resulting in a 21% reduction, leaving on average 303 measurements per stimulus type.", "Subsequently, spectra were frequency corrected and averaged within conditions to produce a spectrum for object and abstract stimuli.", "Analyses were then performed using java Based Magnetic Resonance User Interface (jMRUI; (Naressi et al., 2001)) software version 4.0 (http://www.mrui.uab.es/mrui).", "The fast time domain quantitation option (QUEST — quantitation based on quantum estimation) within jMRUI was used for metabolite concentration estimation.", "To exclude potential blood-oxygen-level-dependent related effects on any concentration levels reported and possible water changes during neuronal activity, results were referenced to the creatine signal, which will have experienced the same blood-oxygen-level-dependent effects in signal as glutamate.", "Creatine is a reliably quantifiable metabolite using 1H-MRS and despite its known role in energy metabolism within muscle it is thought to remain stable over time in healthy human neurons.", "Values for the creatine signal here were found to be very stable for each stimulus type presented throughout the experiment (less than 1% difference in absolute measures, t(11) = 0.279, p = 0.785, d = 0.046).", "Referencing to creatine reduces the need for tissue content correction, as like glutamate, creatine is only detected from the tissue.", "One participant was excluded from the 1H-MRS analyses and subsequent correlations due to poor signal-to-noise and shim (FWHM > 14 Hz) in the 1H-MRS data.", "Statistical tests throughout the study were two-tailed.", "Correlations were made using the Pearson product–moment correlation coefficient.", "Repeated measures of analysis of variances (ANOVA) were used to assess the main effect of stimulus and presentation, and their interaction, on behaviour, evoked oscillatory activity and ERPs.", "Where applicable Greenhouse-Geisser corrections were utilised to adjust for violations of the sphericity assumption.", "A paired t-test was used to determine the difference between abstract and object for glutamate levels across participants.", "The relationship between stimulus averaged evoked gamma-band oscillatory activity and glutamatergic levels was examined where a significant main effect or interaction was found.", "In order to examine the specificity of our results we further explored the relationship between condition averaged evoked oscillatory activity across beta, alpha and theta-band oscillatory activity and mean task glutamate levels.", "ERP components were correlated with glutamate concentration in cases of significant task effects only.", "Associations between variables were assessed in all case using Pearson product–moment correlations.", "All statistical analyses were performed using SPSS and are reported here two tailed.", "One participant was excluded from the overall analyses due to repeated failure to respond within the response time limit and thus their data are not included here.", "The pattern of behavioural results was comparable to previous repetition priming tasks (Gruber and Müller, 2005).", "Participants responded correctly on average of 79% (SD = 13.35%) of the time, but showed no difference in accuracy (t(12) = 1.26, p = 0.231, d = 0.194) between stimulus types for the initial presentation.", "However, overall accuracy (Fig. 1B) was significantly higher (mean difference = 7.39%) for object than abstract stimuli (F(1,12) = 13.45, p = 0.003, ηp2 = 0.529).", "Finally, accuracy of both stimulus types increased with repetition (F(2,24) = 38.45, p < 0.001, ηp2 = 0.762), but there was a trend towards a larger increase in accuracy for object compared to abstract stimuli (stimulus × repetition: F(2,24) = 3.63, p = 0.062, ηp2 = 0.232).", "Analysis of reaction times (RTs) also revealed no difference (t(12) = 1.01, p = 0.333, d = 0.07) between stimulus types for the initial presentation.", "There was however an advantage for object compared to abstract stimuli (F(1,12) = 21.43, p = 0.001, ηp2 = 0.641) and a faster response speed for both stimulus types with repetition (F(2,24) = 136.18, p < 0.001, ηp2 = 0.919; Fig. 1C).", "Increases in speed for repeated stimuli were significantly greater for object compared to abstract stimuli (stimulus type × repetition: F(2,24) = 51.88, p < 0.001, ηp2 = 0.812).", "EEG data revealed that both object and abstract stimuli elicited evoked gamma-band activity at posterior electrodes between 50 and 250 ms post stimulus onset (illustrated in Fig. 2A for the first, second and third stimulus presentation).", "There was a trend towards a significant difference between stimulus types following the initial presentation (t(11) = 1.967, p = 0.075, d = 0.327), with higher evoked gamma-band oscillator activity following object that abstract stimulus types.", "There was no main effect of either stimulus type (F(1,11) = 0.038, p = 0.848, ηp2 = 0.003) or presentation (F(2,22) = 0.070, p = 0.933, ηp2 = 0.006).", "However, evoked gamma-band oscillatory activity decreased with repeated presentations of objects, but increased for repetitions of abstract stimuli (stimulus × repetition: F(2,22) = 4.578, p = 0.042, ηp2 = 0.294; Fig. 2B).", "Utilizing the same analytical approach as our gamma-band analysis (i.e. the same electrodes and a post stimulus onset time window based upon data presented in Fig. 2A), we evaluated the evoked beta (12–30 Hz, 50–200 ms), alpha (8–12 Hz, 100–300 ms) and theta (4–7 Hz, 0–400 ms) activity within our paradigm.", "There was no significant main effect of stimulus type (F(1,11) = 2.320, p = 0.156, ηp2 = 0.174; F(1,11) = 4.076, p = 0.069, ηp2 = 0.270), repetition (F(2,22) = 1.241, p = 0.309, ηp2 = 0.101; F(2,22) = 0.577, ηp2 = 0.294, p = 0.570, ηp2 = 0.050) or interaction (stimulus type × repetition: F(2,22) = .0.045, p = 0.956, ηp2 = 0.004; F(2,22) = 0.156, p = 0.857, ηp2 = 0.014) on beta or alpha, respectively.", "There was a main effect of stimulus type (F(1,11) = 7.393, p = 0.020, ηp2 = 0.402) for theta activity but none for repetition (F(2,22) = 0.193, p = 0.826, ηp2 = 0.017) nor an interaction between these two variables (F(2,22) = 0.132, p = 0.877, ηp2 = 0.012).", "Of note, the abovementioned trend toward a significant effect of stimulus type in the beta-band frequency analysis reflects higher average activity to objects than abstract stimuli across presentations, while the opposite pattern is true for the main effect of stimulus type in the theta-band analysis.", "We found no significant effect for the P1 amplitude.", "N1 amplitude was larger on the left compared to the right side (F(1,12) = 7.052, p = 0.002, ηp2 = 0.352), but showed no other significant effects.", "The late component L1 amplitude however was larger for abstract compared to object stimuli (stimulus type (F(1,12) = 28.08, p < 0.001, ηp2 = 0.684); see Fig. 3) and larger on the right side (laterality (F(1,12) = 7.814, p = 0.015, ηp2 = 0.375)).", "There was no significant difference between first or repeated repetitions of the stimulus.", "The late component L2 was again larger on the right side (laterality F(1,12) = 7.515, p = 0.017, ηp2 = 0.366) with no other significant main effect.", "There were no significant effects on latency.", "For the analysis of glutamate, spectra were averaged separately for object and abstract stimuli across stimulus presentations (one-to-three).", "This produced spectra with sufficient signal to noise to reliably detect glutamate in all conditions for all participants except one.", "1H-MRS data quality is often determined from the Cramer Rao lower bound (CRLB) of the data, which is essentially a ratio of the standard deviation of the residual signal after modelling to the amplitude of the fitted peak.", "All glutamate measures used in our statistical analysis had a CRLB of < 15%, which is considered a reliable cut off for 1H-MRS data, and below the common exclusion criteria of CRLB 20%.", "1H-MRS data showed that average LOC (Figs. 4B,C) glutamate was lower following the presentation of object (M = .5222, SD = .145) than abstract (M = .5822, SD = .128) stimuli (F(1,11) = 8.06, p = 0.016, d = 0.47; Figs. 4B, C).", "This result also demonstrates that ER-MRS can discriminate between these stimulus types and that variation in glutamate concentrations can potentially distinguish between different conditions in a task.", "The reliability of the MRS signal is largely dependent upon the signal-to-noise ratio, which increases with the number of FIDs used to create an average spectrum.", "This has an adverse effect on the power of any analysis that subdivides spectra per condition.", "Nevertheless, consistent with our behavioural and EEG results, we also undertook an exploratory analysis to assess if a similar stimulus type effect was also visible across the three presentations, despite the substantial reduction in the number of spectral averages.", "Tentative (uncorrected) paired t-tests revealed a trend towards stimulus type differentiation at the third presentation, with abstract stimuli eliciting higher glutamate levels than object stimuli (t(11) = 1.92, p = 0.08, d = 0.32).", "No trends for stimulus type differentiation were observed for either the first or second presentation (both t(11) < 1).", "These results support our findings of glutamatergic differentiation between stimulus types (object vs. abstract), but remain tenuous due to the aforementioned reduction in signal reliability and statistical power.", "Importantly, examining the relationship between neurochemical and neurophysiological measures, we found a significant correlation between condition averaged (object and abstract combined) evoked gamma-band oscillatory power and mean glutamate levels (r(11) = .769, p = 0.006; see Fig. 4D).", "This strong correlation reflected a positive relationship between both object (r(11) = .696) and abstract (r(11) = .682) glutamate levels and their respective stimulus specific mean gamma-band oscillatory evoked power.", "In order to ascertain the specificity of our association we assessed the relationship between baseline gamma levels (200 ms prior to stimulus onset) and mean glutamate levels.", "There was no significant correlation between baseline gamma oscillatory activity and glutamate levels (r(11) = .231, p = 0.495).", "Furthermore, to assess whether the correlation coefficient between mean baseline and task gamma-band oscillatory power and glutamate concentrations was significantly different we computed Steiger's Z-test (two-tailed).", "We found a significant difference between the two relationships (Z(8) = 2.12, p = 0.0388), indicating that relative to the baseline period, the stimulus period was associated with a significant increase in gamma-band oscillatory power that was specifically associated with average glutamate levels after the stimulus.", "Moreover, we found no significant relationships between condition averaged beta (r(11) = .075, p = 0.826;), alpha (r(11) = − .135, p = 0.691) or theta (r(11) = −.284, p = 0.397) band oscillatory power, and glutamate levels, respectively.", "Additionally, stimulus averaged ERP amplitude for the area under the L1 was not associated with stimulus averaged glutamate concentrations (r(12) = .258, p = 0.418).", "Thus, we conclude that glutamate concentration is uniquely associated with the amplitude of dynamic stimulus-related gamma-band oscillatory activity.", "Finally, we investigated whether glutamate concentrations across individuals correlated with the peak gamma-band frequency (i.e. specific frequency with the highest power), which has been previously shown to be associated with baseline GABA concentration (Muthukumaraswamy et al., 2009).", "A trend towards a significant correlation with peak gamma-band oscillatory frequency was found (M = 34.25 Hz, r(11) = − .538, p = 0.088), with higher glutamate levels in the LOC associated with a lower peak frequency.", "No significant association between LOC glutamate concentration and peak frequency was found for either beta (r(11) = .077, p = 0.823), alpha (r(11) = .061, p = 0.858) or theta (r(11) = .307, p = 0.358) band analyses.", "Our study provides in-vivo evidence of a relationship between dynamic glutamatergic concentrations and synchronised neuronal oscillations within the gamma-frequency range during a cognitive task in the human brain.", "We show, using a unique combination of ER-MRS and EEG, that levels of excitatory neurotransmitter glutamate correlate significantly with the power of neuronal oscillations within the gamma-band.", "Specifically, we find a relationship between levels of glutamatergic activity in the LOC and stimulus evoked gamma-band amplitude during an object repetition-priming task.", "Our results highlight that stimulus evoked gamma-band oscillatory power is related to glutamatergic concentration in the human brain, and thus provide complementary evidence to previous animal work assessing the underlying neurobiology of gamma-band oscillatory activity both in-vivo and in-vitro (Atallah and Scanziani, 2009; Whittington et al., 1995).", "Additionally, our findings add to recent research assessing the contribution of resting-state GABA levels to the peak inter-individual frequency, but not power, of the induced gamma signal (Muthukumaraswamy et al., 2009).", "Here, we find that dynamic glutamatergic activity levels are associated with specific stimulus-related changes in gamma power, and as a trend to peak evoked gamma-band oscillatory frequency.", "These results are consistent with the proposal that precise temporal tuning (frequency) of activity being determined by the ratio of GABA and glutamate concentrations (Atallah and Scanziani, 2009), while the gain of this neural signal (power) is regulated by modulations in glutamate levels only.", "Taken together, our findings suggest both a unique and potentially complementary role for glutamate levels in modulating gamma-band cortical networks in the human brain.", "Indeed, glutamate may possess a distinct role in driving gamma-oscillatory power; meanwhile, both GABA and glutamate might jointly modulate the peak gamma-band oscillatory frequency, reflecting recent in-vitro research (Atallah and Scanziani, 2009).", "However, the association between glutamate concentration and oscillatory network changes here is limited to the region of 1H-MRS acquisition, the LOC.", "Without data from other areas, generalisation to the rest of the cortex is not possible.", "Importantly, our results do not exclude glutamate changes in other regions of the visual cortex; future studies geared to investigate the generalizability of these relationships would be beneficial.", "Indeed, in contrast to our association between stimulus-related glutamate levels and evoked oscillatory activity in the gamma-range, Gallinat et al. (2006) reported a relationship between baseline hippocampal glutamate concentration and frontal theta oscillations using an auditory target detection paradigm.", "This suggests that the relationship between measures of synchronised oscillatory activity and glutamate concentration may be task specific.", "It would be interesting to investigate if the correlation between theta oscillations and glutamate concentration would still be evident if recorded simultaneously.", "Furthermore, despite demonstrating that our glutamatergic concentration in the LOC and gamma-band oscillatory power correlation was specific to the stimulus, but not the baseline period, this association does not preclude the possibility that other tasks and stimuli or passive viewing of the same stimuli without a task may elicit a similar relationship.", "Future research is needed to clarify the specificity of the relationship evidenced here.", "Repeated presentations of pictures of familiar objects resulted in a decrease of evoked gamma-band activity.", "These effects might be linked to a ‘sharpening’ mechanism within a cell assembly representing a familiar object (cf. Wiggs and Martin, 1998).", "In contrast, the representation of repeated abstract stimuli was associated with an increase in evoked gamma power.", "These findings might be a signature of the formation of a new cortical network representing an object (cf. Martens and Gruber, 2012).", "A similar pattern of results was reported by Gruber and Müller (2005) for the induced but not the evoked gamma-band response.", "A possible explanation for this contradictory finding is that memory effects can be observed in evoked and induced gamma-band oscillatory activity, but task parameters such as instructions may modulate their appearance in distinct analyses (Herrmann et al., 2010).", "For instance, Busch et al. (2008) and Fründ et al. (2008) showed that evoked gamma-band activity in fact is larger for objects compared to abstract stimuli and the response to object stimuli can be suppressed with repetition in cases when an immediate and speeded response is required.", "Indeed participants in the current study, but not the Gruber and Müller (2005) study, were instructed to respond as quickly (and accurately) as possible.", "Comparing RTs between the two studies directly confirmed that participants in the current study responded faster compared to the Gruber and Müller (2005) study.", "Our 1H-MRS results provide evidence for a significantly different glutamate response to stimulus type averaged object and abstract stimuli.", "Tentatively, we also investigated the mean glutamate values for each stimulus presentation, and while these means do decrease for object images, and increase for abstract images (mirroring the changes in gamma-band oscillatory power), they do not reach statistical significance.", "The lack of significance in the interaction between stimulus type and presentation was likely a result of decreased signal to noise ratio, and hence increased variability in the 1H-MRS data averaged for each stimulus presentation.", "Future studies, focusing on either repetition suppression or enhancement effects, with greater acquisitions for each presentation should help to address this issue.", "The degree to which elevated glutamate concentrations, as measured by 1H-MRS, reflect increases in either synaptic glutamate release or glutamatergic cycling as part of the energy metabolism process remains to be determined (Mullins et al., 2005).", "One viewpoint suggests that 1H-MRS measures of glutamate differences reflect oxidative neuronal metabolism (Mangia et al., 2007), as glutamate-glutamine cycling is tightly coupled to oxidative glucose consumption (Rothman et al., 1999).", "However, the timescale of glutamatergic cycling is much slower (1 h for total pool turnover (Shen, 2013)) than the timescale of change in glutamate levels seen here (on the order of a second) and in other studies (Gussew et al., 2010).", "We therefore propose that glutamatergic synthesis cannot account for our results.", "The most parsimonious account for fast changes in glutamate levels across conditions, as shown here, is neurotransmission.", "It is generally accepted that 1H-MRS measures of neurotransmitters reflect total chemical volume within the entire tissue.", "However, it is unknown whether vesicular, extra synaptic or intra synaptic levels of metabolites contribute equally to the 1H-MRS signal.", "Indeed, early work on glutamate MR visibility suggests there is a pool of 1H-MRS invisible glutamate — most likely related to a subcellular neurotransmitter pool (Kauppinen et al., 1994).", "Therefore, we tentatively suggest that the rapid increases in 1H-MRS measured neurotransmitter levels in our study, and other studies (Gussew et al., 2010), are best explained by neurotransmitter release from a 1H-MRS invisible subcellular compartment to a more 1H-MRS visible extracellular compartment, than by an increase in glutamate synthesis.", "While this interpretation of our results is speculative at present, it best fits the current data and previous studies.", "However, it is likely that standard 1H-MRS techniques alone can never provide a definitive answer given the coarse spatial resolution of typical 1H-MRS studies.", "Nevertheless, combining 1H-MRS with other measures such as diffusion weighted spectroscopy (Valette et al., 2005) may allow us to investigate intracellular versus extra cellular changes and may provide evidence for our proposal.", "In addition, with appropriate selection of varying 1H-MRS data acquisition time points in future studies it might be possible to characterise the glutamatergic response in much the same way as the blood-oxygenated-level-dependent (BOLD; fMRI) response has been elucidated, which would go some way to addressing which components of glutamatergic processes are altered here.", "Indeed, one possible limitation of the current study is, relative to the stimulus onset, the time at which the measurement of the glutamatergic signal was acquired.", "The evoked gamma-band power changes occurred between 50 and 250 ms, whereas glutamate concentration measurement started between 950 and 1150 ms post-stimulus onset.", "This raises the question how glutamate concentration following stimulus onset can be related to early-evoked gamma-band power.", "It also raises the issue how such a late measurement of glutamate concentration can be related to object recognition.", "One potential interpretation is that gamma-band oscillatory power drives glutamatergic activity.", "For example, glutamate changes in the LOC 1–2 s following stimulus onset may reflect recurrent neuronal network processes that arise from earlier oscillatory responses measured across the entire visual system.", "However, as the glutamatergic response profile is currently uncharted it is also possible that glutamate levels drive oscillatory activity but that the 1H-MRS signal remains detectable for 1–2 s following an event.", "An alternative and final suggestion may be that the glutamatergic signal changes measured here in response to distinct visual stimuli mainly reflects trait but not state-dependent levels.", "As the 1H-MRS glutamate signal is derived from total tissue glutamate concentration and only a fraction may be implicated in transmission related changes, the contribution of state-dependent changes in glutamatergic processing may primarily reflect largely stable trait processes.", "However, concurrent acquisition of these two measures likely increases the between measure signal-to-noise ratio and improves validity of any significant relationships that may be found.", "Further work is needed to characterize the profile of the glutamate signal to resolve these interpretation possibilities.", "Nevertheless, in all three scenarios increased glutamate in response to abstract relative to object stimuli might indicate the differential computational cost between stimulus types.", "Given the recent surge in research investigating pharmacological manipulations to the glutamatergic system, particularly in the treatment of depression (Duman and Aghajanian, 2012) and schizophrenia (Egerton and Stone, 2012), the task-based event-related approach to 1H-MRS demonstrated here, could provide a unique evaluation of the glutamatergic system under varied psychological conditions.", "The now ubiquitous BOLD contrast imaging provides an indirect measure of brain activity that can, in certain circumstances, produce misleading results.", "For instance, an increase in BOLD activity following acute drug administration could result as a consequence of either decreased regional blood supply or increased brain activity.", "Thus, the ER-MRS methodology evidenced here could provide an adjunct to BOLD imaging, which may be especially relevant to drug development protocols, therapeutic assessment and intervention research.", "While we have focused in the current study on functional glutamate concentration, future research should also directly examine event-related GABA concentrations and oscillatory activity.", "It is likely that the balance between the excitatory glutamate and the inhibitory GABA at the level of interneurons (Mann and Mody, 2010; Whittington et al., 1995) jointly modulate synchronised oscillations, and this would be an interesting area to investigate in future 1H-MRS studies.", "Moreover, concurrent measurements of network activity and neurochemical concentrations offer a potential opportunity to both elucidate the basis of cognition and its impairment in mental disorders.", "For example, schizophrenia has been proposed to result from a deficit in forming dynamic links between neuronal populations (the “disconnection” hypothesis; (Friston and Frith, 1995)), and is also linked to neurochemical dysfunction (Carlsson et al., 1999; Lisman et al., 2008; Stephan et al., 2006).", "There is considerable debate among theorists regarding the relative importance of glutamate and GABA dysfunctions within this disorder (Gonzalez-Burgos and Lewis, 2008; Kantrowitz and Javitt, 2012; Kegeles et al., 2012; Lewis et al., 2005).", "Importantly, the technique outlined here could be used to directly assess the impact of the relative importance of changes in both transmitter concentrations upon task-related changes in neural activity and performance.", "In summary, the results provide in-vivo evidence that dynamic neural network activity during stimulus presentation is associated with glutamate levels within individuals, complementing in-vitro research.", "Furthermore, the present study demonstrates the feasibility of concurrently measuring stimulus and possibly cognitive task-related changes using both 1H-MRS and synchronised oscillatory activity, which, exemplifies a novel neuroscientific methodology for exploring functional associations between basic biological and systems level research.", "We establish that simultaneously recording these measures is possible and allows critical insight into the functional relationship between neurochemical and neurophysiological processes, deepening our understanding of the underlying neurobiology of cognition.", "The authors declare no conflict of interest.", "C.H. conceived and C.H. and P.M. designed the experiments.", "C.H., N.L., M.R., D.P., P.M. performed the experiments.", "M.R., C.H., P.M. were responsible for the technical aspects of the combined measures.", "D.P. programmed the task.", "M.R., DP., N.L. pre-processed the EEG data.", "N.L., C.H., T.G. analysed the EEG data.", "D.P., N.L and P.M. analysed the MRS data.", "All were involved in the writing of the paper." ]
[ "Evoked gamma-band activity changes in response to both object and abstract stimuli.", "Glutamate levels in the LOC differ in response to distinct visual stimuli.", "Glutamate levels correlate with concurrently measured gamma-band oscillatory power.", "ER-MRS can quantify neurochemical concentration changes related to the EEG signal." ]
Age, disability and everyday mobility in London: An analysis of the correlates of ‘non-travel’ in travel diary data
Maintaining everyday mobility is important for health at older age. This paper explores one indicator of lack of mobility: not leaving the home on a particular day, which we term ‘non-travel’. We used travel diary data from London residents between 2005 and 2015 to identify the correlates of non-travel for adults. Rates of non-travel were associated with: female gender, unemployment, lack of access to a car, lack of travel concessions, increasing age, disability and being retired. In a logistic regression analysis, older age was independently associated with non-travel, with those aged 60–69, 70–79 and over 80 more likely than working age adults (odds ratios 1.76; 2.18; 3.88 respectively) to report non-travel than working age adults. London faces similar problems to other global cities, with an increasing older population, and policy obligations to shift further from private car based transport to public and active modes. This study has demonstrated that declining levels of mobility at older age in London are not due solely to leaving the labour market or to disability, and that the availability of transport helps reduce, but does not entirely mitigate, the barriers of older age and impairment. To ensure that cities are as health-promoting as possible, more attention is needed to guarantee transport systems foster mobility at older age.
[ "ENGI", "ENVI", "MEDI", "SOCI" ]
[ "Ageing", "Disability", "Health", "Mobility", "Urban" ]
[ "2213", "2310", "2719", "2739", "3311", "3313" ]
[ "By 2060, people aged 60 or over will make up 22% of the global population, and will outnumber children under the age of 14 for the first time (World Health Organization, 2007).", "Not only will the world's population be older, it will be more urban.", "By 2050, two thirds of the world's population are forecasted to live in cities (United Nations, 2014).", "In the UK, adults aged 65 or over will account for 23% of the population by 2035 (ONS, 2012).", "The increase in life expectancy, however, has not been accompanied by a rise in ‘disability-free life expectancy’ (ONS, 2014).", "In the National Travel Survey, 12% of people in England aged 60–69 and 31% of those over 70 reported mobility difficulties (difficulty travelling by foot or bus) (Department for Transport, 2014).", "More people are living with long-term health conditions and disabilities which affect their ability to travel and be mobile outside of the home.", "Mobility is important for health.", "This paper focuses on one particular aspect of mobility: everyday travel outside the house.", "As Metz (2000) highlights, these ‘short distance and high frequency movements’ outside of the permanent home are the priority for older people, as opposed to larger scale mobility such as residential relocation or international migration.", "The health benefits of this everyday mobility have been well documented (e.g. Boniface et al., 2015; Mackett and Thoreau, 2015; Musselwhite et al., 2015).", "First, in terms of directly contributing to physical activity, most non-car travel entails some ‘active travel’, thus contributing to obesity reduction and other physical health benefits (Saunders et al., 2013; Webb et al., 2012).", "Indeed in London, like many other cities, travel is a key source of physical activity for many residents (Fairnie et al., 2016).", "Second, travel facilitates social inclusion.", "For older adults, social exclusion is a major potential determinant of health, with isolation associated with increased risk of all-cause mortality (Holt-Lunstad et al., 2010), decreased immune system efficacy (Bernard, 2013), and increased risk of cardiovascular disease (Steptoe et al., 2013).", "More generally, there is a large and persuasive body of evidence documenting the psychosocial benefits of mobility in later life, beyond those derived from the instrumental role of transport in facilitating accessibility (Ziegler and Schwanen, 2011).", "Access to the public spaces of the city can have a ‘therapeutic quality’, and be important for fostering a sense of place, belonging and wellbeing (Cattell et al., 2008).", "These public spaces can include the transport system itself, with buses identified as a means of tackling chronic loneliness or isolation by providing a space where opportunistic social interaction is acceptable for older adults (Green et al., 2014).", "Thus transport systems themselves have a role to play in facilitating or inhibiting the wellbeing consequences of mobility at older age, with accessible public transport having the potential (in cities) to foster physical activity and interaction, and to mitigate the loss of mobility risked by older citizens who have to give up give up driving (Mackett, 2015).", "Free bus travel in England for older citizens has been identified as effective in reducing risks for obesity (Webb et al., 2012) and fostering social inclusion (Green et al., 2014).", "The relationship between mobility and health is not, of course, unidirectional: declines in wellbeing can combine with limitations in mobility outside of the home to create a downward spiral for an individual (Ziegler and Schwanen, 2011).", "Ageing itself has therefore been seen as a key risk for declining mobility, with potential for contributing to reduced quality of life at older age (e.g. Metz, 2000; Musselwhite et al., 2015; Schwanen and Páez, 2010; Schwanen et al., 2012).", "However, this link is not inevitable: everyday mobility might be used as an explicit mechanism for structuring life after the transition from work to retirement, and thus contributing to wellbeing (Berg, 2016).", "Given the importance of mobility to health and wellbeing in later life it is important that we understand the different factors which may be associated with increased or decreased propensity to be mobile in urban environments.", "London provides a useful case study for studying the impact of older age and disability on urban mobility.", "By 2035, there will be just over 750,000 Londoners over the age of 75, an increase of over 290,000 from 2015 (GLA Intelligence Unit, 2016).", "Accompanying this increase will be a rise in Londoners experiencing a long-term health condition or disability.", "Currently, 37% of Londoners over the age of 65 report disability, accounting for 45% of disabled London residents (Transport for London, 2014).", "By 2031 it is estimated that there will be an additional 150,000 people in London reporting ‘reduced mobility’ (that is, some kind of disability), bringing the total to over 1 million.", "(London Councils Transport Environment Committee, 2015).", "There have been calls for more evidence on what can improve mobility in later life, an endeavour which must scrutinize the role of transport systems (Musselwhite, 2015).", "Barriers to mobility at older age identified in the literature include inability to drive or access to a family member who can drive (Green and Lakey, 2013; Nordbakke, 2013), giving up driving (Davey, 2007; Mackett and Thoreau, 2015), and financial constraints (Rantakokko et al., 2012).", "Older citizens in London are, theoretically, less vulnerable to these constraints: car ownership and use is lower than elsewhere in the country, and there is relatively good access to frequent public transport services, which is free at the point of use for older adults.", "Over 90% of London's population live within 200 m of a bus stop (Mayor of London, 2010), and public transport use is widespread and less stigmatised than elsewhere (Green et al. 2014).", "Nevertheless, car use is still prevalent, with 57% of Londoners aged 65–69 and 22% of those over 80 reporting driving at least once a week (Transport for London, 2014).", "Further, in general, older residents of cities in both higher and lower income countries report that they do not feel their cities were designed with older people in mind (World Health Organization, 2007).", "Global initiatives such as the World Health Organization's ‘Age Friendly Cities’ aim to improve conditions for urban older adults.", "These initiatives are underpinned by an important point – age and mobility must be considered in relation to the relevant social and built environments.", "The ability of older people to use different types of transportation and be more or less socially included (two important measures of age-friendliness) will differ according to their sociodemographic characteristics and will thus influence how mobile (and potentially how healthy) they are.", "This study aimed to contribute to our understanding of mobility in an urban context by identifying which factors might be associated with ‘non-travel’ in London.", "We utilise the London Travel Demand Survey (LTDS), which measures respondents’ travel for one day, to model associations between non-travel and sociodemographic and transport related characteristics.", "The LTDS is an annual survey capturing information about the travel behaviour of Londoners.", "It is conducted on a rolling basis and is administered by trained interviewers.", "The annual sample is around 8000 households, stratified to yield 250 households for each of the 32 London boroughs and the City of London.", "Data is collected about the household, each individual in the household over the age of 5, and the trips they respectively made on the survey day.", "We focus here on ‘non-travel’, as one indicator of mobility.", "We created the dependent binary variable ‘non-travel’ by assigning a value of one to all respondents who did not leave the house (did not travel) on a particular day, even for a short walking trip.", "All respondents who embarked on at least one trip were assigned a value of zero.", "The LTDS includes respondents who were ‘absent’ on the travel day.", "These are respondents who were outside of the area covered by the survey on the recorded day, either by travelling outside of it on the day itself or remaining outside of it from a previous trip.", "Since these people were away from their homes for the survey day, our analysis included them as having travelled.", "In the LTDS respondents self-select ethnicity from UK 2001 Census categories.", "For this analysis, ethnicity was collated into seven categories (‘White British’, ‘White Other’, ‘Mixed Heritage’, ‘Indian’, ‘Pakistani and Bangladeshi’, ‘Black African/Caribbean/British’, and ‘Other Ethnic Background’).", "Household income, recorded in ten categories, was recoded into quartiles for this analysis: ‘<£14,999’, ‘£15,000-£24,999’, ‘£25,000-£49,999’ and ‘£50,000+’.", "Since our focus was on later life, respondents under the age of 18 were excluded from the analysis.", "Age was then split into five categories: 18–49, 50–59, 60–69, 70–79, and 80+ to approximate different stages in the life course, and distinguish between the ‘young’ and ‘old’ old.", "(Alsnih and Hensher, 2003).", "Employment status was recoded into seven categories: ‘full-time employed’, ‘part-time employed’, ‘self-employed’, ‘in education’, ‘unemployed/unable to work’, ‘retired’, and ‘other economically inactive’.", "Household composition was measured using a variable with five categories: ‘couple with children’, ‘couple without children’, ‘lone parent’, ‘single adult’ and ‘other’.", "This was created from a six-category variable in the dataset which additionally contained a category called ‘single pensioner’ which was incorporated into the ‘single adult’ category to avoid collinearity with age variables.", "We constructed a single binary variable ‘Disability’ (yes/no) from whether a respondent answered ‘yes’ to having a disability or long-term health condition affecting their daily activities or ability to travel and get about.", "This analysis included two car-related variables: whether the household owned or had access to a car, and whether the individual held a driving licence.", "Responses to questions on different types of driving licence (car, motorcycle, minibus etc.) were combined into one binary measure (has/does not have a driving licence).", "To capture access to public transport, we included receipt of travel concessions, and whether residence was in Inner or Outer London.", "Inner London has a population density over twice as high as Outer London (London Data Store, 2014), and (in general) higher transport connectivity (Mayor of London, 2014).", "To maximise sample size, LTDS waves from 2005 to 2015 were combined.", "Cases with missing data for any variable in the model were excluded from the analysis, along with respondents under 18, leaving a sample of 123,562.", "Person level survey weights were used to ensure that the sample was representative of the wider London population.", "These weights make the survey representative for each year by using data derived from the Census and mid-year Greater London Authority population estimates (Fairnie et al., 2016).", "Analyses allowed for the stratification of the sample by London borough.", "We used logistic regression to model the association between ‘non-travel’ and socio-demographic and transport related characteristics.", "The dependent variable was a dichotomous measure of whether the individual travelled on the survey day or not.", "The model included all independent variables available in the LTDS that were, on the basis of existing literature, assumed a priori to potentially have an association with ‘non-travel’.", "The model included individual terms for year, month and day of the week to account for fluctuations in ‘non-travel’ rates over time.", "To further explore the relationship between age and ‘non-travel,’ sensitivity analyses examined interaction effects between age and disability, household structure, travel concessions and location.", "We assessed the joint significance of the interaction effects between age and all chosen variables using Wald tests.", "In a free text box, survey respondents were given the opportunity to provide a reason for their non-travel.", "These replies suggest both the diversity of reasons for not leaving the house, and also the limitations of single day diaries for capturing limited mobility.", "They include some that suggest non-travel days were atypical, such as “Lazy day – playstation”, and “entertaining visitors during Ramadan”.", "Others, however, did suggest that non-travel on the survey day reflected longer term mobility issues: “Hardly ever go out”, “Husband passed away not going out much” and “No driver to take [me] out”.", "Non-travel in London is socially patterned (Table 1).", "On average 16% of Londoners did not leave the house on any given day.", "Propensity to non-travel increases with age (13% of 18–49 year olds, 19% of 60–69 year olds and 40% of those over 80 did not travel) and retirement (with 29% of retirees non-travelling compared with 9% in full-time employment).", "Disability was associated with increased propensity to non-travel (37% of respondents reporting a disability or impairment did not travel, compared with 14% of those who reported no disability) as well as gender (18% of women did not travel compared to 14% of men).", "Household structure was also associated with tendency to non-travel: 13% of people living in households consisting of a couple with children did not travel compared to 17% of people living in a couple without children, and 18% of single adults.", "Owning or having access to a car was associated with a reduced propensity to non-travel compared with having one (14%, 20%).", "To provide more detail on the demographics of the sample, Table A2 in the appendix provides a breakdown of the sample by age.", "Table 2 outlines propensity to non-travel by social and demographic characteristics, giving the odds ratio, 95% confidence interval and p-value for each characteristic in the model.", "Year and day of the week were also included in the analysis (not shown: full results from the model are available as online appendices, see Tables A1 and A2).", "A Hosmer-Lemeshow test suggested that the model is an acceptable fit for the data (p = 0.590).", "There was good evidence that increasing age was associated with non-travel.", "Londoners over the age of 80 were 3.88 times (95% CI 3.42–4.40) more likely to stay at home than 18–49 year olds; 70–79 year olds were 2.18 times (95% CI 1.93–2.46) more likely to non-travel than 18–49 year olds and 60–69 year olds were 1.76 times (95% CI 1.59–1.95) more likely to non-travel than 18–49 year olds.", "There was also good evidence from the model of an association between employment status and non-travel.", "Retired persons were 2.42 (95% CI 2.20–2.66) times more likely to non-travel than those in full-time employment; those who were unemployed or unable to work were 2.83 (95% CI 2.63–3.04) times more likely to non-travel than the full-time employed; and those economically inactive for some other reason were 2.49 times (95% CI 2.32–2.67) more likely to non-travel than those who were full-time employed.", "Respondents who reported having a disability or impairment limiting travel were 1.79 (95% CI 1.70–1.88) times more likely to non-travel than those who did not.", "Women were 1.15 times (95% CI 1.11–1.20) more likely to non-travel than men.", "Londoners of Pakistani and Bangladeshi ethnic backgrounds were 1.47 times (95% CI 1.35–1.60) more likely to non-travel than White British Londoners, and Black African/Caribbean/British Londoners were 1.35 times (95% CI 1.27–1.44) more likely to non-travel than White British Londoners.", "There was good evidence that propensity to non-travel is inversely related to income.", "Those who earned £14,999 or less were 1.20 times (95% CI 1.13–1.28) more likely to non-travel than those earning £50,000 or more.", "Earning between £15,000 and £24,999 per year was associated with a propensity to non-travel of 1.13 times greater (95% CI 1.06–1.20) than those earning £50,000 or more.", "Londoners with a yearly income of between £25,000 and £49,999 were only marginally (1.09 times; 95% CI 1.03–1.15) more likely to non-travel than those in the reference category.", "There was good evidence that receiving travel concessions is inversely related to non-travel, with those who receive the benefit half (0.51; 95% CI 0.47–0.56) as likely to non-travel as those who do not despite the fact that those receiving travel concessions are overall more likely to non-travel than the general population.", "This is perhaps because travel concessions are aimed at Londoners who may find mobility more difficult – those who are unemployed, over 65 or disabled.", "Possessing a driving licence was associated with a decreased propensity to non-travel compared to those without a driving licence (0.64; 95% CI 0.61–0.67).", "However, there was only weak evidence that owning or having access to a car was associated with non-travel (0.95; 95% CI 0.91–1.00).", "Those living in households consisting of couples without children were 1.14 times (95% CI 1.08–1.21) more likely to non-travel than those in households consisting of couples with children.", "Lone parents were a third less likely (0.67; 95% CI 0.60–0.75) to non-travel as those in households consisting of couples with children.", "Single adults living alone were 0.82 times (95% CI 0.77–0.88) as likely to non-travel as people living in households consisting of couples with children.", "Those living in other types of household were 1.14 times (95% CI 1.08–1.21) more likely to non-travel than those living as a couple with children in the house.", "To further unpick the relationship between age and non-travel, sensitivity analyses examined interactions between age and: household structure, travel concessions, disability, gender, and whether the person lived in Inner or Outer London in five separate models (included as web appendices, Tables B1 to B5).", "Wald tests indicate that age interaction terms were jointly significant (at the 5% level) in each of the five models suggesting that the relationship between age and non-travel differs by gender, disability, location, whether the person receives travel concessions or not, and the structure of their household.", "The odds of non-travel appear to increase more rapidly with age among men compared to women (Table B1).", "Men aged 80+ were 2.14 times (95% CI 1.64–2.78) more likely to ‘non-travel’ than men 18–24, while women 80+ were 1.46 times (95% CI 1.27–1.68) more likely to ‘non-travel’ than women 18–49.", "Although the relationship between age and non-travel was more pronounced in non-disabled Londoners than disabled Londoners, the general trend appeared consistent (Table B2).", "The interaction between age and the built environment (Table B3) suggests that the built environment does in fact play a role in the mobility of older adults.", "Those living in Inner London were 2.41 times (95% CI 2.07–2.80) more likely to non-travel than 18–49 year olds when aged 70–79, and 3.86 times (95% CI 3.27–4.54) when aged 80+.", "However, there was little evidence of a relationship between age and ‘non-travel’ in Outer London.", "The odds of non-travel appear to increase particularly steeply amongst those who do not receive travel concessions (Table B4), with 80+ year olds 5.24 times (95% CI 4.33–6.34) more likely to non-travel than 18–49 year olds.", "A sensitivity analysis of the interaction between age and household structure suggests that the most pronounced relationship between age and non-travel is present in households consisting of a couple with children (Table B5).", "Individuals living in a couple with children aged 70–79 are 2.51 (95% CI 1.56–4.06) times more likely to non-travel than 18–49 year olds living in a couple with children, and individuals living in a couple with children over the age of 80 are 4.59 times (95% CI 0.97–21.71) more likely to non-travel than 18–49 year olds living in a couple with children.", "Our findings suggest some sociodemographic and transport related associations of one indicator of everyday mobility in London, non-travel on a given day.", "That is, increasing age, having a disability and not being in paid employment all made it more likely that a person would not leave the house.", "Even for London, with relatively good public transport access, which makes car use less essential, and universal access to free bus travel at older age, increasing age was associated with non-travel.", "Crucially, we have shown that disability and long-term health problems did not explain decreasing mobility at older age, which remained an independent association in the regression model.", "We do not suggest that not leaving the house is necessarily a risk for health and wellbeing: there are good reasons why some benefits of travel outside the house can be provided without leaving it (through virtual technologies, for instance).", "Neither do we claim that having to engage with the rigours of urban life is necessarily good for health; relaxing at home might well confer more benefit, at least some of the time.", "However, given the accumulating evidence of the wellbeing benefits of everyday mobility at older age, our findings do suggest some continuing structural barriers to the determinants of health for older adults.", "There are three potential pathways through which increasing age might put people at risk of declining opportunities for mobility.", "First, retirement is a powerful predictor of decreased mobility.", "This is not inevitable: research from a smaller and less dense urban area in Sweden suggests that after retirement, everyday mobility can (in contrast) become a means of structuring the day by “getting out of the house, either just for a walk or to do errands” (Berg, 2016, p. 53).", "Second, ageing, even when controlling for factors such as disability and retirement, appears to be associated with reduced mobility in its own right, although this may reflect some unmeasured confounding, if those with long-term health problems that affect mobility do not reporting having a disability.", "Third, disability itself has been widely reported as a barrier to mobility (Green and Lakey, 2013; e.g. Imrie, 2012; Nordbakke, 2013; Trailblazers, 2016).", "Travel can provide an important bulwark against loneliness and social isolation.", "In the UK, 20% of people over the age of 52 living alone reported feeling lonely often, and 39% reported feeling lonely some of the time (Beaumont, 2013).", "We found living alone to be associated with a lower propensity to non-travel than living with other adults or children, at all ages.", "This was the case for both single adults and pensioners (the latter of whom are proportionally more likely to be non-travellers), as well as lone parents.", "Single people may be more likely to travel to seek out social contact unavailable within the home, as well as to do shopping for the households.", "Receipt of travel concessions is strongly negatively associated with non-travel.", "For the older adults in our sample, this is largely the Freedom Pass, providing free travel on the public transport system for those over State Retirement Age.", "Although those receiving travel concessions are more likely to non-travel overall, once the factors likely to predict receipt of non-travel are accounted for, receipt protects against non-travel.", "Although there may be selection bias here, with those taking the time to apply for travel passes more likely to be the more mobile, this may have a role in mitigating the effects of household income, which is negatively associated with non-travel.", "That there was little evidence that car ownership or accessibility was associated with reduced propensity to non-travel may relate to the mix and density of London's built environment and relatively good public transport network.", "Holding a driving licence was negatively associated with non-travel; which may reflect some selection bias, in that those who are more mobile more likely to obtain a driving licence.", "That men over the age of 80 are over twice as likely to non-travel as women over the age of 80 is not surprising given their reported lower propensity to draw on social support, relative reluctance to seek health care (Davidson, 2012; Gleibs et al., 2011) and lesser likelihood to attend community support groups compared with older women (Beach and Bamford, 2014).", "These factors may result in increased propensity to non-travel.", "It is more surprising that Outer Londoners over the age of 80 have a much lower propensity to non-travel – we would have expected any relationship to go in the opposite direction since Outer London is more car dependent and thus older people who gave up driving would be left without convenient options for travel.", "One possible explanation is that rates of deprivation amongst older people are much higher in Inner London (Hanna and Bosetti, 2015).", "Although we have tried to control for income and car ownership or access, these measures will not be able capture the many dimensions of deprivation which may influence propensity to leave the house.", "Travel concessions have been successfully used to increase the mobility and inclusion of older people across the UK (Mackett, 2014), so it is no surprise that those receiving travel concessions are less likely to non-travel.", "Although uptake is generally high (92% of 70–79 year olds and 84% of those over 80 in our sample reported receiving travel concessions – see Table A2) more research is needed to understand the minority of people who do not apply.", "Apart from the initial cost of the concessionary travel pass (i.e. deposit) and a preference for car use over public transport, few relevant factors have been identified to date (Rye and Carreno, 2008).", "As we have suggested, this finding may reflect selection bias.", "Further research is also required to examine the potential relationship between household structure and non-travel.", "We can speculate that Londoners over the age of 70 and 80 are less likely to travel when they live with their children if they co-reside because of needs for care.", "It is also possible that living alone is a push factor to seek social contact outside of the home.", "Nevertheless, these suggestive results should be treated with caution as the plethora of categories required to test this interaction resulted in wide confidence intervals.", "Our indicator of ‘non-travel’ does not capture isolation or physical activity levels directly, but does provide some clues at a population level of the effect of socio-demographic and transport related circumstances on propensity to ‘non-travel’.", "The response rates of the LTDS, at between 50% and 55%, are generally in line with other large surveys.", "Although there is evidence to suggest that short walking trips are underreported in travel surveys, this is minimised in the LTDS as participants only have to recall their journeys from the previous day.", "This is important as older adults tend to travel shorter distances in their daily lives (Mercado and Páez, 2009).", "The focus on one day additionally avoids the drop off in trip recording seen towards the end of weekly travel diary surveys (Lepanjuuri et al., 2016).", "However, this record of only one day captures non-travel at the aggregate level (in terms of which groups are most likely to do so), and cannot provide a more finely-tuned analysis of who might never leave the house.", "Nevertheless, the LTDS is a large sample, representative of London's resident population, and this analysis provides useful baseline findings for future comparisons as policies for age-friendly cities are implemented.", "This study has highlighted that, even for a city with relatively good public transport and free travel for older citizens, ageing is independently associated with reduced mobility, beyond the impact of retirement and disability.", "Given the evidence that everyday mobility has benefits for health at older age, this is a major determinant of health.", "In the context of aging populations, and cities which will contain larger populations with disability, policies to foster health and health equity must consider age-friendly environments.", "Transport system continue to favour those who are physically agile, can move quickly and have the cognitive skills necessary to navigate sometimes unpredictable transport provision (Freund, 2001).", "We have demonstrated that, even in London, where older citizens benefit from free public transport and relatively good public transport, age independently predicts ‘non-travel’.", "One important indicator of the success of policies for more age-friendly cities will be reductions in inequalities in mobility for those at older age and those with disabilities." ]
[ "‘Non-travel’ days provide a useful measure of everyday mobility in later life.", "Analysis of the London Travel Demand Survey identifies key correlates of non-travel.", "Illness and disability do not wholly account for non-travel in later life.", "Age is an independent predictor of non-travel even in London.", "Age-friendly transport policy will require more than transport provision and subsidisation." ]
Abnormal corneal epithelial maintenance in heterozygous Mp/+ mutant mice
We investigated the corneal morphology of adult Mp/+ mice, which are heterozygous for the micropinna microphthalmia mutation, and identified several abnormalities, which implied that corneal epithelial maintenance was abnormal. The Mp/+ corneal epithelium was thin, loosely packed and contained goblet cells in older mice. Evidence also suggested that the barrier function was compromised. However, there was no major effect on corneal epithelial cell turnover and mosaic patterns of radial stripes indicated that radial cell movement was normal. Limbal blood vessels formed an abnormally wide limbal vasculature ring, K19-positive cells were distributed more widely than normal and K12 was weakly expressed in the peripheral cornea. This raises the possibilities that the limbal-corneal boundary was poorly defined or the limbus was wider than normal. BrdU label-retaining cell numbers and quantitative clonal analysis suggested that limbal epithelial stem cell numbers were not depleted and might be higher than normal. However, as corneal epithelial homeostasis was abnormal, it is possible that Mp/+ stem cell function was impaired. It has been shown recently that the Mp mutation involves a chromosome 18 inversion that disrupts the Fbn2 and Isoc1 genes and produces an abnormal, truncated fibrillin-2MP protein. This abnormal protein accumulates in the endoplasmic reticulum (ER) of cells that normally express Fbn2 and causes ER stress. It was also shown that Fbn2 is expressed in the corneal stroma but not the corneal epithelium, suggesting that the presence of truncated fibrillin-2MP protein in the corneal stroma disrupts corneal epithelial homeostasis in Mp/+ mice.
[ "MEDI", "NEUR" ]
[ "Corneal epithelium", "Corneal homeostasis", "Limbal epithelial stem cells", "Limbus", "Micropinna microphthalmia mutant", "Mouse", "Mp mutant" ]
[ "2731", "2804", "2809" ]
[ "The corneal epithelium forms the outer barrier layer of the cornea and in adult mice it usually comprises 5–6 cell layers (Smith et al., 2002).", "The main bulk of the cornea is formed by the underlying corneal stroma, which consists of sparsely distributed keratocytes embedded in a collagen and proteoglycan matrix and below this is a single layer of corneal endothelial cells.", "It is widely accepted that the adult corneal epithelium is maintained by limbal epithelial stem cells (LESCs), located in the limbus at the junction of the peripheral cornea and conjunctiva and this has been supported recently by lineage tracing experiments (Amitai-Lange et al., 2015; Di Girolamo et al., 2015; Dorà et al., 2015).", "The LESCs produce transient (or transit) amplifying cells (TACs), which move centripetally across the corneal radius to the centre, within the basal corneal epithelial layer.", "The TACs divide several times in the basal layer and, after a final division, post-mitotic daughter cells move into the suprabasal layer, become differentiated and are finally lost from the surface by desquamation (Beebe and Masters, 1996).", "Maintaining a balance of epithelial cell production and loss is critical for normal corneal epithelial homeostasis (Thoft and Friend, 1983).", "Many mouse mutants, knockouts and conditional gene knockouts affect corneal morphology and disrupt homeostasis but there are few examples where LESC depletion or loss of function has been demonstrated.", "For example, the corneas of PAX6+/− heterozygous individuals with aniridia, show progressive corneal deterioration with encroachment of conjunctival cells, including goblet cells, onto the cornea, and evidence suggests that this is at least partly caused by LESC deficiency (Ahmad, 2012; Nishida et al., 1995; Puangsricharern and Tseng, 1995; Secker and Daniels, 2008).", "A similar progressive corneal deterioration occurs in heterozygous Pax6+/− mice but direct proof of LESC deficiency in these mice remains elusive (Davis et al., 2003; Ramaesh et al., 2003, 2005).", "Analysis of mosaics showed that radial cell movement was disrupted in Pax6+/− mice but not PAX77Tg/- transgenic mice, which have elevated Pax6 levels.", "However, quantitative analysis of stripe numbers in mosaics suggested that LESC clone numbers were reduced in both Pax6+/− and PAX77Tg/- mice (Collinson et al., 2004; Douvaras et al., 2013; Mort et al., 2011).", "There is evidence that LESCs are affected in several other genetic mouse models where corneal epithelial homeostasis is impaired, including Lrig1−/−, an Lhx2 conditional knockout (K14-Cre;Lhx2fl/fl) and Dstncorn1/corn1.", "Nakamura et al. (2014) showed that human LRIG1 gene expression was upregulated in holoclone-type corneal cultures (putative stem cells).", "They then showed that corneal homeostasis was impaired in Lrig1−/− knockout mice and, after multiple debridements, wound healing was delayed and incomplete.", "As LESCs are induced to proliferate in order to repair large wounds (Lehrer et al., 1998), the poor wound healing response suggests LESCs are deficient in Lrig1−/− mice.", "Similar evidence suggests that the abnormal corneal epithelial morphology, implying impaired corneal homeostasis, which was seen in some K14-Cre;Lhx2fl/fl mice also involved a LESC deficiency (Sartaj et al., 2016).", "After successive corneal epithelial debridements, wound healing was incomplete in K14-Cre;Lhx2fl/fl mice and their corneal epithelium contained goblet cells and K15-positive cells.", "In adult Dstncorn1/corn1 mice, corneal epithelial cells proliferated but did not move radially and the corneal epithelium included goblet cells, K8-positive, conjunctiva-like epithelial cells and regions of hypoplasia (Zhang et al., 2008).", "Furthermore, BrdU label-retaining cells were present in the cornea as well as the limbus and the authors proposed that the Dstncorn1/corn1 corneal epithelium was not maintained by LESCs in the limbus but by stem cells, within the corneal epithelium.", "As far as we are aware, the Dstncorn1/corn1 mouse is the only example where differences in label-retaining cell distributions have provided evidence of altered LESC function.", "This approach was also used to try to determine whether the number of LESCs, identified as label-retaining cells, was depleted in Pax6+/− mice but the results were confused by other abnormalities (Douvaras et al., 2013).", "Other genetic models of LESC deficiency are required and, in the present study, we have investigated whether Mp/+ mice have abnormal corneal homeostasis and LESC deficiency.", "Mp/+ mice are heterozygous for the irradiation-induced Mp (micropinna microphthalmia) mutation (Phipps, 1964, 1965).", "Both Mp/+ and Mp/Mp mice have small ears and small eyes but Mp/Mp are more severely affected and usually die at around weaning age.", "At the time of our investigations, the nature of the Mp mutation was not understood but this has now been characterised (Rainger et al., 2013).", "Rainger et al. (2013) showed that the Mp mutation involves a 660 kb inversion on chromosome 18 that disrupts the Fbn2 (fibrillin-2) and Isoc1 (isochorismatase domain containing-1) genes.", "The Mp inversion, produces an abnormal, truncated fibrillin-2Mp (Fbn2Mp) protein and this is thought to cause the abnormal Mp/Mp and Mp/+ ocular phenotypes, which are more severe than either Fbn2−/− homozygotes or Fbn2+/− heterozygotes, for which no ocular defects have been reported (Shi et al., 2013b).", "Rainger et al. (2013) also demonstrated that some Mp/Mp tissues, including the developing corneal stroma, showed the hallmarks of ER stress.", "Cells contained intracellular inclusions, suggesting that Fbn2Mp protein accumulated in the endoplasmic reticulum (ER), reduced the secretion of other proteins and perturbed ER homeostasis.", "This would lead to ER stress and trigger the unfolded protein response, which can also cause cell death.", "The authors, therefore, proposed that this mechanism explained the worse-than-null phenotypes of both Mp/Mp and Mp/+ mice (Rainger et al., 2013).", "The aims of the present study were to determine whether corneal epithelial morphology and maintenance were abnormal in adult Mp/+ mice and if there was evidence of impaired LESC function.", "We characterised Mp/+ corneal epithelial morphology, expression of specific markers and indirectly evaluated permeability.", "We also investigated whether there was evidence for altered rates of corneal epithelial cell turnover and we compared LESC function indirectly in Mp/+ and WT mice by analysing X-inactivation mosaic stripe patterns and BrdU label-retaining cell numbers.", "Animal work was approved by the University of Edinburgh Ethical Review Committee and performed in accordance with UK Home Office regulations under project license number PPL 60/3635.", "Mice of both sexes were used except for the analysis of X-inactivation mosaics when only XLacZTg/- mosaic females were used.", "The Mp mutation (Phipps, 1964, 1965) has now been identified as an inversion in chromosome 18 (Rainger et al., 2013) and is designated In(18Fbn2-Isoc1)Mp or In(18)Mp.", "For simplicity, we have used Mp/+, rather than Mp/Mp+, to denote the heterozygote.", "Heterozygous Mp/+ mice, on a mixed genetic background, were provided by Prof. Ian Jackson and Dr. Joseph Rainger (University of Edinburgh) and crossed to the inbred CBA/Ca strain.", "Mp/+ heterozygotes were distinguished from WT littermates by their small ears and eyes (illustrated in Rainger et al., 2013).", "Heterozygous Pax6+/Sey-Neu (Pax6+/−) mice were maintained on a CBA/Ca genetic background and distinguished from Pax6+/+, wild-type (WT) littermates by PCR and eye size (Quinn et al., 1996).", "H253 strain mice (Tan et al., 1993), ubiquitously expressing the Tg(Hmgcr-lacZ)H253Sest, X-linked nLacZ transgene (abbreviated to XLacZ), were maintained on a mixed C57BL/6 and CBA/Ca genetic background.", "Hemizygous males, hemizygous females and homozygous females are designated respectively XLacZTg/Y, XLacZTg/- and XLacZTg/Tg.", "Both Mp/+, XLacZTg/- and WT, XLacZTg/- X-inactivation mosaics were produced by Mp/+ female × XLacZTg/Y male crosses.", "Pax6+/−, XLacZTg/- mosaics were produced by equivalent crosses between Pax6+/− females and XLacZTg/Y males.", "For acute labelling with BrdU (5-bromo-2’-deoxyuridine; Sigma-Aldrich), 15-week old mice were given single intraperitoneal (i.p.) injections of BrdU (10 mg BrdU/ml in normal saline; 0.2 ml/mouse) at 10:00 a.m. and killed by cervical dislocation following inhalation of gaseous anaesthetic 4, 28 or 52 h later (at 2:00 p.m.).", "Eyes were removed, fixed and processed for immunohistochemistry as described below.", "For BrdU pulse-chase identification of label-retaining cells (LRCs), Alzet mini-osmotic pumps (model 1007D; Palo Alto, CA, USA; from Charles River UK Ltd), containing 0.1 ml BrdU solution (50 mg BrdU/ml in normal saline) were surgically implanted under general anaesthetic at 15 or 30 weeks of age.", "BrdU was delivered continuously (0.5 μl/h) and the pumps were surgically removed after 7 days.", "Mice were killed 10 weeks later (i.e. when mice were 26 or 41 weeks old) and corneal buttons were processed for immunofluorescence as described below.", "Eyes were fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) and some lenses were removed via a posterior incision to facilitate sectioning.", "Eyes were dehydrated in ethanol, embedded in paraffin wax and 7 μm longitudinal sections were cut and mounted on Polysine slides (VWR, Lutterworth, UK).", "The cut surface of the wax block was kept wet during sectioning of eyes with lenses to help preserve the lens morphology.", "Slides were de-waxed in Histo-Clear (from National Diagnostics via Fisher Scientific, Loughborough, UK), rehydrated to 70% ethanol, washed in PBS, stained in haematoxylin, haematoxylin and eosin (H & E) or neutral red and eosin and mounted in DPX (distyrene plasticiser xylene; from BDH via VWR) under coverslips.", "For periodic acid-Schiff (PAS) staining, de-waxed sections were treated with 0.5% periodic acid (Sigma-Aldrich) for 15 min, washed in water, stained with Schiff’s reagent (Merck Millipore) for 5 min and washed in lukewarm water for 5 min.", "Slides were then counterstained with haematoxylin, dehydrated, cleared in xylene and mounted on glass slides with DPX and coverslips.", "For BrdU and keratin 12 (K12) immunohistochemistry, sections were dewaxed, rehydrated and incubated with 3% hydrogen peroxide for 20 min to quench endogenous peroxidase activity (Ramaesh et al., 2005; Shi et al., 1991).", "To avoid loss of sections during antigen retrieval, by heating in a microwave (Shi et al., 1991), slides were immersed in citrate buffer (10 mM sodium citrate pH 6.0) in a covered Coplin jar in a 95 °C water bath for 35 min and then the Coplin jar was removed and allowed to cool for 20 min.", "Slides were washed (3 × 5 min) in PBS-T (0.02% Tween 20 in PBS), treated with blocking serum (10% normal serum, from the species used to raise the secondary antibody, and 1% bovine serum albumin, BSA, in PBS-T) for 1 h at room temperature to block non-specific antibody binding.", "Slides were next incubated with primary antibody overnight at 4 °C in a humidified chamber, washed (PBS-T; 3 × 5 min), treated with blocking serum (10 min) and incubated with biotinylated secondary antibody (45 min).", "Slides were then washed (PBS-T; 3 × 5 min) and incubated with avidin-biotin horseradish peroxidase (ABC complex/HRP kit; Dako K0355) for 1 h, washed (PBS-T; 3 × 5 min) and stained with 3-3’diaminobenzidine (DAB isopac, Sigma D9015) for 4–10 min.", "Finally, sections were counterstained with haematoxylin and mounted in DPX, under coverslips.", "Negative control slides were incubated with normal blocking serum without the primary antibody, but otherwise treated in the same way.", "Two protocols were used for BrdU immunohistochemistry on sections.", "For BrdU protocol 1 (Ramaesh et al., 2005), the primary antibody was mouse monoclonal anti-BrdU antibody (Becton Dickinson 347580), diluted 1:150 in blocking serum (10% normal goat serum and 1% BSA, in PBS-T).", "The secondary antibody was biotinylated goat anti-mouse IgG (Dako E0433), diluted 1:200 in blocking serum.", "For BrdU protocol 2, the primary antibody was sheep anti-BrdU antibody (Fitzgerald Labs, 20-BS17), diluted 1:1000 in blocking serum (10% rabbit serum and 1% BSA in PBS-T).", "The secondary antibody was biotinylated rabbit anti-sheep (Vector BA 6000), diluted 1:500 in blocking serum.", "For K12 immunohistochemistry, the primary antibody was Cytokeratin 12 (Santa Cruz Biotechnology sc-17101), diluted 1:500 in blocking serum (10% normal rabbit serum and 1% BSA in PBS-T).", "The secondary antibody was biotinylated rabbit anti-goat IgG (Vector Labs BA-5000), diluted 1:200 in blocking serum.", "Mid-sections of eyes (identified by counting serial sections) were used for all the measurements and counts.", "Sections were photographed using a calibrated Zeiss Axiovision 4.8 digital camera system on a Zeiss Axioplan 2 microscope.", "The Zeiss measuring tool was used to measure the thickness of the whole cornea, corneal stroma (plus endothelium) and corneal epithelium at peripheral, intermediate and central corneal regions (two locations for each region) in sections close to the mid-section in one eye per mouse.", "The number of corneal epithelial cell layers was also counted.", "For estimates of basal cell packing density, the numbers of nuclei were counted in measured lengths of basal corneal epithelium in digital photographs using Adobe Photoshop CS6 and the results were expressed as the mean epithelial length per basal cell.", "All measurements and counts were made by a single investigator (NJD).", "To analyse cell proliferation and loss in BrdU immunostained sections, the junction of the cornea and limbus was identified near the ciliary body (or anterior to the irido-corneal angle in Mp/+ mice without ciliary bodies).", "Each basal corneal epithelial cell was scored as positive or negative across the entire corneal diameter, which was retrospectively divided into two peripheral regions (P), two intermediate regions (I) and two central regions (C).", "The basal labelling index (LI) was calculated as the percentage BrdU positive cells in the basal layer for the whole diameter and separate P, I and C regions.", "The percentage BrdU-positive cells in the combined suprabasal layers (suprabasal LI) was scored in an equivalent way.", "To compensate for the thinner corneal epithelium in Mp/+ mice, an adjusted suprabasal LI was also calculated, by expressing the number of suprabasal BrdU-positive cells as a percentage of the total cell number in the underlying basal layer rather than the total suprabasal cell number.", "All counts were made by a single investigator (EJL).", "Whole-mount corneal immunofluorescence was based on the method described by Pal-Ghosh et al. (2004), as reported previously (Douvaras et al., 2013).", "Briefly, eyes were fixed in 4 methanol: 1 DMSO, corneal buttons were dissected, and incubated sequentially (with intermediate PBS-T washes) in blocking serum (1% BSA and 1% goat serum in PBS-T), primary antibody (overnight at 4 °C), blocking serum and secondary antibody (overnight at 4 °C).", "Primary antibodies used were rat anti-BrdU (Abcam), rabbit anti-K19 (LifeSpan Biosciences) and rat anti-CD31 (BD Pharmingen).", "Secondary antibodies were goat Alexa 488 Anti-Rat IgG (Invitrogen), goat Alexa 488 Anti-Rabbit IgG (Invitrogen) and goat Alexa 568 Anti-Rat IgG (Invitrogen).", "After incubating with the secondary antibody, corneas were washed (PBS-T; 4 h), counterstained with the fluorescent nuclear stain TO-PRO3 iodide (Invitrogen) containing RNaseA, flattened with radial cuts and mounted on slides with MoWiol 4-88 (Calbiochem) containing 2.5% DABCO (1,4-diazobicyclooctane).", "For flat-mounting, 8 radial cuts were made in the WT corneas to create 8 corneal sectors but only 4 radial cuts were made in the smaller Mp/+ eyes.", "Whole-mount corneas, stained with a fluorescently conjugated antibody, were imaged by acquiring overlapping Z-stacks with a Leica TCS NT confocal microscope and exported to ImageJ (http://rsb.info.nih.gov/ij/).", "The MosaicJ plug-in (http://bigwww.epfl.ch/thevenaz/mosaicj) was used to construct composite images.", "Confocal Z-stack images of whole-mount BrdU immunofluorescence were obtained for each sector and BrdU label-retaining cells were identified as BrdU-positive nuclei in maximum projection images of the Z-stacks.", "ImageJ was used to superimpose a calibrated 340 × 200 μm sampling box on the image across the width of the limbal region in each sector.", "The number of BrdU-positive nuclei within each sampling box was counted and the mean number of LRCs per sampling box (one sampling box per sector) was calculated for each cornea.", "To correct for differences in corneal diameters, the mean number of LRCs per sampling box was multiplied by the corneal circumference (calculated from the mean of 3 diameters) and divided by the length of the sampling box (340 μm) to provide an estimate of the number of LRCs in a 200 μm wide ring around the whole limbal circumference.", "All counts of LRCs were made by a single investigator (PD).", "Dissected whole eyes from XLacZTg/-, X-inactivation mosaic mice were lightly fixed in gluteraldehyde, stained with X-gal, post-fixed in 4% paraformaldehyde then either viewed as whole-mounts or embedded in wax, sectioned, counterstained with neutral red and eosin and mounted in DPX under coverslips (Collinson et al., 2002; Douvaras et al., 2013).", "To compare growth and cell mixing in corneal epithelia from 3 to 10 week old Mp/+, XLacZTg/- and WT, XLacZTg/- mice, mosaic patterns were analysed in histological sections of eyes that had been stained with X-gal.", "Cells in the basal corneal epithelial layer of the mid-section were counted and scored as β-gal-positive or β-gal-negative across the diameter of the cornea by light microscopy and the numbers of cells in alternate β-gal-positive and β-gal-negative patches were recorded.", "The number of patches, mean patch length and median patch length were recorded separately for the β-gal-positive and β-gal-negative cell populations and the percentage β-gal-positive cells was calculated.", "The total numbers of basal cells and patches across the corneal diameter were also recorded.", "All measurements were made by a single investigator (PD).", "To compare patch sizes and evaluate cell mixing, the mean patch length was corrected for effects of different proportions of β-gal-positive cells by dividing it by the function 1/(1-p), where p is the proportion of β-gal-positive cells (Hodson et al., 2011; Roach, 1968; West, 1976).", "Both this corrected mean β-gal-positive patch length and the uncorrected median patch length for the minority cell type (Schmidt et al., 1986) were calculated as described previously (Douvaras et al., 2013).", "Radial stripe numbers in whole-mount eyes, dissected at 15 or 30 weeks, were analysed semi-automatically from digital photographs of the cornea, using the ImageJ plugin ’Clonal Tools’ in batch mode as described elsewhere (Mort, 2009; Mort et al., 2011).", "This provides a ‘corrected stripe number’, which corrects for the probability that stripes would contain multiple adjacent β-gal-positive corneal epithelial clones as described previously (Collinson et al., 2002; Mort et al., 2009).", "Where correction for differences in eye sizes was required, the corrected stripe number was divided by the corneal circumference.", "All measurements were made by a single investigator (RLM).", "GraphPad Prism version 5.0c (GraphPad Software Inc., La Jolla, CA) was used for statistical tests.", "More than two groups were compared by 1-way analysis of variance (ANOVA), followed by Tukey’s post tests or 2-way ANOVA, followed by Bonferroni’s post tests.", "Data that were clearly not normally distributed were analysed by Kruskal-Wallis followed by Dunn’s post tests.", "Two groups were compared by Student’s t-test, Welch’s t-test, corrected for unequal variances, or Mann-Whitney U-tests.", "Rainger et al. (2013) showed that at postnatal day (P) 21 Mp/+ mice, on a C57BL/6 genetic background had no ciliary body, no vitreous chamber, the anterior chamber was greatly reduced, the lens had cataracts, the retina was folded and contained rosettes but iris development appeared relatively normal.", "We confirmed that eyes of our adult Mp/+ mice on a CBA/Ca genetic background were small, had retinal abnormalities and no ciliary body and that the vitreous chamber was either absent or very small.", "However, the lens and iris were more significantly affected than previously described (Fig. 1A-C) and this could be at least partly a consequence of the different genetic backgrounds.", "The iris extended completely across the anterior of the eye without a pupillary opening but did not adhere to the corneal endothelium and the lens was disorganised and sometimes vacuolated.", "One eye was examined from each of two mice at five different ages (4 weeks–12 months) but no consistent relationship between age and severity of the eye abnormalities was noted (data not shown).", "Mp/+ corneas tended to have slightly thinner epithelia than wild-type (Fig. 1D–J) but the thickness of the Mp/+ corneal stromas shown in Fig. 1E, F may be misleading because not all sections illustrated in Fig. 1A–F were mid-sections.", "Mid-sections of 10 WT and 10 Mp/+ corneas, from the series illustrated in Fig. 1G–J, were measured.", "Both the WT and Mp/+ corneas were thicker in the central cornea than at the periphery (Fig. 2A), so the genotypes were compared in three regions: peripheral (P), intermediate (I) and central (C) cornea.", "Although the overall corneal thickness did not differ significantly between genotypes, the stroma (plus endothelium) was slightly thicker in Mp/+ than WT eyes (Fig. 2A, B).", "This difference between genotypes reached significance by 2-way ANOVA for the whole cornea but not for individual regions.", "Conversely, Mp/+ corneal epithelia were significantly thinner than WT in all regions and had significantly fewer cell layers in intermediate and central corneal regions (Fig. 2C, D).", "As expected for the small eye size, the corneal diameter and the number of cells across the corneal diameter were significantly reduced in Mp/+ mice by 15 weeks (Fig. 2E, F).", "The length of corneal epithelium per basal cell (measured diameter divided by the number of basal cell nuclei across the diameter) was also significantly greater in Mp/+ compared to WT corneas (Fig. 2G), indicating that Mp/+ cells were larger and/or more loosely packed at 15 weeks.", "Histological sections cut after staining 3-week old, WT XLacZTg/- mosaic mouse eyes for β-gal showed that staining was restricted to the corneal epithelium, whereas β-gal-positive nuclei were also present in the corneal stroma and endothelium of Mp/+, XLacZTg/- mosaics (Fig. 2I).", "As intact eyes were stained before sections were cut, this difference suggests that the thinner, more loosely-packed Mp/+ corneal epithelium is more permeable to liquids than normal, so stain was able to penetrate to the stroma and endothelium.", "β-gal staining of the corneal stroma and endothelium was also observed in the Pax6+/− XLacZTg/− (Fig. 2I), which has a thin and fragile corneal epithelium (Ramaesh et al., 2003).", "Sections of WT, XLacZTg/- and Mp/+, XLacZTg/- mosaics eyes from 3-, 6- and 10-week old mosaic mice were also used to compare corneal growth in WT and Mp/+ eyes.", "Mp/+ corneas had fewer basal epithelial cells across the corneal diameter than WT at each age (Fig. 2H).", "Separate one-way ANOVAs for each genotype showed significant differences in cell counts across the diameter among ages for WT (P = 0.0003) but not Mp/+ eyes (P = 0.3693), indicating that Mp/+ corneas were smaller in diameter by 3 weeks and failed to grow normally between 3 and 10 weeks.", "The thinner, more loosely packed corneal epithelium of Mp/+ mice could indicate that the rate of corneal epithelial cell turnover is abnormal so we examined incorporation of BrdU by immunohistochemistry.", "Preliminary immunohistochemistry with a mouse monoclonal anti-BrdU and a biotinylated goat anti-mouse IgG secondary antibody (protocol 1) worked well with WT eyes, as reported previously (Ramaesh et al., 2005), but produced heavy background staining with Mp/+ eyes, even without primary antibody or if Mp/+ mice were not injected with BrdU (not shown).", "However, BrdU labelled cells were identified successfully in both WT and Mp/+ corneas by immunohistochemistry using a sheep anti-BrdU antibody (protocol 2), as shown in Fig. 1G-J, and were counted (Fig. 3).", "Four hours after labelling, BrdU-positive cells were confined to the basal layer.", "The basal layer labelling index (LI) increased between 4 and 28 h in both Mp/+ and WT corneas and then declined by 52 h (Fig. 3A).", "There were no significant differences between the genotypes whether the basal layer LIs were calculated for the whole cornea (Fig. 3A) or separately for peripheral, intermediate and central regions (Supplementary Fig. S1A-C).", "At four hours, the basal layer LIs for the whole cornea were very similar for the two genotypes: 6.32 ± 1.32% (mean ± 95% confidence interval) for WT and 7.15 ± 2.12% for Mp/+.", "This implies that there was no major effect on the rate of cell proliferation.", "Only basal cells divide and incorporate BrdU so suprabasal cells only become labelled when basal cells move to the suprabasal layers.", "This is irreversible and is the first step towards cell loss, so more rapid accumulation of BrdU-positive cells in the suprabasal layers in one group would suggest a higher rate of cell loss, unless the total number of corneal epithelial cells (and thus the thickness of the epithelium) changed progressively over time.", "By 28 h BrdU-positive cells appeared in the suprabasal layers of both genotypes.", "The suprabasal LI was slightly higher in Mp/+ than WT eyes at both 28 h and 52 h (Fig. 3B) but the difference was only significant when peripheral, intermediate and central regions were analysed separately for each genotype at 28 h (P = 0.0221 for genotype differences by 2-way ANOVA; Supplementary Fig. S1D-F).", "However, when the LI was adjusted to compensate for the reduced number of Mp/+ suprabasal layers there were no significant differences between genotypes, either for the whole cornea (Fig. 3C) or for the three separate regions (Supplementary Fig. S1G–I).", "At 28 h, the adjusted suprabasal LIs for the whole cornea were 2.99 ± 1.07% for WT and 4.66 ± 2.14% for Mp/+.", "At this time, in WT corneas 23.4 ± 6.4% of BrdU-labelled cells were in the suprabasal layers and for Mp/+ corneas this was slightly higher (30.4 ± 6.7%) but this difference was not significant by student’s t-test (P = 0.1712).", "Thus, despite some trends for a small increase in suprabasal labelling, there was no evidence that the Mp/+ genotype had a major effect on corneal epithelial cell proliferation or loss.", "Although, this does not exclude the possibility that cell turnover is increased marginally in Mp/+ corneas, a larger experiment would be required to investigate this possibility.", "It is widely accepted that corneal epithelial cells move centripetally across the corneal radius from stem cells in the limbus (Buck, 1985; Di Girolamo et al., 2015; Nagasaki and Zhao, 2003).", "The radial striped patterns in adult XLacZTg/-, X-inactivation mosaics reflect this movement of corneal epithelial cells.", "Mp/+, XLacZTg/- mosaics corneas showed similar patterns to WT, XLacZTg/- eyes with radial stripes extending between the limbal region and central cornea (Fig. 4A, B), implying that normal radial movement occurs in Mp/+ eyes.", "We used two indirect methods to investigate whether the thinner Mp/+ corneal epithelium was associated with limbal epithelial stem cell (LESC) deficiency.", "First, we compared striped patterns quantitatively in Mp/+, XLacZTg/- and WT, XLacZTg/- X-inactivation mosaics, as described elsewhere (Collinson et al., 2002, 2004; Douvaras et al., 2013; Mort et al., 2009, 2011).", "The observed number of β-gal-positive and β-gal-negative radial stripes was corrected to adjust for the expected frequency of adjacent stripes of the same cell population (β-gal-positive or β-gal-negative), as described in the Materials and Methods.", "The resultant corrected stripe number is not likely to be numerically equal to the number of LESCs because LESCs are expected to be arranged in small coherent clonal groups.", "However, the corrected stripe number is an estimate of the number of coherent clones of active LESCs (Collinson et al., 2002; Mort et al., 2009).", "Differences in corrected stripe numbers between Mp/+ and WT eyes are, therefore, expected to be proportional to the differences in numbers of active LESCs, as long as the mean number of LESCs per coherent clone does not differ significantly between genotypes.", "The percentage of β-gal-positive cells did not differ significantly between Mp/+, XLacZTg/- and WT, XLacZTg/- genotypes for the two ages studied (15 and 30 weeks; Fig. 4C).", "The corrected stripe numbers and, by implication, the number of coherent clones of LESCs declined with age in both genotypes (Fig. 4D).", "These age differences were statistically significant for the Mp/+ XLacZTg/- mosaics but not the WT, XLacZTg/- controls but this may be because few WT, XLacZTg/- corneas were analysed at 15 weeks.", "Despite the much smaller eye size, the corrected stripe number per eye was not lower in Mp/+, XLacZTg/- mosaics than WT, XLacZTg/- controls at either 15 or 30 weeks (Fig. 4D).", "After allowing for the much smaller circumferences of Mp/+, corneas, the corrected stripe number per mm of circumference, at the corneal-limbal border, was significantly higher in Mp/+ XLacZTg/- mosaics than WT, XLacZTg/- controls at both 15 and 30 weeks (Fig. 4E).", "This suggests that active coherent clones of LESCs were distributed more densely around the limbal circumference in Mp/+ eyes.", "However, a higher concentration of LESC clones need not imply a higher concentration of LESCs, if β-gal-positive LESCs were more randomly distributed in Mp/+ eyes, so there were fewer LESCs in each coherent clone of LESCs.", "This could occur, for example, if β-gal-positive and β-gal-negative cells were initially more finely intermixed in the developing Mp/+ ocular surface than in WT (Fig. 1 in Douvaras et al., 2013).", "To test whether cell mixing differed before the LESCs were activated and the stripes emerged (at around 5 weeks; Collinson et al., 2002), the sizes of β-gal-positive patches in the basal layer of the corneal epithelium were compared in 3-week old Mp/+, XLacZTg/- and WT, XLacZTg/- X-inactivation mosaics (Fig. 2I), as described in the Materials & Methods.", "Neither the corrected mean lengths of β-gal-positive patches nor the uncorrected median lengths of the minor cell population in the corneal epithelium differed significantly (Fig. 4F, G).", "Thus, there was no evidence that cells were more finely mixed in the Mp/+ocular surface before stem cells were activated and so no evidence that β-gal-positive LESCs in Mp/+ eyes were likely to be arranged in smaller coherent clonal groups than in WT eyes.", "It, therefore, remains possible that LESCs were distributed more densely around the limbal circumference in adult Mp/+ eyes.", "In any case, the analysis of stripe numbers clearly provides no evidence for a depletion of active LESCs in Mp/+ mice.", "The second indirect method we used to compare stem cell numbers in WT and Mp/+ mice was to compare the numbers of label-retaining cells (LRCs).", "These were expected to be predominantly putative slow cycling or quiescent stem cells (Cotsarelis et al., 1989; Ksander et al., 2014; Lehrer et al., 1998; Li and Clevers, 2010; Pajoohesh-Ganji et al., 2006; Zhao et al., 2009).", "We exposed WT and Mp/+ mice to BrdU for 7 days at 15 or 30 weeks of age and identified LRCs in the limbal region by whole-mount BrdU immunofluorescence after a 10-week chase period (at 26 or 41 weeks).", "LRCs were identified by confocal microscopy in each sector of flat mounted cornea buttons (Fig. 5A–D) and counted in a 340 × 200 μm sampling box superimposed over the limbal region.", "LRCs were restricted to the limbal region in WT eyes and almost exclusively restricted to the limbal region in Mp/+ eyes (Fig. 5B, D).", "The mean number of LRCs per limbal sampling area did not differ significantly between ages for either genotype but it was significantly greater in Mp/+ than in WT eyes, for both ages (Fig. 5E).", "As Mp/+ eyes are smaller than WT eyes, we also estimated the number of LRCs within a 200 μm wide ring (the width of the sampling box) around the whole limbal circumference.", "The mean number of LRCs remained greater in Mp/+ eyes when expressed as LRCs per limbal circumference for both age groups but this was only significant for those labelled at 15-weeks (Fig. 5F).", "The comparison of LRC numbers clearly provided no evidence that Mp/+ LESC numbers are depleted.", "Histological sections of three WT and three Mp/+ eyes from 15-week old mice were immunostained for the corneal epithelial marker keratin 12 (K12) and both genotypes showed strong K12 staining throughout the central corneal epithelium (Fig. 6A, B).", "K12 staining was present in the peripheral corneal epithelium but not in the limbus of WT eyes and the staining boundary was approximately level with the ciliary body (Fig. 6C).", "K12 staining was also strong in the peripheral corneal epithelium of Mp/+ eyes but there was a short length of weak and patchy staining at the limbal-corneal boundary (Fig. 6D).", "However, as Mp/+ eyes had no ciliary body it was difficult to determine whether the weak staining was in the peripheral cornea or the limbus.", "Although most Mp/+ peripheral corneal epithelial cells were strongly K12-positive, there were some isolated unstained cells, suggesting the presence of goblet cells or other atypical corneal epithelial cells in the Mp/+ eye (Fig. 6D).", "To determine whether these isolated K12-negative cells in the peripheral corneal epithelium of Mp/+ eyes were goblet cells, some corneas were stained for polysaccharides with periodic acid-Schiff (PAS) reagent.", "Eyes from two mice at each of 5 ages (4 weeks, 9 weeks, 15 weeks, 6 months and 12 months) for each genotype were stained for PAS.", "No PAS-positive goblet cells were detected in the WT corneal epithelium at any age but some PAS-positive goblet cells were present in the peripheral corneal epithelium in sections of eyes from 3 of the Mp/+ mice (both 6-month eyes and one of the two 12-month eyes), as shown in Fig. 6E, F.", "This suggests that the isolated cells, undetected by K12 immunohistochemistry, were goblet cells.", "We next used confocal microscopy to visualise double immunofluorescence of keratin 19 (K19) and the blood vessel marker, CD31.", "Blood vessels are usually absent from the cornea and restricted to a characteristic limbal vascular ring in the limbal stroma (Morrison et al., 1995), which helps identify the limbus.", "K19 is a marker of the limbal epithelium and conjunctiva but is not usually expressed in the mouse corneal epithelium (Yoshida et al., 2006).", "K19-positive cells in the Mp/+ ocular surface (Fig. 7A-C) extended further from the main vessels of the limbal vascular ring than in the WT mice (Fig. 7D) and they appeared to encroach into the peripheral cornea.", "Furthermore, in Mp/+ eyes, some small blood vessels appeared to enter the Mp/+ cornea and loop back to the main limbal vascular ring (Fig. 7A-C).", "This differs from WT, where blood vessels do not extend far towards the cornea from the main limbal vascular ring (Fig. 7D; Fig 5 in Douvaras et al., 2013; Zhao et al., 2009).", "It is now known that the Mp mutation results in the accumulation of abnormal Fbn2Mp protein, which causes ER stress in Mp/+ cell types that normally express Fbn2 (Rainger et al., 2013).", "Although Fbn2 is not expressed in the corneal epithelium it is expressed in some ocular tissues during development of WT embryos (Rainger et al., 2013; Shi et al., 2013a).", "Rainger et al. also showed that Fbn2 was expressed in several tissues of fetal Mp/Mp eyes, including the corneal mesenchyme, and that protein was retained intracellularly rather than secreted (Rainger et al., 2013).", "Mp/+ cells that express Fbn2 will produce the abnormal Fbn2Mp protein as well as Fbn2.", "Thus, proteins will be retained within the cell, causing ER stress and affecting the composition of the extracellular matrix.", "Cell types that do not express Fbn2 may also be affected if they interact with Fbn2Mp-positive cells.", "Although Fbn2 is not expressed in the corneal epithelium, Mp/+ corneal epithelial development and maintenance could be affected indirectly by ER stress in adjacent tissues.", "The expression of Fbn2 in the developing corneal mesenchyme and irido-corneal angle (Rainger et al., 2013; Shi et al., 2013a) suggests that ER stress is likely to occur in the corneal stroma, limbal stroma, corneal endothelium, trabecular meshwork and Schlemm’s canal.", "Thus, ER stress might perturb Mp/+ corneal epithelial homeostasis if ER stress in the corneal stroma affects the corneal epithelium or if ER stress in the limbal stroma affects the LESC niche and so compromises LESC function.", "The main aims were to determine whether corneal epithelial maintenance was abnormal in adult Mp/+ mice and if LESC function was compromised.", "The significant trend for the Mp/+ corneal stroma to be thicker than normal could be a direct effect of ER stress within the corneal stroma or endothelium.", "For example, this might be mediated via poor regulation of fluid retention by the corneal endothelium.", "Further investigations are required to determine whether ER stress has a direct effect on the Mp/+ corneal endothelium or corneal stroma and also identify any quantitative or qualitative changes in keratocytes or stromal lamellae.", "The Mp/+ corneal epithelium was thinner than normal, cells were loosely packed and results suggested that the barrier function was compromised.", "The presence of PAS-positive goblet cells in the peripheral corneal epithelium of some older Mp/+ mice but not WT mice could reflect incursion of conjunctival cells and LESC deficiency (Nishida et al., 1995), abnormal differentiation (Ramaesh et al., 2003) or possibly a response to wounding (Pajoohesh-Ganji et al., 2012).", "Regardless of how they arise, the presence of goblet cells is abnormal.", "Together, the evidence for a thin and loosely packed corneal epithelium, which accumulates goblet cells in older mice, implies that corneal epithelial homeostasis was abnormal.", "Investigations with the K12 and K19 markers indicate that the Mp/+ ocular surface is abnormal.", "The Mp/+ corneal epithelium had a short length of weak and patchy K12 staining at the limbal-corneal boundary, suggesting differentiation of cells in this region was abnormal.", "However, it was not clear whether this region was in the peripheral cornea or limbus.", "Conversely, K19-positive epithelial cells occupied a broader region than normal and this region was defined by an unusually extensive network of capillaries, extending from the Mp/+ limbal vascular ring.", "However, this does not appear to be equivalent to neovascularisation as the blood vessels looped back to the Mp/+ limbus, rather than extending towards the centre of the cornea.", "The normal striped pattern in adult Mp/+ XLacZTg/- mosaics implies that the radial movement of corneal epithelial cells was unaffected in Mp/+ eyes, unlike some other genotypes, which either produce abnormal stripes or no stripes in mosaic systems (Collinson et al., 2004; Mort et al., 2012; Zhang et al., 2008).", "This evidence for normal cell movement in the Mp/+ corneal epithelium is also consistent with the notion that the enlarged vasculature in Mp/+ eyes differs from neovascularisation.", "This is because corneal neovascularisation is often associated with abnormal corneal epithelial cell movement (Zhao et al., 2011).", "For example, both occur in Dstncorn1/corn1 and Pax6+/− mice (Collinson et al., 2004; Mort et al., 2011; Ramaesh et al., 2003; Zhang et al., 2008; Zhao et al., 2011).", "The distributions of K12, K19 and blood vessels are consistent with the possibility that the Mp/+ limbus is wider than normal.", "If so, the expanded domain of K19-positive cells and larger network of blood vessels would be entirely within an enlarged limbus.", "Further studies are required to characterise the Mp/+ limbus more fully and identify whether, in Mp/+ eyes, (i) the blood vessels and K19-positive cells enter the peripheral cornea, (ii) the corneal-limbal boundary is poorly defined, perhaps with an abnormally differentiated zone that includes both K12-positive and K19-positive cells, or (iii) the Mp/+ limbus is wider than normal.", "Quantitative studies of X-inactivation mosaic mice showed that the mean corrected stripe number per eye was similar in Mp/+ and WT mosaic corneas, despite the large difference in eye size and corneal circumference.", "This implies that Mp/+ and WT corneal epithelia are maintained by a similar number of LESC clones, which are distributed more densely around the smaller Mp/+ limbal circumference.", "This might occur, for example, if similar numbers of LESCs are specified in Mp/+ and WT eyes early in development (before the corneas diverged in size) but clones of LESCs become more densely distributed around the circumference because the Mp/+ cornea stops growing.", "This could result in either more LESC clones per area of limbus (perhaps with fewer LESCs per clone) or the formation of a wider limbus, as discussed above, with a smaller circumference and a more normal concentration of LESC clones and LESCs per area of limbus.", "In either case, the number of LESC clones per mm of circumference would be greater than usual in Mp/+ eyes.", "Although adult stripe patterns in X-inactivation mosaics only provide indirect information about active stem cells, it is clear that these results provide no evidence for depletion of active LESCs in Mp/+ mice, even without taking into account the smaller Mp/+ limbal circumference.", "There was a trend for the mean corrected stripe number per cornea and, by implication, the number of coherent clones of LESCs to decline with age in both Mp/+ and WT mosaic corneas.", "This has been shown previously for WT mosaics (Collinson et al., 2002; Mort et al., 2009) and could occur if (i) more LESCs become quiescent during ageing, (ii) some LESCs are lost (so numbers of active LESCs and LESC clones decrease) or (iii) some LESCs are replaced by neighbouring LESCs by stochastic neutral drift (so LESC clone numbers decrease but LESC numbers remain unchanged; Mort et al., 2012).", "The label-retaining cell (LRC) method was originally used in the ocular surface to help identify the limbus as the location of putative stem cells (Cotsarelis et al., 1989).", "Our observation that Mp/+ mice had significantly more BrdU LRCs per sampling area than WT mice clearly provides no evidence for depletion of LESCs in Mp/+ mice.", "Although most LRCs are likely to be putative stem cells, it is not a specific method and other slow-cycling cell types might be included as LRCs.", "For example, when short chase times (4 weeks) were used in rats, some Langerhans cells were included as LRCs (Chen et al., 2007).", "Also, any labelled cell that stops dividing when it terminally differentiates and is retained in the field that is imaged, would be identified as an LRC.", "We cannot rule out the possibility that Mp/+ LRCs included slow dividing or differentiated cell types that are less numerous among WT LRCs.", "For example, if proliferating and differentiating vascular cells were present in the enlarged Mp/+ limbal vascular ring this might contribute to the increased number of LRCs in the Mp/+ limbus but is unlikely to be the full explanation.", "Although not all LRCs are LESCs, they are normally present in the limbus but not the cornea (Cotsarelis et al., 1989; Douvaras et al., 2013; Ksander et al., 2014; Lehrer et al., 1998; Pajoohesh-Ganji et al., 2006; Zhao et al., 2009).", "LRCs in the WT mouse limbus are restricted to a 100–200 μm wide region of the ocular surface (Zhao et al., 2009).", "If, the Mp/+ limbus were wider than normal, the 200 μm wide sampling box would probably include more limbal tissue when Mp/+ LRCs were counted.", "Thus, the higher counts for Mp/+ eyes need not imply a higher density of LRCs per area of limbus.", "However, as Mp/+ eyes have more LRCs per circumference (not just per mm of circumference), it is possible that the Mp/+ limbus is both wider and has a higher density of LRCs.", "Many types of adult stem cells are quiescent (Li and Clevers, 2010) and lineage tracing suggests that LESCs cycle though phases of quiescence and activity (Dorà et al., 2015).", "Stem cells may be identified as LRCs either because they divide slowly or because they enter quiescence.", "The greater number of LRCs per sampling area, observed in the Mp/+ limbus, might suggest that the Mp/+ limbus contained a higher proportion of quiescent LESCs than WT.", "However, whether a greater proportion of quiescent LESCs results in more LRCs will depend on the balance of two opposite effects (a relative decrease in the number of cells that become labelled and an increase in the proportion of labelled cells that retain the label for 10 weeks).", "Evidence from X-inactivation mosaics suggests that the Mp/+ limbus contains more active LESC clones per mm of circumference and, overall, it seems likely that the total number of LESCs per mm of circumference is greater in Mp/+ than WT eyes.", "This is also consistent with the possibility, suggested earlier, that the limbus and LESCs occupy a wider region of the ocular surface in Mp/+ than WT eyes.", "If the Mp/+ eye does have more LESCs, they seem barely able to keep up with the demand for corneal epithelial cells because the corneal epithelium is thin and loosely packed even though the Mp/+ cornea is greatly reduced in diameter.", "However, we found no convincing evidence that corneal epithelial cell proliferation was reduced or cell loss was much greater in Mp/+ eyes.", "It remains possible, though, that corneal maintenance is compromised because the Mp/+ LESCs function poorly.", "Further investigations with markers, such as ABCB5, that identify LESCs and probably early TACs (Ksander et al., 2014), or Cre-loxP lineage tracing (Amitai-Lange et al., 2015; Di Girolamo et al., 2015; Dorà et al., 2015) may help test the predictions that the Mp/+ limbus is abnormally wide and contains more LESCs than normal but they function poorly.", "Together, our investigations indicate that heterozygous Mp/+ mice have a novel combination of corneal phenotypes.", "The thin, loosely packed Mp/+ corneal epithelium and the presence of goblet cells imply that corneal epithelial maintenance is impaired.", "The distribution of K19-positive and K12-positive cells and limbal blood vessels suggest either that the limbal-corneal boundary is poorly defined or the limbus is wider than normal.", "Indirect analyses with label-retaining cells and mosaic patterns provided no evidence that Mp/+ LESC numbers were depleted but suggested that they may be more numerous than usual yet function poorly.", "Further investigations are required to characterise the Mp/+ limbus more fully, test the predictions about Mp/+ LESCs and clarify to what extent ER stress in the corneal and/or limbal stroma contributes to the observed abnormalities of corneal epithelial morphology and maintenance.", "None of the authors have any conflicts of interest.", "PD, NJD, RLM and EJL performed the experiments, PD, NJD, RLM, EJL and JDW analysed the data and prepared the figures, JDW and REH designed the experiment and supervised the work, JDW and PD wrote the first draft and all authors contributed to the preparation of the final manuscript." ]
[ "Heterozygous mutant Mp/+ mice have small, abnormal eyes.", "The corneal epithelium is thin, loosely packed and has goblet cells.", "Corneal epithelial cell turnover and radial cell movement appear normal.", "The cornea-limbal border is poorly defined and the limbus appears wider than normal.", "Indirect tests suggest stem cells are not depleted and numbers might be increased." ]
A review of optimisation techniques used in the composite recycling area: State-of-the-art and steps towards a research agenda
The increased use of carbon fibre and glass fibre reinforced polymer in industry coupled with restrictions on landfill disposal has resulted in a need to develop effective recycling technologies for composites. Currently, mechanical, thermal and chemical approaches have been use to recycle composites. This paper seeks to examine the applications of engineering optimisation techniques in the composite recycling and re-manufacturing processes and their relevant systems, providing an overview of state-of-the-art. This paper is based on a comprehensive review of literature covering nearly all the research papers in this area. These papers are analysed to identify current trends and future research directions. The composite recycling is a relatively new area, and the modelling and optimisation work for composite recycling and re-manufacturing techniques and their relevant systems is still in its infancy. Currently, the optimisation work developed in composite recycling mainly focus on the applications of design of experiments methods. These approaches have been applied to improve the quality of recyclates such as carbon fibres. Some of the soft-computing algorithms have been applied to optimise the re-manufacturing at the system level. Based on the existing research, the area of optimisation for composite recycling and re-manufacturing haven't been well explored despite the fact that many opportunities and requirements for optimisation exist. This means significant amount of modelling and optimisation work is required for the future research. More significantly, considering optimisation at the early stage of a system development is very beneficial in terms of the long term health of the composite recycling industry.
[ "BUSI", "ENER", "ENGI", "ENVI" ]
[ "Composite", "Optimisation", "Recycling" ]
[ "1408", "2105", "2209", "2300" ]
[ "In the last 30 years, fibre reinforcement resins, thermoplastics as well as thermosets have been increasingly used in a wide range of applications such as the automotive, aerospace and transportation industries.", "However, the major drawback of composites is that they are difficult to recycle.", "This is based on their inherent nature of heterogeneity, especially for the thermoset-based polymer composite (Yang et al., 2012; Ye et al., 2013).", "The traditional way to deal with the composite waste is disposal in landfills or incineration.", "These methods are still acceptable due to the relatively low amount of production waste and the fact that most Carbon Fibre Reinforced Composites (CFRCs) produced so far are still in operation.", "However, the cost of the traditional approaches is high.", "For instance, the cost for disposal of CFRCs waste, where not illegal, is around 0.2 GBP/kg (Pimenta and Pinho, 2011).", "By the year 2030, around 6000–8000 commercial planes are expected to reach their end-of-life (Ye et al., 2013).", "This will bring a large amount of composite waste.", "On the other hand, the amount of production waste will increase as the demand for composites and the amount of composites parts produced is growing rapidly.", "For instance, in Europe and the USA, the annual generation of CFRCs scrap is around 3,000t (Ye et al., 2013).", "Hence, the demand for developing more cost effective and sustainable composite recycling approaches is growing.", "Besides future problems with composite disposal, it is also economically beneficial to implement composite recycling.", "For instance, carbon fibres are an expensive raw material.", "The price of it as of 2015 is 20 USD/kg.", "Recycling activities provide the potentiality to use cheap carbon fibres for applications which do not require high strength, and will also ultimately open up new sustainable and secure sources of carbon fibre material.", "To securely use the recycled carbon fibres, relevant applications and specific standards are needed to be developed (Oliveux et al., 2015a).", "Comparatively, it is easier to recycle thermoplastic matrix composites.", "Since they can be re-shaped by re-melting and remoulding (Yang et al., 2012).", "For the thermoset matrix composites, current research on recycling technologies mainly focuses on mechanical (Oliveux et al., 2015a; Howarth et al., 2014), thermal (Pickering, 2006) and chemical recycling (Yang et al., 2012).", "In the mechanical recycling process, the composites are milled or ground to particles with a length from 10 mm to 50 μm.", "The thermal recycling processes can combust the resin matrix, thereby recovering the glass or carbon fibres.", "Chemical recycling uses the dissolution reagents to depolymerise the matrix of composites.", "Some of these technologies have reached an industrial scale.", "For instance, the Filon Product Ltd. in United Kingdom use grinding to recycle Glass Fibre Reinforced Composites (GFRCs), ELG Carbon Fibre Ltd. in United Kingdom use pyrolysis, Adherent Technologies Inc. in USA use a wet chemical breakdown of composite matrix resins to recover fibrous reinforcements (Oliveux et al., 2015a) and SGL Carbon in Germany employs a solvolysis process to recycle carbon fibres which can be used in the roof of the new BMW i models and in the rear seat of BMW i3 (Gardiner, 2014; SGL, 2016).", "The recyclates can be used as filler or reinforcement in new composites.", "The existing composite recycling techniques have their own advantages and drawbacks.", "The emerging trend is: mechanical recycling is more suitable for glass fibre reinforced composites while thermal and chemical approaches are more for carbon fibre reinforced composites, since glass fibres are tend to be damaged during thermo-chemical processes (Yang et al., 2012).", "The composite recycling is still an immature area.", "This means the developing techniques mentioned above still need to be optimised to produce higher quality recyclates and improve resource efficiency.", "Within the existing work, some research focuses on achieving higher quality products in less time.", "The authors normally try to optimise the recycling techniques with the design of experiment approaches (Meyer et al., 2009; Ye et al., 2013).", "The basic objective is to find the values of a group of parameters which lead to the optimal decomposition rate and decomposition time.", "Some other research considers the resource efficiency, energy efficiency for instance, for the aforementioned recycling processes.", "The existing work of reducing recycling energy consumption has focused so far on searching for more energy efficient operating parameters (Howarth et al., 2014).", "How to optimally use the recyclates to improve the performance, or reduce the cost of the re-manufactured product is also a very important issue worth investigating.", "Research works have also been developed focusing on this topic (Meira et al., 2014; Pohlak et al., 2010).", "Optimisation is important for the development of composite recycling techniques as it can help to improve recyclate quality and process efficiency, as well as reduce cost.", "Currently, there is no paper focusing on the state-of-the-art of the applications of optimisation techniques on composite recycling.", "The aim of this paper is to examine a number of optimisation approaches used in the composite recycling area and outline the future research issues that optimisation in composite recycling as a practice should cover.", "This review is done in the framework of the Efficient X-sector Use of Heterogeneous Materials in Manufacturing (EXHUME) project.", "The purpose of this EPSRC funded project is to establish a sustainable and cost-effective strategy for deconstructing, recycling and remanufacture of composite materials.", "In the remaining of the paper, the methodology used to deliver this review and the optimisation techniques used in the mechanical, thermal and chemical recycling are presented in Section 2; then, in Section 3, optimisation approaches used at the re-manufacture stage is presented; finally, the research gaps and important research opportunities for the future are proposed in Section 4.", "According to Roy et al. (2008), the engineering optimisation is a process of identifying the right combination of the product and associated process parameters for the best solution.", "Traditionally, the process is often done manually.", "The manual approach can be very time consuming, and the searching ability of it is limited and can often lead to sub-optimal solutions.", "Some automatic searching approaches have been developed in recent decades such as Genetic Algorithms (Holland, 1975) and Swarm Intelligence (Blum and Li, 2008) and offer alternative methods to the more traditional manual approaches.", "The mathematical modelling for a specific process is normally developed before the application of optimisation techniques.", "The purpose of this paper is to search the applications of modelling and engineering optimisation in the composite recycling and re-manufacturing areas.", "The composite recycling or re-manufacturing system can be studied at different levels.", "These levels range from the entire factory to the recycling techniques (the y axis in Fig. 1).", "As shown in Fig. 1, each of these levels has its corresponding optimisation objectives (the x axis).", "At the recycling techniques level (mechanical, thermal and chemical recycling methods), improving the quality of recyclates, increasing the recycling rate (decomposition rate) and shortening the processing time are the main issues for consideration.", "At the equipment level, resource efficiency is the main performance indicator to be improved.", "This means resources consumption such as energy and water need to be reduced for recycling certain amount of composites.", "Furthermore, at the enterprise level, the trade-off between quality, resource and time within the whole recycling/re-manufacturing system are to be realised.", "Fig. 1 also presents the potential approaches to realise the optimisation at each level.", "For instance, at the enterprise level, production planning and scheduling methods can be used to achieve the trade-off between quality, resource and time (Liu et al., 2015).", "These approaches are not absolutely independent.", "They overlap each other, filling the whole process for the composite recycling or re-manufacturing systems.", "The model developing and optimisation techniques can be applied on any level.", "The review of existing research works on composite recycling presented in this paper is based on the levels shown in Fig. 1.", "The search strategy consisted of looking for relevant studies within scientific literature sources, represented by academic studies published in peer-reviewed journals and conferences.", "The online databases including Web of Science and ScienceDirect have been searched to identify the relevant articles published on the topics of applications of optimisation techniques on composite recycling with different keywords combination as shown in Table 1.", "The keywords are selected based on some comprehensive review papers about the current status of recycling of composites (Oliveux et al., 2015a; Pimenta and Pinho, 2011; Yang et al., 2012).", "According to these reviews, mainly, keywords used for recycling methods are defined and shown in Table 1.", "Every keyword in each category (‘Keywords used for recycling methods’, ‘Keywords used for materials’, ‘Keywords used for modelling and optimisation’) show in Table 1 is combined with each other to form a string for searching.", "For instance, ‘Mechnical + Carbon Fibre + Optimisation’ is a representative string for literature search.", "Seven and three keywords are selected for the category of ‘Keywords used for recycling methods’ and the category of ‘Keywords used for materials’ respectively since they can cover nearly all names for recycling technologies.", "‘Model’ is selected as a keyword besides ‘optimisation’ and ‘optimise’ in the category of ‘Key words used for modelling and optimisation’.", "Since for the engineering optimisation problems, normally a mathematical model is developed before any optimisation techniques can be applied.", "As a result, research work that focuses on modeling the composite recycling process can be defined as the basement for applying optimisation techniques.", "Besides the search based on the three keywords categories, some extended search using ‘Recycling method’ + ‘Polymer’ + ‘Optimisation/Optimise/Model’ combination have been accomplished.", "There are mainly two reasons for extending the search.", "Firstly, the publication identified in the first round of search is scarce.", "Secondly, the techniques used to optimise the recycling approaches when they are applied for other purposes are potential useful references for their application in composite recycling.", "The numbers of optimisation research works identified on each level are shown in Fig. 1.", "The number of research works identified in each keywords category is show in Table 2.", "All research reported in this paper is based on analysis of published papers referred to in the paper.", "The research work focusing on optimisation techniques applied in composite recycling and re-manufacturing and its relevant system is identified in the literature search.", "They will be presented in the rest of this section and Section 3.", "Molnar (1995) evaluated the potential of using mechanical method to recycle Kevlar® aramid and carbon fibre reinforced scraps and cured prepreg composites.", "During the mechanical recycling process, materials are ground or milled to finer products ranging from 10 mm down to 50 μm in size after a first crushing or shredding into smaller pieces (Oliveux et al., 2015b; Morin et al., 2012).", "The recyclates can be separated into resin-rich powders and fibres of various lengths that are still embedded in resin (Oliveux et al., 2015b; Palmer et al., 2009).", "The resin-rich powders can be reused as active fillers in either bulk molding compound (BMC) or sheet molding compound (SMC) composites (Morin et al., 2012; Pickering, 2006; Schinner et al., 1996; Palmer et al., 2010; Pimenta and Pinho, 2011).", "Recovered fibrous fractions can be reused in thermoset composites (Morin et al., 2012).", "However, the re-use of the mechanical recyclates is limited by the cost and quality issue.", "Virgin fillers such as calcium carbonate or silica have very low costs (Oliveux et al., 2015b).", "Meanwhile, the most important obstacle for using the fibrous fractions as the reinforcement is that the bonding between the recyclates and the new resin is poor.", "This would cause the decreasing mechanical properties of the final product, such as tensile strength of the fibre, Young modulus and surface quality (Oliveux et al., 2015b).", "The mechanical recycling technique is the most mature one among all recycling methods.", "It is mainly used to recycle GFRC.", "The applications for recycling CFRC also exist.", "Based on the literature search, the optimisation works considering the relationship between recyclates quality and machining parameter variance are scarce.", "Schinner et al. (1996) used the milling approach to recycle the carbon fibre reinforced polyether-ether-ketone (PEEK) resin.", "The authors found that cutting mills, gave more homogeneous fibre length distribution and longer fibres than hammer mills, although the cutting blades wore out faster than the former.", "Currently, the mechanical recycling approach has not been optimised on the system level.", "This means the trade-off between the recyclates quality, cost and resource consumption has not been investigated.", "Howarth et al. (2014) developed a bottom-up model to describe the electrical energy consumption of the milling process when it is used as an option for carbon fibre reinforced composite recycling.", "The energy demand of CFRPs recycling using milling can be theoretically calculated when the material removal rate is known.", "It has been discovered that at a recycling rate of 10 kg/hr, the recycling energy consumption (2.03 MJ/kg) is significantly less than the embodied energy of virgin carbon fibre (183–286 MJ/kg) (Howarth et al., 2014).", "This model suggested that there is potential environmental benefit of using recycled fibres in short fibre composites where mechanical performance is less important.", "Based on this model, further optimisation approaches for mechanical recycling to improve the energy efficiency can be developed.", "For instance, the application of heuristics can be considered to achieve multi-objective optimisation in terms of recyclates quality, energy consumption and processing time.", "Based on the above, for the mechanical recycling methods, modelling work has been developed in this area.", "However, there is still a lack of application of optimisation approaches.", "Pyrolysis (Meyer et al., 2009; Pickering et al., 2000; Kaminsky, 2010; Yang et al., 2012; Torresa et al., 2000) is a thermal decomposition method for polymers at high temperature from 300 °C to 800 °C in the absence of oxygen.", "It allows the recovery of long, high modulus fibres.", "In some circumstances, a higher temperature can be applied, however, this will result in some serious degradation of the recyclates.", "Pyrolysis therefore appears to be more suitable to recover carbon fibres, since glass fibres retain less than 50% of their mechanical properties at the minimal temperature of 400 °C (Oliveux et al., 2015a).", "When using pyrolysis as a recycling treatment of polymer matrix composites, the matrix is transformed into smaller molecules at temperatures above 350 °C in an oven.", "These smaller molecules evaporate from the material and, can be used as energy source for the process due to their high calorific value.", "Some pyrolytic carbon (residue) bonded to the fibre surface remains in the oven together with the fibres.", "The amount of residue can influence the mechanical properties of the reclaimed fibres such as fibre strength, electrical properties and fibre-matrix adhesion.", "The amount of residue strongly depends on process parameters as oven atmosphere, temperature, heating rate and others.", "Controlling the value of these parameters in the pyrolysis reactor is crucial for achieving the complete depolymerisation and higher cleanliness level of the recovered fibres.", "To obtain the recovered fibres with properties close to new fibres, Meyer et al. (2009) have used thermogravimetric analysis (TGA) to investigate and optimise different process parameters during pyrolysis to remove the residue as much as possible without oxidation of the carbon fibre itself.", "Based on lab scale pyrolysis experiments, the variation of pyrolysis temperature, oven atmosphere and isothermal residue time had been studied.", "It was found that in the synthetic air environment, within the temperature range from 580 °C to 600 °C, nearly all the matrix can be removed.", "For the oven atmospheres, nitrogen and synthetic air environments have been investigated based on a fixed heating rate of 10 °C/min.", "When the temperature is between 400 °C and 550 °C, the residues exist in both atmospheres.", "However, the nitrogen environment provides better results in this temperature range.", "Above 550 °C the results are changing, as synthetic air provides the condition for residue to be oxidised.", "It is safe to conclude that pyrolysis in nitrogen is tending toward residue production, even at very high temperatures.", "With synthetic air a complete removal of the former matrix can be achieved at around 600 °C.", "However, at 650 °C and above, the carbon fibres start to be oxidised.", "This results in a strong decrease of the mechanical properties of the fibres.", "Consequently, temperatures higher than 650 °C should be avoided when oxygen is used.", "The influence of isothermal residue time at maximum pyrolysis temperatures between 400 °C and 600 °C was examined.", "In a nitrogen atmosphere, an increase in the matrix weight loss was detected for the first 30 min at 400 °C.", "This increase became much smaller at 500 °C.", "It even disappears at 600 °C.", "Hence, the reaction is time dependent only in the lower temperature range.", "Therefore, neither high temperatures above 600 °C, nor long residue time of more than 30min are required when pyrolysis is performed in nitrogen atmosphere.", "Comparatively, the oxidation reaction is much more time dependent.", "An increase in weight loss with residue time was observed in synthetic air, and therefore the exposure time of the carbon fibres in this temperature should be short in order to avoid oxidative fibre damage.", "The above research work focuses on the process level of recycling/re-manufacturing techniques.", "It had been delivered based on experiments.", "This means there is a lack of mathematical model to describe the pyrolysis process used for CFRP recycling, and no automatic optimisation techniques have been applied.", "This work only considered to optimise the mechanical properties of the recyclates based on parameter tuning.", "The cost and environmental impact factors have not been considered yet.", "Cheung et al. (2011a,b); Lam et al. (2011) had developed a series of modelling and optimisation work for pyrolysis process which is used for treating waste tyres.", "These researches can be used as reference for pyrolysis based composite recycling to develop models and optimisation techniques.", "Furthermore, this work has included energy consumption as one of the objective to be optimised.", "Lam et al. (2011) have found that the heating rate and the operation temperature can affect the overall energy consumption, the product quality and yield of the pyrolysis process.", "Based on the fact that pyrolysis is an overall endothermic process but performs exothermically at its early stage, Cheung et al. (2011a,b) proposed an idea to reduce the pyrolysis energy consumption in order to reach its full potential to produce bio-fuels.", "This approach suggests trapping the exothermic heat released in the beginning of the pyrolysis process and using it to fulfil the energy requirement of the endothermic reactions at the end of the process.", "Cheung et al. (2011b) developed a mathematical model that considers the mass loss kinetics, exothermic kinetics, and heat flow together based on tyre pyrolysis experiments.", "A multi-stage pyrolysis operation approach is then proposed with the aid of the developed model.", "The approach starts the pyrolysis with a heating stage to initialise the exothermic reactions.", "This is then changed to an adiabatic stage where the captured exothermic heat is used to facilitate the endothermic reactions afterwards.", "A significant energy saving has been achieved by employing the multi-stage operation strategy comparing with the conventional operation strategy.", "In the industrial pyrolysis process, the heating rate is difficult or often not necessary to be kept constant (Lam et al., 2011).", "This is termed as ‘dynamic heating’ that the heating rate varies at different reaction stages.", "Lam et al. (2011) studied the pyrolysis with dynamic heating to understand the effect of heating rate on production rate, energy consumption and product quality, etc.", "Based on experiments, the production rate and reaction heat of tyre pyrolysis at different heating rates are obtained.", "A transient model considering the effect of dynamic heating was then developed and compared with the conventional static heating model.", "It has been shown that a higher heating rate favours the production of volatiles fractions and shifts the overall pyrolysis heat flow to more endothermic.", "Based on the aforementioned work, the authors (Cheung et al., 2011a,b) proposed a four-stage strategy for the tyre pyrolysis, which has the sequence of heating, adiabatic, heating and adiabatic.", "The approach is verified by the model, and it is capable to save about 22.5% energy consumption with a 100% increase in completion time compared to the conventional strategy.", "Oyedun et al. (2012) proposes an optimisation method to tune the operating parameters in the developed four-stage pyrolysis.", "Finally, this approach can achieve a 29% reduction in energy usage with just 36% increase in completion time.", "Some of the modelling work for pyrolysis had been delivered at this stage, however, the optimisation work still based on experiments.", "None of the advanced automatic optimisation techniques had been applied.", "Ye et al. (2013) used Taguchi method (Roy, 2001) to optimise the steam thermolysis which is used for recycling the epoxy based CFRP materials.", "Steam thermolysis is a combination of vacuum pyrolysis and mild gasification.", "The process is operated with a maximum temperature of 600 °C and under atmospheric pressure.", "Superheated steam is used as the soft oxidant.", "Operational parameters including temperature, isothermal residue time and steam flow-rate have been investigated.", "Two levels for each of them were tested to determine their influence on the decomposition rate of the polymer matrix and the mechanical property of the recycled carbon fibres.", "Taguchi method is used in this research to reduce the number of experiments based on the full factorial design to a practical level.", "Normally, two types of factors are defined in the Taguchi method: control factors and noise factors.", "An inner design constructed over the control factors is used to find the optimum settings (Ye et al., 2013).", "It can be referred to Roy (2001) for more details about Taguchi method and its relevant applications.", "The analysis of variance and standard least squares linear regression are used to analysis the experiment results.", "It has been identified that the decomposition rate of matrix is directly related with the mass ratio of steam/sample, the heating temperature, and the presence of steam at higher temperature during the later stage of the thermal treatment.", "The influences of the process parameters on the tensile strength of the recovered carbon fibres, however, cannot be determined within attributed levels.", "Fluidised-bed process is a pyrolysis-based approach using a bed, of silica sand for example, fluidised by hot air.", "It enables a rapid heating of the materials and release the fibres by attrition of the resin (Oliveux et al., 2015a; Pickering et al., 2000).", "This method has been applied to recycle both GFRCs and CFRCs.", "Pickering et al. (2000) has implemented a rotating sieve separator to separate fibres from fillers of GFRCs recylates.", "The tensile strength of glass fibre was reduced by 50% at 450 °C, while the reduction achieved 80% at 500 °C.", "The strength degradation of carbon fibres is around 25% at 550 °C (Oliveux et al., 2015a; Pickering et al., 2000).", "Microwave-assisted pyrolysis has also been used as a composite recycling method (Lester et al., 2004).", "This method costs less energy since the material is heated in its core so that thermal transfer is very fast (Oliveux et al., 2015a).", "However, both of the processes have not been optimised.", "Based on above, mainly, the manual approaches such as Taguchi method and sensitive analysis have been applied to optimise the thermal recycling approaches.", "Solvolysis is a chemical treatment using a solvent to degrade the resin (Oliveux et al., 2015b; Yoon et al., 1997).", "The solvolysis process can reclaim both the clean fibres and fillers as well as depolymerised matrix in the form of monomers or petrochemical feedstock (Yang et al., 2012; Hydea et al., 2006).", "Based on a wide range of solvents, temperature, pressure and catalysts, solvolysis can offer a large numbers of possibilities.", "Generally, for solvolysis, lower temperatures are required to degrade the polymer compare to pyrolysis (Oliveux et al., 2015a).", "Water or alcohol is normally used as the solvent, which is relatively environmentally friendly (Yang et al., 2012).", "The solvolysis recycling process can be used for both GFRCs and CFRCs.", "The reclaimed fibre retains most of its mechanical properties (Yang et al., 2012).", "To reach higher dissolution efficiency and faster dissolution rate, catalysts are normally used, and the solvent properties are also been tuned and optimised.", "Normally, the factorial design method is used to tune the solvent properties (Oliveux et al., 2015a; Kleinert et al., 2009).", "The solvolysis process has not been optimised on the system level.", "Actually, expensive reactors which can withstand high temperatures and pressures as well as corrosions are required when supercritical conditions are used (Kritzer, 2004).", "As a result there is a trade-off problem between the cost of facilities and the solvent properties that needs to be solved before applying the solvolysis to industrial scale.", "Perrin et al. (2008) used the experimental design and statistical process control techniques to find the optimal values of solution concentration and agitation time for maximizing the dissolution of CaCO3 to reclaim glass fibres from the SMC waste.", "In this research, the authors aimed at obtaining the longest fibres and the highest fibre content in the final recyclates.", "The optimisation approaches applied to the chemical recycling methods are very similar to the ones applied to the thermal recycling approaches.", "They are still manual approaches such as factorial design.", "Optimal usage of the recyclates during the re-manufacturing is another very important issue in the composite recycling and re-manufacturing system.", "According to the existing work, researchers have considered to use the recyclates to achieve cost reduction while guaranteeing the necessary mechanical performance.", "Meira et al. (2014) uses the mechanical recycling method to reclaim the thermoset based glass fibre reinforced polymer rejects derived from the pultrusion manufacturing industry.", "The recyclates are used as aggregates and reinforcement replacements into concrete-polymer composite materials.", "The authors found that the recyclate content and size grade, and the effect of the incorporation of an adhesion promoter can be considered as factors which influence the flexural and compressive strengths of final composite.", "The authors used the Fuzzy Boolean Nets method to find the best balance between material parameters that maximizes both flexural and compressive strengths of final composite based on limited experiment result.", "Fuzzy Boolean Nets is a universal approximator with excellent generalisation capability which is able to predict the response of parameter-dependent systems (Carvalhoa and Tomé, 2007).", "This technique has been selected for the aforementioned study due to its good performance based on sparseness of available trial data (Meira et al., 2014).", "It can be referred to Carvalhoa and Tomé (2007) for more information about the working procedure of this technique.", "Shah et al. (2010) developed a reinforcement learning algorithm (Gosavi, 2003, 2007) to help the plant manager to optimally use the recycled materials, thereby reducing the cost of the final products.", "Reinforcement learning allows machines and software agents to automatically determine the ideal behaviour within a specific context, in order to maximise its performance (Gosavi, 2003).", "The reason for using this technique to optimise the above problem is that it can generate near-optimal solutions to stochastic dynamic programs without probability transition (Shah et al., 2010).", "Gosavi (2003) introduced how a typical reinforcement learning algorithm works.", "Normally, it is environmentally and costly beneficial to use recyclates.", "However, the service level can be negatively influenced by using recyclates since they usually need additional pre-processing which results in an increasing in the production time.", "Hence, it is necessary to use virgin materials when the supply of finished products is running low, to satisfy service levels.", "The authors modelled the aforementioned material selection as a semi-Markov decision problem.", "The developed reinforcement learning algorithm identified an optimal strategy of switching between the two sources of material.", "The simulation result shows a 65% cost improvement over a policy that uses only virgin materials.", "This research provides a general material selection strategy when recyclates are used as one type of the raw material.", "It is a very good reference on the optimisation work on the workshop level when automatic optimisation techniques are employed.", "Zhao et al. (2008) investigated the feasibility of manufacturing wood-rubber functional composite panels with a polymeric methylene diphenyl diisocyanate and urea-formaldehyde combination binder.", "The response surface method was used to determine the significant independent variables among board density, pressing time and pressing temperature and their influence on the board performance including internal bond strength, modulus of rupture and modulus of elasticity.", "A mathematical simulation or response surface models were developed to predict the board properties.", "This work is not directly related with the optimisation research in composite recycling.", "However, the response surface method used in it can be used as a reference for the design optimisation research focusing on using recyclates during re-manufacturing.", "Pohlak et al. (2010) developed a multistage optimisation procedure to find the optimal thickness of the glass fibre polyester resin layer in the composite bathtub.", "The objectives of the optimisation procedure are to minimise the total cost of the bathtub and the production time.", "The ratio of polyester resin to the curing agent has significant influence on curing time, and both objectives considered.", "The curing time decreases with increasing layer thickness.", "Compare to structure with constant thickness, the structure with optimal thickness distribution of the reinforcement layer has several advantages.", "The stresses and displacements are reduced several times and have more uniform distribution.", "This work can be very beneficial for research focusing on optimising the product quality at the design stage when using recyclates as the reinforcement.", "In the recyclates re-manufacturing sector, both of the manual and automatic searching approaches (Fuzzy Boolean Nets and reinforcement learning algorithm) has been applied as the optimisation approaches.", "More automatic searching approaches are expected to be applied in this sector since the re-manufacturing is more focused on the system level.", "Based on the literature presented in this paper, it can be found that on most levels of composite recycling and re-manufacturing system, the application of optimisation techniques can be identified.", "However, these applications are still scarce considering the potentialities to be optimised.", "Meanwhile, most of the techniques applied are the experimental design approaches to tune the parameters.", "The following research gaps are identified:", "There is a lack of mathematical models to depict the influence of variance of processing parameters on the quality of recyclates for all recycling approaches.", "Based on the models, more efficient optimisation techniques can be applied.", "Currently, most of the existing optimisation work on the composite recycling technology level is developed based on experiments and experiences.", "The development of mathematical models for these recycling techniques is crucial for researchers to better understand and optimise them.", "There is a lack of mathematical models to depict the influence of variance of processing parameters and other relevant factors on the cost and environmental impact of all recycling approaches.", "Cost and environmental issues during the composite recycling is very important, therefore the developed approaches should not only be technically feasible, but also cost effective and environmentally friendly.", "There is no work that focuses on optimising the composite recycling on the workshop level.", "Based on existing research in the sustainable manufacturing area, operations research and scheduling methods had been proved to be very effective tools to reduce energy consumption and environmental impact of a specific manufacturing system.", "However, in the literature identified in the composite recycling area, energy and cost saving haven't been considered on the workshop level.", "The optimisation work on this level is very important, since it can considerably reduce the negative impact of composite recycling system without changing the legacy system too much.", "Even if a new factory for recycling composites were going to be developed, considering optimisation on the operations level would also be very beneficial.", "The re-use of recyclate is as significant as recycling the composites.", "The work focuses on the optimal usage of the recyclates to improve the quality, or reduce the cost of a specific product is scarce.", "The feasible and economical applications of recyclates still need to be considered at the current stage of investigation.", "At the same time, the modelling and optimisation techniques can be applied at any level in the re-manufacturing system.", "For instance, the optimal parameter combination used for the re-manufacturing technologies is worth to be investigated; the optimal combination of virgin and recycled fibres could also be considered; on the workshop level, optimising the dispatching of recyclates for production can also effectively reduce the production cost.", "In terms of the optimisation techniques, in the existing research, very little optimisation algorithm (automatic approaches) has been applied to complete the optimisation tasks.", "The design of experiments methods are widely used in the existing research.", "However, more preferable results could have been achieved if techniques like Genetic Algorithm (Holland, 1975) or Artificial Neural Networks (Yegnanarayana, 2009) had been applied.", "The efficiency of the composite recycling and re-manufacturing system can be largely improved by applying the optimisation algorithm.", "This area is worth to be investigated in the future." ]
[]
Chemotactic behavior of egg mitochondria in response to sperm fusion in mice
Mitochondria are the powerhouses of eukaryotic cells and their positioning contributes to fertilization and early developmental processes. We report that sperm fusion triggers Ca2+ oscillations and mitochondrial movement toward fused sperm (mitochondrial chemotaxis) in mouse eggs. Mitochondria functioned in Ca2+ storage and were colocalized with endoplasmic reticulum (ER) during Ca2+ oscillations. Mitochondria then moved toward the fused sperm. Sperm extracts lacking nuclei induced Ca2+ oscillations, but did not promote mitochondrial chemotaxis. Our results suggest that sperm fusion motivates Ca2+ oscillation-independent mitochondrial chemotaxis. This phenomenon indicates that egg mitochondria interact with sperm materials, presumably nuclear substances, and their network tethers egg and sperm nuclei at the early stage of zygote formation.
[ "MULT" ]
[ "Cell biology", "Developmental biology", "Molecular biology", "Zoology" ]
[ "1000" ]
[ "Mitochondria are important organelles for ATP production in eukaryotic cells and the regulation of cell functions [1]; accordingly, they are important determinants of the success of fertilization and early embryonic development.", "They are not autonomous, but form an interconnected network that varies in structure according to cell functions [2].", "Mitochondrial replacement has recently emerged as a promising strategy to prevent the transmission of mitochondrial diseases and infertility [3].", "Accordingly, understanding mitochondrial dynamics has substantial implications for basic biology and medical science.", "Mitochondrial fusion and fission generate mitochondria with distinct morphologies and interconnected networks [4].", "In animals, mitochondria are strictly inherited from a single parent, usually the mother.", "After fertilization, sperm mitochondria are eliminated by diverse mechanisms, including ubiquitination-proteasomal degradation [5], mitophagy [6], and allophagy [7].", "However, the biological significance of the uniparental transmission of mitochondria remains mysterious.", "When cells are exposed to external stimuli, calcium ions (Ca2+) are released from intracellular stores, such as the endoplasmic reticulum (ER), leading to oscillatory rises in the intracellular Ca2+ concentration (Ca2+ oscillation) or transient rises [8].", "Accordingly, Ca2+ is categorized as a second messenger involved in transducing external signals to intracellular events [9].", "After fertilization, mammalian eggs transition from the metaphase-arrested state of the second meiotic division (MII), termed egg activation, and this is initiated by Ca2+ oscillation [10].", "In ascidians, sperm-triggered Ca2+ oscillations are transduced into mitochondrial Ca2+ signals that stimulate mitochondrial respiration [11].", "Mitochondria may function to absorb intracellular Ca2+ by the concentration of heterogeneously distributed mitochondria in eggs after sperm fusion, leading to Ca2+ oscillations.", "Furthermore, Ca2+ oscillations may be coordinated by the interplay between ER and mitochondrial activities in eggs.", "However, the precise links between Ca2+ oscillations, mitochondrial movement, and sperm fusion have not been established.", "To study mitochondrial dynamics during Ca2+ oscillations, we monitored mouse eggs after sperm fusion using eggs labelled with fluorescent biomarkers.", "A red-fluorescent dye that stains mitochondria in living cells in a membrane potential-dependent manner (MitoTracker Red CMXRos), a visible light-excitable calcium indicator (Oregon Green 488 BAPTA-1, AM), and a photostable probe selectively stains the ER in living cells (ER-Tracker Blue-White DPX) were purchased from Invitrogen Corp (Waltham, MA, USA).", "Egg and sperm nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen Corp.).", "Trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), a potent uncoupler of oxidative phosphorylation in mitochondria that disrupts membrane potential and ATP synthesis by transporting protons across cell membranes [12], was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).", "Female C57BL/6N mice (8–12 weeks old; purchased from Japan SLC Inc., Shizuoka, Japan) received intraperitoneal injections of 5 IU of pregnant mare's serum gonadotropin (PMSG; Merck4Biosciences, Darmstadt, Germany) followed by 5 IU of human chorionic gonadotropin (hCG; Merck4Biosciences) 46–48 h apart.", "Eggs at metaphase II were collected from the oviduct of females 14–16 h after hCG administration.", "To collect cumulus-removed, zona-intact eggs (zona-intact eggs), cumulus cells were dispersed from eggs by incubation for 10 min at 37 °C in TYH medium or M2 medium containing hyaluronidase (300 μg/ml; Merck4Biosciences).", "The eggs were then incubated in TYH medium or M2 medium.", "To collect eggs lacking the zona pellucida (zona-free eggs), ovulated eggs were incubated with collagenase (WAKO#034-10533; Wako Pure Chemical Industries Ltd., Osaka, Japan) at a final concentration of 0.1 mg/ml for 5 min at 37 °C in TYH medium or M2 medium.", "Sperm collected from the epididymides of 8- to 12-week-old B6C3F1 male mice were capacitated by incubation in TYH medium for 30 min in an atmosphere of 5% CO2 in air at 37 °C before insemination.", "All mice were housed under specific pathogen-free controlled conditions.", "Food and water were available ad libitum.", "The procedures for animal experiments were in accordance with the principles and guidelines of the Institutional Animal Care and Use Committee of the National Research Institute for Child Health and Development and were approved by this committee (No. A2004-004-C13).", "Zona-free eggs were incubated in medium containing 500 nM MitoTracker Red CMXRos, 5 μM Oregon Green 488 BAPTA-1, AM, and 5 μg/ml DAPI for 30 min at 37 °C, and were washed twice in TYH medium.", "The eggs were transferred to a 2-μl drop of TYH medium in glass-based dishes (35 mm; IWAKI Glass Co. Ltd., Tokyo, Japan) and fixed by covering with a square coverslip (18 mm × 18 mm) spotted with white Vaseline.", "TYH medium was added to the eggs from the slit of coverslips.", "The sperm (final concentration, 5 × 102 sperm/ml) were then added to the TYH medium containing eggs.", "The eggs were monitored with CSU-Frontier (Yokogawa Corp., Tokyo, Japan) by incubation in TYH medium in an atmosphere of 5% CO2 in air at 37 °C.", "The serial Z-stack images were reconstructed as 3-dimentional (3D) movies.", "Zona-intact eggs were incubated in M2 medium or Ca2+-free M2 medium containing 500 nM MitoTracker Red CMXRos and 5 μM Oregon Green 488 BAPTA-1, AM for 30 min at 37 °C, and were washed twice in new media.", "The eggs were transferred to a glass-based dish (35 mm) and covered with a square coverslip (18 mm × 18 mm) sealed with double-faced tape (NITOMS, Inc., Tokyo, Japan).", "The eggs were filled with M2 medium or Ca2+-free M2 medium.", "The eggs were monitored using a confocal laser microscope (LSM510Meta; ZEISS, Jena, Germany).", "After 5 min of incubation, FCCP was added to the medium at a final concentration of 1 μM.", "After 10 min of incubation, fresh M2 medium or Ca2+-free M2 medium was added to eggs and the medium containing FCCP was wiped with a paper towel.", "The eggs were monitored for an additional 10 min.", "The serial Z-stack images were reconstructed as 3D movies.", "Zona-intact eggs were stained with 500 nM MitoTracker Red CMXRos and 1.7 μM ER-Tracker Blue-White DPX for 30 min in TYH medium, and were washed twice in fresh TYH media.", "After the eggs were washed, they were transferred to a 3-μl drop of TYH medium and monitored using a confocal laser microscope (LSM510Meta).", "The sperm (final concentration, 5 × 102 sperm/ml) were then added to the medium containing the eggs.", "After 3 h of insemination, fertilized eggs were observed using a confocal laser microscope (LSM510Meta).", "The serial Z-stack images were reconstructed as 3D movies.", "Zona-free eggs were collected from 8 to 12-week-old female C57BL/6N mice (Cd9+/+) and Cd9−/− mice.", "As reported previously [13, 14], Cd9-deficient eggs are defective in sperm fusion, but sperm adhesion occurs normally.", "Thus, we determined whether sperm adhesion contributes to mitochondrial chemotaxis by monitoring zona-free Cd9-deficient eggs adhered to sperm in comparison with wild-type eggs adhered and fused to sperm.", "They were incubated in M2 medium containing 5 μM Oregon Green 488 BAPTA-1, AM and 5 μg/ml DAPI for 30 min, and were washed twice for 10 min in fresh M2 medium.", "After the eggs were washed, they were transferred to glass-based dishes (35 mm), and fixed by covering with a square coverslip (18 mm × 18 mm) spotted with white Vaseline.", "The 15-μl TYH medium was added to the eggs from the slit of coverslips.", "The sperm (final concentration, 5 × 102 sperm/ml) were then added to the medium containing the eggs.", "Then, the eggs were monitored using CSU-Frontier (Yokogawa Corp.) by incubation in M2 medium in an atmosphere of 5% CO2 in air at 37 °C.", "The serial Z-stack images were reconstructed as 3D movies.", "Sperm were collected from the epididymides of 8- to 12-week-old B6C3F1 male mice and washed 3 times in phosphate-buffered saline.", "After the sperm were washed, they were suspended in intracellular-like medium (ICM) containing 120 mM KCl, 20 mM HEPES, 0.1 nM EGTA, 10 mM Na-β-glycerophosphate, 0.2 mM PMSF, and 1 mM DTT.", "The sperm suspension was treated 5 times by ultrasonication for 20 s and centrifuged for 30 min at 4 °C at 12,000 × g.", "The supernatants (mouse sperm extracts) were frozen at −80 °C.", "The mSE was injected into the zona-intact eggs in Ca2+-free HEPE-CZB medium.", "The volume of injected mSE was set to 1–5% of the egg volume.", "The serial Z-stack images were reconstructed as 3D movies.", "In addition, mSE were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% acrylamide gels and stained with Coomassie Brilliant Blue (CBB) and silver stain solutions.", "Zona-free eggs were stained with 500 nM MitoTracker Red CMXRos, 1.7 μM ER-Tracker Blue-White DPX for 30 min in TYH medium, and were washed twice in fresh TYH media.", "The eggs were treated with 5 μM nocodazole or 24 mM latrunculin A for 10 min.", "After treatment, the eggs were transferred to glass-based dishes (35 mm), and fixed by covering with a square coverslip (18 mm × 18 mm) spotted with white vaseline.", "The 15-μl TYH medium was added to the eggs from the slit of coverslips.", "The sperm at the final concentration of 5 × 102 sperm/ml were then added to the medium containing the eggs.", "The eggs were monitored with CSU-Frontier (Yokogawa Corp., Tokyo Japan) by incubating them in M2 medium in an atmosphere of 5% CO2 in air at 37 °C.", "In addition, cumulus-intact eggs were collected from C57BL/6N and treated with 24 mM latrunculin A.", "After washing, the sperm at the final concentration of 1 × 105 sperm/ml were added to the eggs, and the rate of eggs at the pronuclear stage was estimated by confocal-microscopic observation.", "Comparisons were evaluated by one-way analysis of variance following by post-hoc Scheffe's tests, Mann–Whitney U-tests, or Fisher's exact tests.", "Statistical significance was defined as P < 0.05.", "Results are expressed as means ± standard error of the mean (SEM).", "We examined the relationship between mitochondrial activity and the intracellular Ca2+ concentration after sperm fusion (Fig. 1).", "Mitochondrial membrane potential is critical for maintaining the physiological function of the respiratory chain to generate ATP [15].", "Since the eggs are covered with cumulus cells and the egg coat, i.e., the zona pellucida, the time of onset of sperm fusion cannot be precisely controlled.", "Therefore, to standardize this time, we used cumulus cell- and zona-free eggs.", "When eggs were monitored from the time of sperm addition (set to 0 min) (Fig. 2A and Movie 1), the first Ca2+ rise was recorded at 80 min and repetitive Ca2+ rises continued until 140 min.", "In this experiment, eggs were scanned every 5 min; accordingly, the number of Ca2+ rises was less than previous estimates for eggs scanned at 1-min intervals [10, 16].", "As reported previously [10, 16], Ca2+ was evenly distributed throughout the egg cytoplasm (80 and 105 min in Fig. 2A).", "Mitochondrial membrane potential increased moderately after sperm addition and returned to the steady state after the end of Ca2+ oscillations.", "This pattern appeared to be correlated to the period of Ca2+ oscillations.", "We examined the relationship between mitochondrial membrane potential and intracellular Ca2+ concentration.", "In particular, we examined unfertilized eggs transiently treated with FCCP.", "As shown in Fig. 2B and Movie 2, FCCP treatment and removal reduced and increased mitochondrial membrane potential in cumulus-free and zona-intact eggs (zona-intact eggs), corresponding to the increase and reduction of the Ca2+ concentration at 5 and 20 min.", "To restrict Ca2+ reservoirs to the egg cytoplasm, we next incubated eggs in Ca2+-free M2 medium (Fig. 2C and Movie 3).", "In the Ca2+-free M2 medium, the patterns in FCCP-treated zona-intact eggs were quite similar to those of eggs in conventional M2 medium at 7.5 and 20 min.", "Membrane contact sites between ER and mitochondria are hotspots for Ca2+ signaling [17, 18, 19].", "To examine the interaction between ER and mitochondrial membranes, we compared their localization in zona-intact eggs before and after sperm fusion.", "Mitochondria and ER were labeled with MitoTracker Red CMXRos and ER Tracker Blue-White DPX, respectively (Fig. 2D).", "ER was patchily distributed mainly underneath the plasma membrane, whereas mitochondria were localized mainly in the inner space (upper images in Fig. 2D).", "The ER colocalized with mitochondria at 3 h after insemination (middle and lower images in Fig. 2D).", "To perform long-time observation of living eggs without cellular damage, the eggs were scanned every 5 min.", "Although Ca2+ rises were frequently missed under this condition, Ca2+ oscillation was predicted to serially occur beyond 300 min (Fig. 2A), because Ca2+ oscillation corresponds to mitochondrial activity.", "Additionally, mitochondria partially colocalized with ER even in unfertilized eggs.", "Thus, we assumed that ER-mitochondria colocalization might be involved in long-lasting Ca2+ oscillation (Fig. 2E).", "To examine the mitochondrial distribution during Ca2+ oscillation, we monitored zona-free eggs stained with MitoTracker Red CMXRos and Oregon Green 488 BAPTA-1, AM before and after sperm addition (Fig. 3A, B and Movie 4).", "At 10 min after sperm addition, the sperm fused to the egg plasma membrane (arrowheads in Fig. 3A), and the first Ca2+ rise was observed.", "At 70 min, polar body extrusion began.", "Mitochondria began to move from 110 min and reached sperm nuclei at 155 min (dotted lines in Fig. 3A).", "The final Ca2+ rise occurred at 185 min after sperm addition.", "Moreover, fluorescence increased only for sperm DNA, and not for egg DNA or egg cytoplasm, from 65 to 95 min after sperm addition (Fig. 3C).", "Concurrently, we monitored mitochondria in unfertilized eggs (Fig. 3D and Movie 5).", "Mitochondria were stationary throughout the monitoring period in the eggs without sperm fusion (dotted lines in Fig. 3D).", "The fluorescence (dotted circles in Fig. 3D) reduced gradually (Fig. 3E, F).", "In non-mammalian vertebrates, sperm adhesion induces a transient Ca2+ rise in eggs, leading to egg activation [20].", "As reported previously [13, 14], Cd9-deficient eggs are defective in sperm fusion, but sperm adhesion occurs normally.", "Thus, we determined whether sperm adhesion contributes to mitochondrial chemotaxis by monitoring zona-free Cd9-deficient eggs adhered to sperm in comparison with wild-type eggs adhered and fused to sperm.", "Eggs were stained with Oregon Green 488 BAPTA-1, AM.", "Eggs were preloaded with DAPI, and we were able to easily determine the beginning of sperm fusion by DAPI transfer from eggs to the sperm nuclei [21].", "In wild-type eggs, a Ca2+ rise occurred before DAPI incorporation into the sperm DNA (box in Fig. 4A).", "We next monitored the Ca2+ concentration in Cd9-deficient eggs (Fig. 4B), but no Ca2+ rise was detected.", "This result implies that sperm fusion increases the Ca2+ concentration in the egg cytoplasm, leading to mitochondrial chemotaxis.", "To examine the role of sperm materials in mitochondrial chemotaxis, sperm extracts lacking nuclei were injected into zona-intact eggs and mitochondrial chemotaxis was monitored (Fig. 4C and Movie 6).", "The mouse sperm extracts were injected into the egg in the left region (indicated by arrowheads in the figure).", "The first Ca2+ rise was observed at 5 min after mSE injection.", "The sperm extracts induced Ca2+ oscillations at 10, 20, 90, 115, and 140 min (Fig. 4D), but not mitochondrial chemotaxis (dotted circles in Fig. 4C), and mitochondrial activity was reduced gradually (Fig. 4D).", "To explore the components that trigger mitochondrial redistribution, we examined the components included in the mSE after ultrasonication.", "The sperm heads were sustained microscopically in spite of ultrasonication (Fig. 5A).", "After centrifugation, nucleotides were below the detectable level (Fig. 5B).", "Otherwise, multiple proteins were detected in the mSE (Fig. 5C).", "Form this result, we hypothesize that in the absence of detectable DNA/RNA in the ultrasonicated sperm heads, these molecules contribute to the mitochondrial attraction.", "To investigate the contribution of cytoskeletons to mitochondrial redistribution, we observed zona-free eggs treated with cytoskeletal disruptors (Fig. 6).", "We used two types of the disruptors, nocodazole that induces microtubule depolymerization [22], and latrunculin A that disrupts microfilament organization by binding to monomeric G-actin [23].", "After zona-fee eggs were treated with nocodazole or latrunculin A for 10 min, they were incubated with the sperm.", "In nocodazole-treated eggs, the sperm fused to the egg at 20 min after insemination, and the first Ca2+ rise occurred.", "Despite no second polar body extrusion and chromosomal separation, maternal mitochondria moved and reached the sperm nucleus at 200 min after insemination (Fig. 6B and D).", "Ca2+ rises occurred 5 times from 30 to 90 min, but their peaks were small (Fig. 6B).", "This result suggests that mitochondrial redistribution is independent from microtubule organization.", "In latrunculin A-treated eggs, the sperm fused to the egg at 65 min after insemination, and the first Ca2+ rise occurred.", "Similarly to the nocodazole-treated eggs, the second polar body extrusion was disabled in the latrunculin A-treated eggs (Fig. 6C and E).", "Notably, latrunculin A treatment completely inhibited mitochondrial redistribution and lead to the formation of mitochondrial aggregates (Fig. 6E).", "This result suggests that microfilaments regulate mitochondrial redistribution.", "To investigate the effect of latrunculin A treatment on pronuclear formation, we counted the rate of pronuclear zygotes.", "The cumulus-intact eggs were treated with latrunculin A for 10 min and incubated with sperm.", "When the rate of pronuclear zygotes was counted, 14.3% of inseminated eggs developed to the pronuclear zygote in the latrunculin A-treated eggs, compared to 59% of inseminated eggs.", "Latrunculin A treatment had no effect on pronuclear formation in uninseminated eggs.", "This result indicates that microfilaments also contribute to pronuclear formation (Fig. 6F).", "We explored the relationship between mitochondrial activity and the intracellular Ca2+ concentration in eggs (Fig. 6G).", "First, we showed that sperm fusion-triggered Ca2+ oscillation is linked to mitochondrial activity, presumably via interactions with the ER.", "Second, sperm-egg fusion triggers Ca2+ oscillation-powered mitochondrial chemotaxis (mitotaxis) toward the fused sperm.", "Finally, mitochondrial redistribution depends on actin microfilament.", "In many organisms, a transient intracellular Ca2+ rise occurs in eggs after sperm fusion and is essential for the resumption of meiosis and egg activation [24].", "Ca2+ is released from mitochondria in sea urchins [25], and ATP molecules produced in mitochondria function in pacemaker Ca2+ oscillations in ascidians [26].", "Our results show that mitochondria also play a crucial role in Ca2+ signaling as a Ca2+ reservoir in mice (Fig. 2B, C).", "In yeasts, the multi-subunit ER-mitochondria encounter structure, which tethers the ER and mitochondria, is both spatially and functionally linked to sites of mitochondrial division [17, 18, 19].", "As reported previously [26, 27], small patches of ER localize underneath the egg plasma membrane in unfertilized eggs and partially colocalize with mitochondria, as supported by our results (Fig. 2D).", "In addition, our results suggest that in fertilized eggs, the ER is internalized, form a large cluster, and colocalizes with mitochondria (Fig. 2D, E).", "In mammals, PIP2 hydrolysis generate IP3, which activates IP3 receptor-mediated Ca2+ release from ER [28].", "Ca2+ flux from the ER to mitochondria is a major determinant of some mitochondrial functions [29].", "Since the dysfunction of this Ca2+ flux causes several human diseases [30], its tight regulation is crucial for cellular homeostasis.", "Our results indicate that ER and mitochondria may cooperatively function in fertilized mouse eggs as well.", "Microinjection of sperm extracts is known to induce Ca2+ oscillation, despite the absence of sperm fusion [16].", "However, in our study, sperm extracts were unable to promote mitotaxis (Fig. 4C, D).", "The sperm structures were sustained after ultrasonication, and nucleotides were below the detectable level in the mSE (Fig. 5).", "These results indicate that the mSE lacks a major part of genomic DNA and RNA, implying that sperm nucleotides attract egg mitochondria after sperm fusion.", "Microfilaments directly implicate in mitochondrial ATP production and distribution during oocyte maturation [31].", "Furthermore, our result revealed that microfilaments regulate mitochondrial redistribution.", "Compatibility between the mitochondrion and nucleus is essential for somatic cell nuclear transfer [32], and optimal component pairing is essential for efficient cellular respiration [32] and defeat of ROS [33].", "Egg mitochondria are expected to have a selective advantage for paternal mitochondria.", "Our study improves our understanding of the molecular mechanisms underlying the matching of mitochondria with sperm nuclei after fertilization.", "Maki Iwai: Performed the experiments; Wrote the paper.", "Yuichirou Harada: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.", "Rinako Miyabayashi, Woojin Kang, Akihiro Nakamura, Natsuko Kawano, Yoshitaka Miyamoto: Performed the experiments.", "Mitsutoshi Yamada, Toshio Hamatani, Mami Miyado, Keiichi Yoshida, Hidekazu Saito, Mamoru Tanaka, Akihiro Umezawa: Analyzed and interpreted the data.", "Kenji Miyado: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.", "This work was supported by the Grant-in-aids for Scientific Research from the Japan Society for the Promotion of Science (No. 26670733 and No. 26293363 to K. Miyado, No. 17K16850 to K. Yoshida, No. 16KK0192 to Y. Miyamoto, and No. 18K16824 to M. Iwai).", "The authors declare no conflict of interest.", "No additional information is available for this paper.", "Supplementary content related to this article has been published online at https://doi.org/10.1016/j.heliyon.2018.e00944." ]
[]
Shades of white: diffusion properties of T1- and FLAIR-defined white matter signal abnormalities differ in stages from cognitively normal to dementia
The underlying pathology of white matter signal abnormalities (WMSAs) is heterogeneous and may vary dependent on the magnetic resonance imaging contrast used to define them. We investigated differences in white matter diffusivity as an indicator for white matter integrity underlying WMSA based on T1-weighted and fluid-attenuated inversion recovery (FLAIR) imaging contrast. In addition, we investigated which white matter region of interest (ROI) could predict clinical diagnosis best using diffusion metrics. One hundred three older individuals with varying cognitive impairment levels were included and underwent neuroimaging. Diffusion metrics were extracted from WMSA areas based on T1 and FLAIR contrast and from their overlapping areas, the border surrounding the WMSA and the normal-appearing white matter (NAWM). Regional diffusivity differences were calculated with linear mixed effects models. Multinomial logistic regression determined which ROI diffusion values classified individuals best into clinically defined diagnostic groups. T1-based WMSA showed lower white matter integrity compared to FLAIR WMSA-defined regions. Diffusion values of NAWM predicted diagnostic group best compared to other ROI's. To conclude, T1- or FLAIR-defined WMSA provides distinct information on the underlying white matter integrity associated with cognitive decline. Importantly, not the “diseased” but the NAWM is a potentially sensitive indicator for cognitive brain health status.
[ "BIOC", "MEDI", "NEUR" ]
[ "Aging", "Alzheimer's disease", "Diffusion tensor imaging", "MRI", "White matter", "White matter hyper intensities" ]
[ "1302", "1309", "2717", "2728", "2800" ]
[ "The authors have no actual or potential conflicts of interest.", "White matter signal abnormalities (WMSAs) are a common observation on magnetic resonance imaging (MRI) scans in older individuals as well as patients with dementia.", "WMSA appear dark (i.e., hypointense) on T1-weighted images and bright (i.e., hyperintense) on fluid-attenuated inversion recovery (FLAIR) images, showing a generally larger volume on FLAIR images compared with T1-weighted images.", "Their prevalence and severity increase with age and are associated with cognitive dysfunction and an increased risk of Alzheimer's disease (AD) (Abe et al., 2002; Burns et al., 2005; DeCarli et al., 1995; de Leeuw et al., 2001; Erten-Lyons et al., 2013).", "While the underlying pathology of WMSA is still not fully understood, they are considered to be an important proxy for vascular-associated brain tissue damage (Benedictus et al., 2014; Salat, 2014; Wardlaw et al., 2013a,b).", "Meta-analysis of 19 studies correlating postmortem observations of WMSA with pathology data revealed that the suspected pathogenic mechanisms are heterogeneous, including ischemia/hypoxia, hypoperfusion and altered cerebrovascular autoregulation, blood-brain barrier leakage, inflammation, degeneration, and amyloid angiopathy (Erten-Lyons et al., 2013; Gouw et al., 2008, 2011).", "The wide range of potential etiologies underlying WMSA reflects the currently accepted idea that WMSA have a more global effect on white matter integrity, reaching beyond the WMSA itself.", "Variability in microstructural changes in the normal-appearing white matter (NAWM) have been shown to be associated with various neuropsychological measures (Vernooij et al., 2009), suggesting that changes in NAWM integrity based on diffusion tensor imaging (DTI) may be more closely associated with early symptomatology and related early pathology in neuronal tissue cognitive decline than WMSA load (Altamura et al., 2016).", "The Rotterdam study showed that WMSA may impact the health of the NAWM, showing a relationship between WMSA burden and microstructural white matter changes in the NAWM, as measured with DTI (Vernooij et al., 2008).", "In this study, the relation between regional decreases in fractional anisotropy (FA) and age was nearly completely explained by white matter atrophy or white matter lesions.", "Interestingly, Leritz et al. applied a similar approach but defined the WMSA using T1 contrast and reported that WMSA do not fully explain the association between age and microstructural white matter changes in the NAWM.", "The latter authors suggested that the differences in the amount of explained variance by WMSA may be dependent on the MRI contrast on which WMSAs are defined (Leritz et al., 2014).", "This could suggest that WMSA identified on either T1 or FLAIR images reflect different neural markers or represent similar pathologies but have a different sensitivity to the severity of the pathology.", "Studies in multiple sclerosis (MS) patients, showed that the T1 sequence is more specific for atrophy (Sailer et al., 2001) and that T1 WMSA are histologically correlated with axonal loss and demyelination (Bitsch et al., 2001).", "However, log-transformed WMSA volumes determined on FLAIR images are on average 1.14 times higher than those on T1-weighted images, but both volumes are highly correlated in both aging and MS (Coutu et al., 2016) (Klistorner et al., 2016).", "WMSA are also important for the clinical outcome of patients, as large WMSA have been linked to dementia (Prins and Scheltens, 2015).", "But distinguishing cognitively healthy individuals from those with incipient to mild cognitive impairment (MCI) based on WMSA is more challenging.", "Studies correlating the WMSA burden with cognition have shown both negative and positive associations as well as no associations (Bombois et al., 2007; Burns et al., 2005; Debette et al., 2007; de Mendonca et al., 2005; Jacobs et al., 2012; Smith et al., 2000; Tullberg et al., 2004; ).", "DTI may be a more promising technique to identify subgroups early on in the disease as it is more sensitive to microstructural changes.", "Variability in microstructural properties in the NAWM has been shown to be associated with various neuropsychological measures in the general population (Vernooij et al., 2009) and in patients with dementia (Altamura et al., 2016).", "This suggests that NAWM integrity measures based on DTI may be more closely associated with early symptomatology and related early pathology in neuronal tissue than WMSA load.", "The NAWM may indeed contain valuable predictive information for pathology, as changes in the DTI properties of the penumbra of WMSA have been shown to predict future formation of WMSA (Amlien and Fjell, 2014; Maillard et al., 2013).", "It is the aim of this study to investigate whether the microstructural properties of white matter tissue measured with DTI within WMSA, differs between those identified on T1-weighted images, from those identified on FLAIR images in memory-clinic patients, which received a clinical diagnoses of AD, MCI, subjective cognitive decline (SCD), and in normal controls.", "Furthermore, based on the previously discussed literature, we expect that the white matter integrity in the NAWM and the border surrounding the WMSA will be higher compared to the integrity in the FLAIR- or T1-defined regions.", "Finally, to test the hypothesis that the discrimination of diagnostic groups by DTI measures may be dependent on the choice of the region of interest (ROI), we also investigated the probability of diffusion values in these different ROIs to distinguish groups based on clinical diagnoses.", "The results of this study can provide more insight into the difference in white matter integrity of WMSA defined on T1 versus FLAIR in neurodegenerative disorders and can contribute in tailoring more sensitive biomarkers based on DTI data.", "A total of 85 patients (SCD, n = 26), MCI patients (n = 43) and patients with AD (n = 16) were recruited at the memory clinic of Maastricht University Medical Center.", "In addition, 18 healthy participants without signs or complaints of cognitive impairment were recruited via advertisements from the general population to serve as cognitively normal controls (CN).", "All patients underwent a standard diagnostic workup, including clinical history taking, medical and neurological examination, blood draw for clinical chemistry, functional evaluation using the Clinical Dementia Rating scale (Hughes et al., 1982), rating scales for depression and neuropsychiatric symptoms, a neuropsychological test battery and neuroimaging.", "The diagnoses were made by a senior staff member (F.R.J. Verheij) using the core clinical criteria for MCI and AD (Albert et al., 2011; McKhann et al., 2011).", "Criteria for SCD diagnosis included the self-reported presence of subjective cognitive complaints but absence of impairment in any of the measured cognitive domains (normal scores on age-, gender-, and education-adjusted performance [within 1.5 SD of the mean] on the standardized cognitive tests).", "Controls were required to have a clinical dementia rating of 0, no cognitive complaints and no evidence of cognitive deficits on testing.", "For all healthy controls, general demographic data, educational level, medical history, and neuropsychological-testing data were recorded.", "Exclusion criteria for all participants were age <51 years, macrovascular abnormalities, large cerebral infarction, hemorrhage, a history of psychoactive medication use, abuse of alcohol or drugs, past or present psychiatric or neurological disorders (i.e., Parkinson's disease, epilepsy, stroke, multiple sclerosis, brain surgery, brain trauma, electroconvulsive therapy, heart disease or brain infections), presence of depressive symptoms as indicated by the Hamilton Depression Rating Scale (Hamilton, 1960) (HDRS17; score ≥17), or contraindications for scanning.", "An experienced neuroradiologist reviewed the magnetic resonance images to ensure that none of the participants had radiological findings consistent with pathology in our exclusion criteria.", "This study was carried out in accordance with the rules and regulations of institutional research and ethics committees of the Maastricht University Medical Center, and written informed consent was obtained from all participants before participation.", "All participants were scanned on a single MRI system (Philips Achieva 3.0 T) using an 8-channel head coil.", "T1-weighted images were acquired with repetition time (TR) = 8.2 ms, echo time (TE) = 3.7 ms, flip angle = 8°, number of slices = 180 (sagittal), matrix size = 240 × 240, and voxel size = 1.0 mm isotropic.", "The T2-weighted images were collected with TR = 2500 ms, TE = 100 ms, flip angle = 90°, number of slices = 48 (axial, no slice gap), matrix size = 512 × 512, and voxel size = 0.5 × 0.5 × 3.0 mm.", "The FLAIR images were acquired with TR = 11000 ms, TE = 125 ms, inversion time (TI) = 2800 ms, flip angle = 90°, number of slices = 48 (axial, no slice gap), matrix size = 512 × 512, and voxel size = 0.5 × 0.5 × 3.0 mm.", "The field of view across the various sequences was kept constant to improve registration and anatomical localization.", "The diffusion-weighted images were acquired with TR = 8250 ms, TE = 80 ms, number of slices = 70 (axial), 61 directions with b value = 1000 (s/mm2), one b = 0 scan, matrix size = 128 × 128, and voxel size = 2.0 × 2.0 × 2.0 mm.", "WMSA were identified using a semiautomated method (in-house developed software package GIANT) (Jacobs et al., 2014).", "First, the FLAIR and T2 scans were corrected for intensity nonuniformities (Sled et al., 1998), followed by a coregistration of the T2 scans with the corresponding FLAIR scans using FMRIB's linear image registration tool from the FMRIB's Software Library (FSL) version 5.0.4 (www.fmrib.ox.ac.uk/fsl).", "The axial FLAIR- and T2-weighted images were displayed side by side allowing for visual inspection and easy identification of WMSA, while they were traced manually.", "These manual WMSA traces served as input to train the segmentation algorithm.", "The actual segmentation was then performed semiautomatically by identifying each WMSA region in each slice of the simultaneously displayed FLAIR- and T2-weighted images to provide a seed point to initiate a region-growing algorithm.", "Manual corrections were performed slice-by-slice where necessary by a single-trained rater W.M. Freeze), who performed excellent (intra class correlation >0.98 [Shrout and Fleiss, 1979]) when compared to an experienced neuroradiologist on 2 independent data sets.", "Intrarater variabilities were computed for a subset (n = 10) (intra class correlation >0.99).", "Cortical reconstruction and volumetric segmentation was performed with the FreeSurfer image analysis suite (version 5.1), which is documented and freely available for download online (http://surfer.nmr.mgh.harvard.edu/).", "The technical details of these procedures are described in prior publications (Dale and Sereno, 1993; Dale et al., 1999; Fischl and Dale, 2000; Fischl et al., 1999a,b, 2004a, 2004b; Jovicich et al., 2009; Segonne et al., 2004).", "The T1 hypointensities were labeled using a probabilistic procedure subsequently extended to label WMSA (Fischl et al., 2002).", "All images were visually checked after processing and if necessary, we edited voxels where the algorithm overestimated or underestimated the gray/white matter boundaries or where the brain areas were erroneously excluded during skull stripping.", "The DTI data were processed with the freely available MR diffusion toolbox Explore-DTI version 4.8.3 (www.exploredti.com) (Leemans and Jones, 2009).", "In brief, the diffusion-weighted data were corrected for motion- and eddy current-induced distortions, incorporating the B-matrix rotation to preserve the diffusion gradient orientation information correctly (Leemans and Jones, 2009).", "This was done in 1 step together with the correction for echo-planar imaging and susceptibility distortions, using the T1 data as the undistorted template to unwarp the diffusion data using rigid transformations (Irfanoglu et al., 2012).", "The diffusion tensor was estimated from the aforementioned data (in isotropic T1 space) using “RESTORE”, a robust nonlinear least-squares method (Chang et al., 2005).", "From this diffusion tensor, the DTI metrics FA, mean diffusivity (MD), radial diffusivity (RD), and axial diffusivity (AxD) were derived.", "Diffusion images were thresholded at individual level of FA > 0.20 to minimize a bias due to partial volume effects of voxels located on the border of white matter and cerebral spinal fluid or on the border of white and gray matter (Jones et al., 2013).", "To extract DTI metrics from the WMSA data, the T1 data were coregistered linearly to each individual's FLAIR-weighted scan data by using FMRIB's linear image registration tool from FSL version 5.0.4.", "Using the derived warp field, the DTI data were subsequently warped to the FLAIR data.", "All warping was done using affine transformations.", "The same procedure was applied to the FreeSurfer data to warp the T1-weighted hypointensities to the FLAIR data.", "All registrations were checked visually to ensure correctness.", "DTI metrics were then extracted from the WMSA determined on the FLAIR images, T1-weighted images, and the overlapping voxels of the FLAIR- and T1-based WMSA, FLAIR not T1, and T1 not FLAIR (see Fig. 1).", "In addition, a border zone surrounding the union of T1 and FLAIR WMSA (T1∪ FLAIR) was determined by dilating this region of WMSA by 1 voxel.", "Voxels located in the ventricles were excluded from this border zone.", "The remaining white matter (i.e., white matter tissue map of FreeSurfer excluding the union of T1 and FLAIR WMSA and its dilated border) was labeled as NAWM, and DTI metrics were also extracted from this region.", "This resulted in diffusion metrics extracted from 7 regions: the FLAIR-defined WMSA, FLAIR not T1 (FLnotT1), T1 not FLAIR (T1notFL), T1-defined WMSA, the overlap between T1- and FLAIR-defined WMSA, the border of WMSA, and the NAWM.", "In this context, the WMSA as defined in T1 or FLAIR or a subset thereof is referred to as a ROI.", "WMSA volumes were adjusted for Intracranial volume (ICV) by means of the scaling factor generated by FSL-SIENAX, which correlates well with manual estimations (Keihaninejad et al., 2010).", "The Mini Mental State Examination (Folstein et al., 1975) was used as a general cognitive screening.", "Neuropsychological testing for specific cognitive domains included the verbal 15-word learning test for measuring episodic memory (Dutch version of Rey auditory verbal memory learning test delayed word free recall [number of correctly reproduced words 20 minutes after the last learning trial] and cued retrieval presenting 15 new and 15 old words).", "The verbal semantic fluency test measuring language (Van der Elst et al., 2005) (number of correctly named animals within 1 minute) and the Stroop color-word test as a measure of executive functioning (time to read Stroop color-word test card 3) (Klein et al., 1997; Van der Elst et al., 2006b,c).", "The letter digit substitution test was used for assessment of information-processing speed (van der Elst et al., 2006a).", "Medial temporal lobe atrophy (MTA) score was assessed blinded using a qualitative visual rating scale (Scheltens et al., 1992).", "Coronal T1-weighted images were rated on using a 5-point scale ranging from 0 (no atrophy) to 4 (severe atrophy) based on the height of the hippocampal formation and surrounding cerebrospinal spaces.", "Participants given a MTA score of 2 or higher (both hemispheres summed) were considered as positive for AD risk (Clerx et al., 2013; Korf et al., 2004).", "In the control group, 6 of 18 (33%) scored 2 or higher (5 persons had an MTA score of 2 and 1 person had a score of 3).", "All (100%) MCI and AD patients had an MTA sum score of 2 or higher (n = 16 with 2 and n = 20 with 3, n = 13 with 4, n = 10 with >5).", "At the time of the inclusion, we followed the clinical core criteria of Albert et al. (2011), and these criteria suggested that the patients included in this study (amyloid pathology unknown, presence of neurodegeneration) have an intermediate probability to have cognitive deficits due to AD pathology (Albert et al., 2011).", "Of the SCD patients, 14 of 26 (54%) had MTA sum score of 2 or higher (n = 9 score 2, n = 1 score 3, n = 3 score 4, n = 1 score 5).", "Applying age-dependent MTA cutoffs as recently suggested in the literature (Ferreira et al., 2015; Rhodius-Meester et al., 2017) would still classify 95% of our cognitively impaired patients as having a higher likelihood of having AD pathology (2 of 18 CN, 13 of 26 SCD, 42 of 43 MCI, and 14 of 16 AD patients would be above the cutoff).", "We would like to note that the research criteria for preclinical and prodromal AD have recently changed and indicate that MTA scale-derived topographical biomarker evidence are associated with increased likelihood to progress to AD in the context of elevated levels of amyloid pathology (Dubois et al., 2016).", "Unfortunately, we did not have amyloid pathology data available.", "Statistical analyses were conducted in R version 3.2.4 (Foundation for Statistical Computing, Vienna, Austria.", "ISBN 3-900051-07-0, http://www.R-project.org/) and Stata 13 (Stata Corp, College Station TX, USA).", "One-way analyses of variance with post hoc t-tests were performed to assess group differences for continuous variables.", "Fisher's exact tests were used to test for differences in proportions between groups for categorical variables.", "All pairwise t-tests were corrected for multiple comparisons using the Bonferroni method.", "To investigate whether the diffusivity values differed depending on the different ROIs, we conducted linear mixed effects analyses with a random intercept for each participant using the restricted estimated maximum likelihood method for parameter estimation within the LME4 package (Bates, Maechler & Bolker, 2015).", "This approach allowed us to control for interdependency from the multiple measurements (ROIs) per participant.", "Age, sex, education level, diagnosis, and ICV-adjusted WMSA volume were used as covariates for the total group analyses.", "These models were repeated adding the scores on the HDRS (total) as a covariate.", "As the results were similar, we opted to provide the results without HDRS in the model, as this allowed to include all participants (n = 17 missing values on the HDRS).", "To investigate whether the DTI differences between ROIs were dependent on the diagnostic status of participants, we added the interaction term “ROI by diagnosis” to the linear mixed effect model.", "Probability values were obtained by a parametric bootstrap test and a Kenward-Roger modification of F-tests for linear mixed effects models (Halekoh and Højsgaard, 2014; Kenward and Roger, 1997).", "Bonferroni correction was used to account for multiple comparisons (20 comparisons).", "To investigate the ability of the diffusion values underlying the ROIs to predict diagnostic group belongingness, we applied multinomial logistic regression using the maximum likelihood method for parameter estimation, with diagnosis as outcome measure, and DTI metric in the ROI as predictor of interest.", "Age, sex, education level, and ICV-adjusted WMSA volume were added to the model as covariates.", "For each ROI, we ran a different model.", "Model fit, using the Nagelkerke R2 and Akaike information criterion (AIC), was evaluated for each model.", "As the models are non-nested, likelihood ratio testing was not possible; hence, we used the weighted delta Akaike information criterion with finite sample size correction (ΔAICc), which accounts for finite sample size, to compare the performance of the various models in combination with the evidence ratio (Burnham et al., 2011).", "Demographic and cognitive group characteristics are shown in Table 1.", "There were significant differences between the 4 groups for age (F(3,99) = 12.79, p < 0.0001), Mini Mental State Examination (F(3,96) = 18.41, p < 0.0001) and HDRS (F(3,82) = 8.36, p < 0.0001).", "As expected, there were also significant group differences in cognitive performance on the verbal-learning test total and delayed score (F(3,99) = 31.04, p < 0.001; F(3,99) = 32.54, p < 0.001, respectively), letter digit substitution test (F(3,92) = 11.20, p < 0.001, [CN>AD]), fluency F(3,95) = 11.31 p < 0.001, and Stroop card 3 time (F (3,95) = 6.7, p < 0.01).", "There were no significant group differences in education.", "WMSA volumes differed between diagnostic groups for FLAIR (F(3,99) = 4.08, p = 0.008, post hoc pairwise t-test: AD>CN, p = 0.006), T1 (F(3,99) = 3.38, p = 0.02, post hoc pairwise t-test: AD>CN, p = 0.01), Overlap (F(3,99) = 3.73, p = 0.01, post hoc pairwise t-test: AD>CN, p = 0.009), and Border (F(3,99) = 4.24, p = 0.007, post hoc pairwise t-test: AD>CN, p = 0.02, AD>SCD, p = 0.01).", "Cognitively impaired participants (MCI and AD) had significantly higher scores on the MTA visual rating scale compared to cognitively nonimpaired participants (CN and SCD).", "On average, across all groups, 14.8 % of the T1 WMSA voxels were not localized within the FLAIR WMSA voxels.", "It should be noted that there was a large variability in the proportion of T1 WMSA voxels outside the FLAIR WMSA area (range: 3%–41%).", "The volume of the FLAIR-defined WMSA was approximately 2.17 higher than the T1-defined WMSA volume, and the log-transformed T1 and FLAIR WMSA were highly correlated (Pearson's r = 0.898, p < 0.001).", "The adjusted threshold for significance after correction for multiple comparisons was p < 0.0025.", "The linear mixed effect analyses across the entire sample showed higher mean FA values in the NAWM compared to the T1, T1notFL, Overlap, FlnotT1, and FLAIR-defined WMSA (p < 0.0005).", "No significant differences were found in FA values between the Border zone and NAWM (p = 0.61) and between FLAIR and FLnotT1 (p = 0.023).", "FLAIR-based WMSA FA values were significantly higher than T1-based WMSA FA values (p < 0.0005).", "The FA values in the Overlap region were lower than the FA values in the FLAIR-defined WMSA and the Border zone (p < 0.0005) but not the T1-defined WMSA (uncorrected p = 0.014).", "For the MD, AxD, and RD values, the NAWM values were lower than the T1-defined WMSA, Overlap, FLAIR, and the Border zone values (p < 0.0005), and the Overlap lies between diffusion values in the FLAIR- and T1-defined WMSA and different from both (p < 0.0001).", "Mean MD in FLAIR-defined WMSA was lower compared to T1-defined WMSA, Overlap, and higher than the Border (p < 0.0005) (see Fig. 2 and Table 2 for the FA and MD results; the results for AxD and RD are provided in the Supplemental Tables 1 and 2, average diffusion values per group are listed in Supplementary Tables 4a and 4b).", "Adding the interaction term “ROI by diagnosis” to the model showed that the FA differences between the NAWM and T1-defined WMSA (T1 and T1notFL), FLAIR-defined WMSA (FLAIR and FLnotT1), or Overlap were larger for CN than for the SCD, MCI, or AD group (p < 0.0005).", "The difference between the FA of the Border zone and the NAWM and FLAIR compared to FLnotT1 was not different across the 4 groups.", "Interaction effects with the NAWM as reference region are listed in Table 2, while the interactions with other ROIs as reference region are provided in the Supplemental Data Tables 1 and 2.", "These interactions revealed effects in the same direction.", "No group differences were observed for the comparison between FA values in T1-defined WMSA, T1notFL, and the Overlap region.", "For the MD metric, we observed only a significant interaction effect when comparing the MD values of the NAWM with those of the T1-based WMSA, indicating that the difference in MD values between these 2 regions was larger for CNs than for MCI and AD patients (p < 0.0005 after multiple comparison correction for results see Table 2).", "The interaction effects for the axial and RD are provided in the Supplemental Data (Table 2 supplemental).", "As the interaction “ROI by diagnosis” in the previous analyses showed mainly significant associations for the FA values, we performed the multinomial logistic regression models on the FA values to investigate the potential of using FA values to predict diagnosis.", "These models showed that the FA values in every ROI were able to predict the difference between CN and SCD.", "A 0.1 unit increase in the FA of the NAWM was associated with 7.27 log odds decrease in being SCD compared to CN.", "Interestingly, the FA log odds values of SCD are in similar ranges as the AD patients in the Overlap, NAWM, and Border zone (see Supplemental Table 3).", "The ability to correctly differentiate CN from the other patients groups was only significant for FA values in the NAWM and the Border zone (see Supplemental Table 3), showing that as the FA value increases with 0.1 unit in the NAWM or the Border zone, the log odds to be MCI compared to control decreased with 3.28 or 2.29, respectively.", "For the AD group, these log odds are even higher (a decrease of 7.58 vs. 4.55, respectively).", "These results do not change when using NAWM volume (controlled for ICV) instead of WMSA volume as a covariate (data not shown).", "Fig. 3 shows the expected probability of belonging to a specific diagnostic group at certain FA values in each ROI.", "The plots showed that for NAWM and the Border zone, higher FA values were more strongly associated with the CN group, whereas lower FA values were associated with the AD group.", "The MCI group showed higher probabilities in the midrange FA values.", "As the NAWM and the Border zone were able to differentiate all diagnostic groups, we compared the Nagelkerke R2, the ΔAICc, and the evidence ratio to determine the best model.", "A lower AIC indicates a better model, whereas the ΔAICc compares the relative merit of each model (Supplemental Table 3).", "The ΔAICc of NAWM and Border zone 10.60, between the Border zone and the FLAIR 15.45, between the FLAIR and the T1 11.41, and between the T1 and Overlap 8.97, and T1 not FL versus T1 8.9 indicating a higher likelihood of information loss when comparing to the next best model, whereas between FLnotT1 and FLAIR, the ΔAICc of 1.6 indicates that the models are relatively equal in merit.", "The evidence ratio of weighted AIC's indicated 4.6 × 105 times stronger evidence for the NAWM model relative to the FLAIR model and 201.1 times stronger relative to the Border zone.", "In this study, we aimed to investigate differences in microstructural properties of T1- and FLAIR-defined WMSA in a total of 103 participants within the range of cognitively healthy to AD dementia.", "Previous work suggested that the pathology underlying WMSA may differ in type or severity, depending on the type of MRI contrast that is used to define WMSA (Bitsch et al., 2001; Leritz et al., 2014; Sailer et al., 2001).", "Our results add important information to the existing literature by showing that first, the diffusion properties underlying T1-defined WMSA are more abnormal than those defined on the FLAIR images, additionally T1notFL voxels are different from general T1 voxels, while FLnotT1 are not different from general FLAIR, suggesting that these 2 MRI contrasts provide different information regarding the underlying pathology.", "Second, even though most ROIs showed differences in all DTI metrics across the entire group, an interaction effect with diagnosis was only observed in the FA values.", "This may suggest that the FA metric may be more sensitive to subtle differences in white matter health compared to the MD.", "Third, diffusion values from the NAWM can predict group adherence of individuals of varying cognitive impairment with higher probabilities than the other white matter ROIs.", "Even though T1-weighted WMSA were characterized by the most abnormal diffusion values, they also provide the lowest classification accuracy.", "These results illustrate a need for a better understanding of what the underlying pathology for WMSA may constitute and how it can be detected and defined in various MRI sequences.", "WMSA can be defined on both T1- and FLAIR-weighted images, although aging and Alzheimer's research has mainly focused on FLAIR-weighted WMSA.", "The considerable heterogeneity in pathology within WMSA and between WMSA and the penumbra of the WMSA areas, suggests that not all WMSA reflect the same degree or type of white matter damage (Gouw et al., 2008, 2011; Maillard et al., 2011, 2014; Viswanathan, 2014).", "Our study shows that T1 and FLAIR signal abnormalities are highly colocalized phenomena as most, but not all, T1-defined WMSA are localized within the FLAIR-defined WMSA.", "While volumes of T1-defined WMSA correlated highly with the FLAIR-defined WMSA, T1 WMSA was characterized by more aberrant diffusion values compared to the FLAIR-defined WMSA.", "We did not observe a different pattern with regard to the various diffusion metrics across the ROIs for all groups, but rather observed differences in the magnitude of the diffusion values, which may indicate that T1-defined WMSA, reflect more severe rather than different pathology compared to FLAIR-defined WMSA.", "Postmortem neuropathology combined with high-field MRI determined that myelin is a dominant source of MRI contrast in T1 maps (Stuber et al., 2014).", "In addition in MS, T1 lesions are thought to be a combination of permanent demyelination and loss of tissue matrix (Klistorner et al., 2016).", "The reported high correlation of T1- and T2-weighted WMSA volumes in the latter study (r = 0.94) is very similar to those computed from a subsample of the Alzheimer's Disease Neuroimaging Initiative population (r = 0.96) (Coutu et al., 2016) and our findings (r = 0.89) and suggest that even though the volumes are similar, the tissue integrity is more severely affected in T1-defined WMSA compared to the FLAIR ones.", "It is important to note that the pathology and symptomatology underlying MS is different than that in AD, though neuroinflammatory responses can be a component of this signal, as neuroinflammation also plays an important role in disease progression of AD (Calsolaro and Edison, 2016).", "As for FLAIR, a recent series of 134 premortem MRI scans and subsequent autopsy observed that FLAIR sequence-identified WMSA overestimated demyelination in the periventricular and perivascular regions but underestimated it in the deep WM during normal brain aging (Haller et al., 2013).", "This is possibly due to increased blood-brain barrier permeability in the perivascular and periventricular areas leading to a higher high local water concentration.", "Maillard et al. (2014) reported normalizing FLAIR signal over time in stagnant WMSA, possibly due to a resolving inflammation.", "FLAIR WMSA could therefore be a mix of white matter damage, peri-inflammatory processes as well as proxies of increased vascular and blood-brain barrier permeability (Young et al., 2008), whereas T1 WMSA would involve areas with more severe demyelinating damage.", "It may therefore be speculated that T1 WMSA, especially outside the FLAIR boundaries, reflect more consolidated WMSA.", "We found that the NAWM is negatively affected in patients; this is consistent with literature (Maillard et al., 2014; Maniega et al., 2015; Topakian et al., 2010).", "A new intriguing finding is that the difference in FA values between the NAWM and the other ROIs seems to be related to the diagnosis, while there was no such clear association for MD.", "We did not find overall or group differences between the NAWM and the Border zone.", "While the Border zone has the highest probability of capturing voxels of the penumbra as they are closest to the WMSA, the average signal could be influenced by including voxels of the NAWM.", "The interpretation of the underlying biology is speculative, and care should be taken not to interpret the findings beyond what can reasonably be deducted from the tensor model.", "Any small nonproportional change in any of the eigenvectors could hypothetically result in a large change in FA especially when the magnitude of the vectors is small.", "Very early stages of deteriorating cellular health and its resulting minute microstructural changes could possibly introduce a reduction in anisotropy that would be more apparent in the FA but much less so in MD, RD, or AxD.", "This may explain why the FA values in Overlap and T1 not FLAIR and T1-defined WMSA are in the same range for all diagnostic groups, including the healthy controls, while showing a clear difference in the distribution of the MD values between healthy controls and patients excluding theT1 not FLAIR group.", "Possible underlying biological processes can be neurofibrillary loss, glial damage, increased fluid in the extracellular and perivascular space, but more work is needed to better understand the relation between biology and diffusion.", "Even though the diffusion values for T1-defined WMSA were the most aberrant, the diffusion properties of the NAWM show promise as a predictor for diagnostic adherence.", "The fact that the mean FA in NAWM was a better predictor of group adherence than the mean FA in the Border zone, suggests that more variance can be found in the NAWM that is not adjacent to any WMSA.", "These results are not conclusive as the groups are small, and there are confounders that the current data available do not allow us to adequately control for.", "Despite these limitations, the results of the multinomial logistic regression are consistent with previous studies showing that subtle white matter alterations can occur in the NAWM in clinically healthy older individuals (Firbank et al., 2003; Maillard et al., 2014; Maniega et al., 2015; Vernooij et al., 2009).", "Our results now show similar findings in a memory-clinic population.", "Replication in larger populations will be needed to reach more definitive conclusions.", "Notably, we included SCD individuals in our sample to have a broad range of individuals and spanning the entire range from healthy aging to AD dementia.", "Interestingly, the diffusion values of the SCD group in our sample behaved quite similarly as the AD group.", "Studies including individuals with SCD reported higher volumes of temporal WMSA, lower hippocampal volume, and more amyloid-β compared to controls (Amariglio et al., 2015; Schultz et al., 2015; Stewart et al., 2008; van der Flier et al., 2004).", "Inconsistent findings have been reported regarding alterations in the microstructural properties of white matter in SCD individuals.", "So far, the few studies performed within SCD, observed either local lower FA and higher MD values in medial temporal lobe regions in SCD individuals compared to controls (Ryu et al., 2017; Wang et al., 2012) or no differences between both groups (Kiuchi et al., 2014; Wang et al., 2012).", "One study reported that DTI was a better predictor for the progression of SCD to AD than cerebral spinal fluid markers (Selnes et al., 2013).", "These inconsistencies may be related to the criteria used to define SCD (Jessen et al., 2014) or the recruitment strategy (Perrotin et al., 2017).", "We adopted a broad definition of SCD, allowing everybody entering the outpatient memory clinic presenting with complaints but no objective memory deficits (Cognitive tests within 1.5 SD of mean corrected for age, education, and sex) and no clear medical reason underlying these deficits into the study.", "Therefore, this group of subjective memory complaints represents a heterogeneous group in terms of the underlying etiology of their complaints.", "We did not have information on amyloid burden or genetic risk factors in these individuals, but 53 % of the MTA scores of the SCD group were 2 or higher, indicative for ongoing neurodegenerative processes, potentially AD related.", "Furthermore, correcting for the level of depressive symptoms, which was higher in SCD individuals, did not change our results, suggesting that mood was most likely not driving these relationships.", "There are some limitations to this study.", "As this study population was acquired in a clinical setting, the spatial resolution of the FLAIR sequence was based on commonly used clinical parameters.", "By warping all images to FLAIR resolution, we may have lost some information.", "Replication of these results using high-resolution isotropic FLAIR sequence could provide more detailed information.", "Furthermore, the used DTI sequence does not take into account the effects of crossing and kissing fibers, more complex models using multi shell acquisition schemes could be considered for future studies.", "The results were derived using 2 separate software packages (GIANT and FreeSurfer) to extract FLAIR and T1 WMSA.", "Although GIANT is designed to closely interact with FreeSurfer, this could possibly induce a bias.", "As we did not collect amyloid or tau data on these individuals, we cannot totally rule out that some of our cognitively normal participants are in a preclinical AD stage and cannot fully guarantee that the cognitive impairment in the MCI and AD groups was due to Alzheimer pathology.", "We also did not incorporate other measures of vascular health, such as blood pressure, cholesterol, or diabetes, as these were not available for all participants.", "Cardiovascular risk factors have been shown to be linked independently to WMSA and DTI metrics (Jacobs et al., 2013), and therefore may relate differently to T1- or FLAIR-defined WMSA.", "Even though, our results survived stringent multiple comparisons corrections, the sample size is moderate and replication in larger cohorts is needed.", "The underlying pathology of WMSA is heterogeneous, and our study showed that different MRI contrasts used to identify WMSA can provide more information about the underlying WM integrity.", "T1- and FLAIR-defined WMSA are microstructurally different, potentially reflecting differences in the type or severity of white matter pathology in individuals with cognitive decline and in healthy older adults.", "Diffusion values of the NAWM have potential for use as an early and sensitive indicator for brain health decline in AD.", "Further investigation is warranted, especially in SCD individuals and in larger longitudinal samples." ]
[]
Perceptions and expectations of autonomous vehicles – A snapshot of vulnerable road user opinion
Public perceptions play a crucial role in wider adoption of autonomous vehicles (AVs). This paper aims to make two contributions to the understanding of public attitudes toward AVs. First, we explore opinions regarding the perceived benefits and challenges of AVs among vulnerable road users – in particular, pedestrians and bicyclists. Second, the paper evaluated whether interaction experiences with AVs influence perceptions among vulnerable road users. To explore this, we examined survey data collected by Bike PGH, a Pittsburgh based organization involved in programs to promote safe mobility options for road users. Analysis of the data revealed that respondents with direct experience interacting with AVs reported significantly higher expectations of the safety benefits of the transition to AVs than respondents with no AV interaction experience. This finding did not differ across pedestrian and bicyclist respondents. The results of this study indicate that as the public increasingly interacts with AVs, their attitudes toward the technology are more likely to be positive. Thus, this study recommends that policy makers should provide the opportunities for the public to have interaction experience with AVs. The opportunities can be provided through legislation that allows auto manufacturers and technology industries to operate and test AVs on public roads. This interactive experience will positively affect people's perceptions and help in wider adoption of AV technology.
[ "BUSI", "PSYC" ]
[ "Autonomous vehicles", "Bicyclist", "Pedestrians", "Perception", "Safety", "Vulnerable road users" ]
[ "1403", "1405", "3202" ]
[ "Autonomous vehicles (AVs) are capable of sensing their environments and navigating different traffic conditions with little or no human input (Skeete, 2018).", "By reducing the input of a human operator, AVs have the potential to reduce traffic congestion while improving fuel efficiency, reducing air pollution, and mitigating climate change (Forrest and Konca, 2007.", "; Tientrakool et al., 2011; Atiyeh, 2012.", "; Shladover et al., 2012.", "; Plumer, 2017.", "; LaFrance, 2018).", "Research findings reveal that human factors are responsible for more than 90% of all crashes involving automobiles (Automated Driving Systems, 2017).", "Many industry experts believe that the transition to AVs will significantly improve road safety (Forrest and Konca, 2007; Plumer, 2017; Fagnant and Kockelman, 2018; Reimer, 2014; Hayes, 2011; Silberg et al., 2012).", "On March 18, 2018, however, an Uber struck and killed a pedestrian in Arizona.", "This crash garnered worldwide media attention as the vehicle was operating with a self-driving system at the time of the collision.", "Questions related to the safety performance and acceptance of these technologies (autonomous vehicle, self-driving) immediately surfaced – especially in relation to their ability to interact with vulnerable road users, a term generally used to refer to pedestrians, bicyclists, and motorcyclists (Barth, 2017; Fairley, 2018; Levin, 2018).", "We present a study of safety perceptions of AVs among vulnerable road users and report how they relate to exposure to the technology.", "Recent advances in AV technology by automotive companies (e.g., General Motors, Ford, Daimler, Renault-Nissan) and tech companies (e.g., Google, Uber, Waymo) have accelerated the development, testing, and deployment of AVs within a much shorter time than previously anticipated (Muoio, 2018).", "Researchers, private organizations, and industry experts have made efforts in predicting the adoption rates of AVs (Bansal and Kockelman, 2017; Hars, 2018; Laslau et al., 2014; Lavasani et al., 2016; Levinson and Krizek, 2015; Litman, 2018; Mosquet et al., 2015; Rowe, 2018; Silberg et al., 2012).", "Several factors such as regulations, willingness to pay, and technology prices play a crucial role in wide adoption of AVs (Hohenberger et al., 2016; Payre et al., 2014).", "Many researchers have also explored the role of public perceptions and acceptance in greater adoption of AVs (Fagnant and Kockelman, 2018; Fairley, 2018; Litman, 2018; Hohenberger et al., 2017; Bansal and Kockelman, 2017; Lavasani et al., 2016; Gold et al., 2015; Heide and Henning, 2006).", "This logically follows from the fact that the public will ultimately play a crucial role in purchasing vehicles with AV-related technology and supporting (or opposing) policies and electing politicians that will make it easier for AVs to share the roadways with other users.", "Understanding public perceptions of AVs is thus a crucial component of knowing the rate at which the technology will become implemented (Hengstler et al., 2016) For instance, Casley et al. (2013) designed a survey to assess public feelings, beliefs, expectations, and predictions toward AVs.", "Secondary traits of AVs such as their expected productivity, efficiency, environmental impact were appealing to the public.", "A survey conducted by Schoettle and Sivak (2014) found that respondents in the United States, United Kingdom, and Australia (a) have positive initial opinion of this technology, (b) have high expectations on benefits derived from AVs, (c) express concern about riding in AVs, and security issues related to AVs, and (d) desire to have self-driving technology in their vehicle without paying extra.", "Similar results regarding the acceptance, concerns, and willingness to pay for AVs were reported in a study of over 5000 respondents from 109 countries conducted by Kyriakidis et al. (2015) (Kyriakidis et al., 2015).", "A series of focus-group meetings conducted with people from Los Angeles, Chicago, and Iselin by KPMG (2013) found public acceptance of AVs to vary by geographic location and gender.", "Females are more interested in AV technologies than males, with females emphasizing perceived benefits of self-driving vehicles, and males expressing concerns about being restricted by speed limits.", "This contradicts the results form Hulse et al. (2018) - males displayed greater acceptance to AV technology.", "Interestingly, this study found that the oldest respondents (60+ year-old) and the youngest (21–34 year-olds) expressed the highest willingness to pay for self-driving technologies.", "According to a research by Howard and Dai (2014), survey respondents viewed safety and convenience as the most attractive features of AVs whereas, liability and cost were the least attractive elements (Howard and Dai, 2014).", "In a study of over 3500 London residents, Begg (2014) found 60% of respondents agreed or strongly agreed that AVs would improve safety for road users.", "Payre et al. (2014) observed that older people were more likely to accept AVs but less likely to pay for this technology.", "Underwood (2014) conducted a survey at Automated Vehicle Symposium 2014 and gathered opinions of 217 AV experts.", "The AV experts believed that social and consumer acceptance were the least difficult barriers while liability and regulations were the most difficult barriers to the adoption of AVs.", "Finally, Deb et al. (2017) found in a study that 67% of respondents expected AVs to improve the overall transportation system, but over 40% stated they will not feel comfortable if their child, spouse, parents, or their loved ones cross roads in the presence of AVs.", "Males, younger respondents, and respondents from urban areas were more receptive toward AVs.", "A review of existing literature reveals some interesting limitations in the current understanding of public perception and acceptability of AVs:", "Presently, encounters with AVs on the roadways represent largely a hypothetical future for most people.", "Therefore, their indications of acceptability and/or expectations may not be solid ground for future adoption of AVs, given that an individual's beliefs about how they will feel about future events can be highly inaccurate (Wilson and Gilbert, 2005).", "Indeed, Sims et al. (2018) have shown that having a prior experience with a travel mode can lead to a more accurate prediction about it (Sims et al., 2018).", "Only few respondents to past surveys regarding attitudes and beliefs related to AVs involve people who have directly interacted with AVs.", "Given this lack of experience or interactions with AVs, most respondents only have a vague idea about how they will actually value these technologies when they arrive, which can lead to both seriously overestimating or underestimating their value (Vilimek and Keinath, 2014).", "Further, the past surveys have largely ignored the assessment of attitudes and beliefs of vulnerable road users (non-motorists).", "To overcome the limitations of the existing literature, this paper aims to: 1) explore opinions regarding the perceived benefits and challenges of AVs among vulnerable road users (i.e., pedestrians and bicyclists); and 2) evaluate whether interaction with AVs changes perceptions among vulnerable road users.", "Finally, we provide insights to aid policy makers for better public acceptance and wider adoption of AV technology.", "The study analyzes data obtained from a third-party organization, Bike PGH.", "Bike PGH conducted a survey and gathered responses from Pittsburgh residents on their experiences, perceptions, and level of support to AVs deployed in Pittsburgh, Pennsylvania.", "In 2017, United States Department of Transportation (USDOT) designated ten AV proving grounds to encourage testing of new technologies, which included Pittsburgh, PA.", "Since September 2016, Uber's self-driving cars have been tested on the streets of Pittsburgh by providing rides for the public.", "By March 2017, there were 20 Uber AVs in Pittsburgh serving over 800 trips per week (Bhuiyan, 2018).", "Bike PGH, an organization promoting safety and accessibility for bikers and pedestrians, conducted a survey to better understand the attitudes and beliefs of Pittsburgh, Pennsylvania residents regarding sharing roads with AVs.", "Bike PGH conducted the survey in two stages: a) first, exclusively to the donor members of the Bike PGH group, and b) second, a general public survey.", "The general public survey aimed at gathering responses from the Pittsburgh residents.", "Bike PGH promoted the survey through their website, social media, and news articles.", "The data was then provided to Western Pennsylvania Regional Data Center and made public.", "Bike PGH gathered 321 responses from their members and 798 responses from general public.", "Initial results of the data were presented by Bike PGH (AV Survey Results, 2018).", "However, most of their analysis concentrated on comparisons of responses between Bike PGH members and general public.", "Their results clearly showed that Bike PGH members have more positive attitudes and beliefs regarding AVs than the general public.", "Because our interests were to examine a more representative sample, in the present study analyses were limited to the 798 responses from survey of the general public (only 384 were needed for 95% a confidence level).", "Descriptive statistics of the original survey data are summarized in Table 1.", "Among the survey respondents, 46% stated that they had interacted with an AV while using sidewalks and crosswalks, and 43% have not had any interactions with an AV as a pedestrian.", "Among the respondents, 11% were not sure if they had interactions with AVs as pedestrians.", "Thirty-five percent of the respondents reported having interacted with AVs while riding a bicycle.", "A reasonable number of respondents had interactions/experiences with AVs, which is necessary for the kind of comparisons made in this study.", "Approximately half of the respondents (49%) fully approved of Pittsburgh serving as a proving ground for AVs.", "Only 10% of the respondents fully disapproved, and 13% reported neutral attitudes.", "Combing both approve and somewhat approve, close to 70% of the people approved of their city serving as a proving ground for this technology.", "Respondents expressed their observations or circumstances during their interaction with AV.", "Since the data was freely provided on the public domain, the data providers converted the descriptive variable into 5 level categorical variable.", "Among the respondents who had interaction experience with AVs, 70% stated that they didn't find any difference between a human driver or experienced no negative interaction with an AV.", "Around 12% of the respondents who had interactions, experienced AVs more cautious or had difficulty anticipating AV movement.", "Only 6% of the respondents perceived AVs as being safer than humans.", "Among the respondents, 62% reported that AVs have the potential to reduce both fatalities and injuries.", "Only 8% of the respondents clearly stated no and another 30% reported ‘Maybe’ or ‘Not Sure’ (Table 1).", "Of the survey respondents, 70% felt that authorities should lay regulations regarding how AVs are tested.", "Regarding beliefs about the safety of sharing roads with human drivers as pedestrians or bicyclists, the respondents' responses were diverse; 4% of the respondents rated Pittsburgh streets as very safe, 39% stated moderately safe, and only 6% felt their city roads very unsafe.", "Among the respondents, 21% rated Pittsburgh streets very safe, and 36% stated safe.", "Interestingly, respondents rated sharing roads with humans (mean = 2.95 on the scale given in Table 1) as less safe than sharing the roads with AVs (mean = 3.53).", "Table 2 displays public's opinions on how safe they feel using Pittsburgh's streets with human drivers and AVs.", "Respondents had more positive beliefs regarding the safety of sharing the road with AVs if they had past interactions with AVs (M = 3.70) than if they had not (M = 3.35).", "This difference was statistically significant (p = 0.0003).", "In contrast, respondents shared similar attitudes about the safety of sharing roads with human drivers regardless of whether they had experience interacting with AVs (M = 2.92) or had not (M = 3.00).", "This difference was not statistically significant (p = 0.23).", "This discrepancy is important as it indicates that exposure to AVs was associated specifically with differences in the perceived safety of AVs but did not systematically affect the perceived safety of human drivers.", "Respondents with interactive experience have significantly high expectations than respondents without interactions with AVs.", "In general, public expectations were high on safety benefits from AVs.", "This observation is similar to previous findings of Schoettle and Sivak (2014), Casley et al. (2013), and Deb et al. (2017).", "Table 3 displays vulnerable road users' opinion on Pittsburgh as the proving ground for AVs by their interactive experience with AV.", "Since the responses are ordinal (1-disapprove, 2-somewhat disapprove, 3-neutral, 4-somewhat approve, 5-approve).", "The mean reported approval of Pittsburgh as an AV proving ground was 4.10 for respondents who had interacted with AVs as pedestrians, whereas approval was only 3.67 for respondents with no interaction experience as pedestrians; a one-way ANOVA indicated this difference was statistically significant at a 95% confidence level (p = 0.0002).", "Similarly, respondents reported more positive attitudes toward AVs if they had interacted with AVs as bicyclists (M = 4.06) than if they had not (M = 3.78); this difference was again significantly different (p = 0.02).", "These results indicate that interacting with AVs as a vulnerable road user was associated with increasing acceptance of this technology.", "Table 4 provides the mean perceptions of respondents concerning on potential safety benefits with AVs.", "Among pedestrians who had interactive experience, 67% stated that AVs have great potential to reduce traffic fatalities and injuries.", "Only 6% of the same group felt AVs can't improve safety on roads.", "As tested by the chi-square statistic, the interactive experience of pedestrians had statistically significant effect on their perceptions about AVs safety potential to reduce injuries and fatalities at 95% confidence level (p-value is 0.037).", "Similarly, bicyclists who had interactive experience were significantly more likely to perceive that AVs have great potential to reduce traffic injuries and fatalities.", "Irrespective of the type of vulnerable road user, respondents described being more likely to envision the safety benefits of AVs if they had past interactions with AVs.", "The respondents of the survey also expressed their opinions on imposing regulations for AV testing and Table 5 presents those results.", "Respondents who have interacted with AVs as pedestrians, 65% of them stated that authorities should lay regulations for AV testing, whereas 76% of the pedestrians who haven't interacted said regulations are necessary.", "The difference was statistically significant at 95% confidence level.", "The same result was found among bicyclists.", "Approximately 10% of the pedestrians and bicyclists who had not interacted with AVs responded that regulations are not necessary, whereas this percentage increased to 16%, and 18% among the pedestrians and bicyclists who had interacted with AVs.", "Public perception plays a crucial role in the rate of new technology acceptance and adoption by personals choices to adopt and willingness to support government actions to support changes.", "Presently, the rate at which AVs become a part of the transportation system is determined in no small part by political decisions regarding how to regulate the technology, support testing, and assess liability for accidents, and these decisions in turn are considerably influenced by the attitudes of political constituents.", "This paper investigated opinions of a specific subset of stakeholders, vulnerable road users, on the benefits, experiences, and perceived challenges with AVs.", "This paper further evaluated whether interaction experiences with AVs could change perceptions among vulnerable road users and also provide policy recommendations for wider acceptance of this technology.", "The results of this study provide some of the first evidence that interactions with AVs of vulnerable road users increase perceptions of safety and approval of this technology.", "This suggests that as the public has increased opportunities to interact with AVs – even without considering further advances beyond current technologies – attitudes toward the technology would be expected to improve.", "Presently, there is considerable geographic variation in the extent to which people may have such experiences, due to wide variation in the regulations concerning the ability to operate AVs on public roads.", "According to the Insurance Institute for Highway Safety (IIHS) by January 2018, 23 states and District of Columbia have enacted some form of legislation or executive orders on autonomous vehicles: seven authorize testing; seven simply authorize a study or funding, or defined terms; nine states and DC authorize full deployment (Automation and Crash Avoidance, 2018).", "Across the US, there are only a handful of places where AVs are tested or operated on public roads.", "After the fatal pedestrian crash involving Uber, Arizona Governor Doug Ducey issued an Executive Order to suspend all the testing of Uber autonomous vehicles on public roadways in Arizona (Schwartz, 2018).", "This crash attracted attention from all over the country and raised more questions related to the safety performance and acceptance of the AV technology.", "According to Groves and Kalra (2017), even with the current concerns and obstacles surrounding AVs, the testing and deploying of AVs on the public road should be promoted before the AV technology is nearly perfect at the SAE level 5.", "Early deployment of AVs would save many lives instead of waiting until they become nearly perfect (Groves and Kalra, 2017; Skeete, 2018).", "In view of the findings of this study, it is recommended that, policy makers should provide opportunities for the public to have interaction experience with AVs.", "The opportunities can be provided through legislation that allows auto manufacturers and technology industries to operate and test AVs on public roads.", "This interactive experience will positively affect people's perceptions and help in wider adoption of AV technology.", "The data used for this study was obtained from a study conducted by Bike PGH, an organization that advocates for safer communities for bikers and pedestrians.", "Given the sample collecting strategy through social media, the sample might over-represent bicycle-users relative to the total Pittsburgh population.", "The results of the study still provide important evidence that experiences with AVs are associated with systematic differences in how AVs are perceived.", "This research was conducted using internal funds from the Alabama Transportation Institute at the University of Alabama." ]
[ "Public perceptions play a crucial role in wider adoption of autonomous vehicles (AVs).", "As the public increasingly interacts with AVs, their attitudes toward the technology are more likely to be positive.", "We recommend policy makers to pass legislation that will authorize testing and operation of AVs on public roads.", "Interaction experience with AVs will positively affect people's perceptions and help in wider adoption of the technology." ]
Modelling of industrial biopharmaceutical multicomponent chromatography
The development and validation of a chromatography rate model for an industrial multicomponent chromatographic bioseparation is presented. The model is intended for use in a process scenario to allow specific variables critical to product quality to be studied. The chromatography provides impurity clearance whilst producing a complex product composed of six closely related variants of a dimer protein therapeutic (~30. kDa), with their monomer subunits in a specific ratio. Impurity removal is well understood, however, achieving the correct monomer subunit ratio can pose a purification challenge. We utilise a stepwise approach to develop a model for studying the effect of feed material variability on product quality. Scale down experiments are completed to quickly generate data for estimating model parameters, before an iterative procedure is employed where the industrial process is used to refine parameters in a sequential manner, until model predictions exhibit satisfactory agreement with experimental data. Final model predictions were in good agreement with experimental product quality (within 3%). The results demonstrate how good understanding of an industrial process can help facilitate model development when an exhaustive description is not required, despite considering a chromatographic bioseparation with crude feed material and challenging purification objectives. © 2013 The Authors.
[ "CENG", "CHEM" ]
[ "Chromatography modelling", "Hydrophobic interaction chromatography", "Process systems modelling", "Purification process development", "Quality by design", "Therapeutic protein" ]
[ "1500", "1600" ]
[ "Advances in healthcare over the past half century have been of immense benefit to the quality of life for an increasing world population.", "The rapid growth in protein therapeutics has played a key role in this, and is predicted to continue with several hundred clinical candidate proteins currently estimated in company pipelines (Kelley, 2009) of which many serve significant unmet medical needs (Shukla et al., 2007).", "However, despite several decades of effort to improve R&D efficiency and performance, the process for bringing a new biopharmaceutical product to market remains an expensive, time-consuming, and risky proposition (Lightfoot and Moscariello, 2004).", "Chromatographic separations are the workhorse of therapeutic protein purification (Kelley, 2007), but their design and operation is a challenging task.", "An optimal, safe and economic process must be found quickly somewhere in an extremely large parameter space which simply cannot be explored in depth using traditional experimental methodologies.", "Downstream process development currently depends heavily upon empirical experimentation interpreted using heuristic knowledge to arrive at an acceptable process (Lightfoot and Moscariello, 2004).", "The amount of material available to work with is often limited, and the work must be completed within constricted timelines to meet time to market constraints.", "Furthermore, US Food and Drugs Administration (FDA) regulations require that the basic separation scheme is fixed prior to clinical trials, early on in the overall development process.", "The FDA is now encouraging the use of quality by design (QbD) principles during process development and operation (US Food and Drug Administration, 2006).", "Key to a QbD approach is a thorough understanding of process inputs and their impact on performance, the relationship between the process and the products’ critical quality attributes (CQA), and the association between the CQA's and a product's clinical properties (Jiang et al., 2010).", "The expected benefit from a QbD approach is an increase in the assurance of product quality, and in turn, the FDA will allow manufacturers greater flexibility to operate with lower regulatory burden, enabling continuous process improvement, as well as greater robustness.", "Mechanistic modelling can be a useful tool for studying the impact of process parameters on process performance and product CQA's.", "Altering the values of process parameters may be difficult or even impossible to accomplish experimentally, e.g. feed stream composition, but is trivial in a model based approach.", "In addition, simulations can be completed quickly and efficiently which is valuable in an industrial scenario where time and material is often limited, and the fundamental knowledge gained by their application can be used to better understand, and reduce, processing risks.", "The equations used to mathematically describe the chromatographic purification of proteins are well understood (e.g. Kaczmarski et al., 2001; Guiochon, 2002; Brooks and Cramer, 1992; Seidel-Morgenstern, 2004).", "The systematic development of a chromatographic model has been described for many different systems, including ion exchange (Melter et al., 2008), hydrophobic interaction (McCue et al., 2008; Nagrath et al., 2011), and protein A chromatography (Ng et al., 2012).", "The issue of efficient model calibration has been thoroughly addressed (Teoh et al., 2001; Persson et al., 2006; Susanto et al., 2006; Osberghaus et al., 2012a).", "Mechanistic models of chromatography have been successfully employed to simulate numerous case studies (Kaczmarski et al., 2002; Mollerup et al., 2007; Osberghaus et al., 2012b).", "In addition, there are useful examples of using chromatography models for optimisation (Degerman et al., 2006, 2007; Ng et al., 2012), scale up (Gerontas et al., 2010), design space characterisation (Degerman et al., 2009; Gétaz et al., 2013b), as well as robustness, uncertainty and sensitivity analysis (Jakobsson et al., 2005; Borg et al., 2013).", "As a result of the progress in modelling chromatography that has been made over the last decade, systems with crude feed material, containing product, product-related impurities (e.g. oxidation, deamidation, acetylation, dimerisation), and process-related impurities (e.g. antifoam, DNA, protein, virus) have recently been considered (e.g. Gétaz et al., 2013a; Nfor et al., 2013).", "The complexity of the industrial feed material in these studies means that the model development procedures involve conducting an extensive range of detailed experiments which may not be suitable in certain scenarios.", "One such scenario in industry is where the majority of process development has already taken place, but there remains a desire to develop understanding of a key feature of a bioseparation.", "The experimental effort required to develop an exhaustive model may discourage a mechanistic modelling approach considering time and material constraints.", "In this work, a chromatography model for predicting product quality in an industrial multicomponent bioseparation is developed and validated.", "The model is intended for use in a process scenario to allow specific variables critical to product quality to be studied.", "The chromatography utilises a hydrophobic interaction retention mechanism to purify a multicomponent product from a complex mixture of impurities.", "Process parameters were predefined prior to this work.", "Impurity removal is well understood and therefore a model description of this feature of the chromatography is not required.", "However, the step must also deliver the multicomponent product composed of six closely related variants of a dimer protein therapeutic (∼30 kDa) with their monomer subunits in a specific ratio.", "Variability in the feed material poses a purification challenge, and consequently, there is a risk that the products’ monomer subunit ratio will not meet product quality specifications incurring significant losses.", "Therefore, a model which can study product quality as a function of the load material concentration and composition is developed and validated in this work.", "A systematic procedure is used to determine key model parameter values, first using targeted experimental studies to quickly generate experimental data for estimation of model parameters, before employing an iterative procedure where laboratory scale column runs of the industrial process using real feed material are used to refine parameters in a sequential manner until model predictions exhibit satisfactory agreement with experimental data.", "We demonstrate how good understanding of an industrial process can facilitate model development, despite considering a chromatographic bioseparation with crude feed material and challenging purification objectives.", "Minimising the time and amount of material required to develop, validate, solve and determine model solutions is extremely important, as process development timelines are typically very constricted (Steinmeyer and McCormick, 2008).", "Especially so, as many simulations may be required to map the impact of process parameters on process performance and product CQA's (Degerman et al., 2006; Karlson et al., 2004).", "Thus, we aimed to minimise the necessary experimental and computational effort required to model the HIC and our ultimate goal was not to develop the most exact model description, but to develop a model with sufficient accuracy for use in industry.", "An equilibrium dispersive model was used to simulate the HIC (Guiochon et al., 1994).", "This model assumes that the mass transfer kinetics between the mobile phase moving through the column bed and the particles is infinitely fast.", "Thus the concentration of component i in the mobile phase is equal to the average concentration of component i in the intra-particular mobile phase, and the axial dispersion coefficient is replaced with an apparent axial dispersion coefficient which includes the contribution from the mass transfer kinetics.", "The model has the following additional assumptions; (i) the column is one-dimensional (radially homogeneous), (ii) the chromatographic separation is isothermal and adiabatic, (iii) the compressibility of the mobile phase is negligible and thus the velocity profile is flat, and (iv) the mass transfer parameters are independent of component concentration.", "The boundary conditions for Eq. (1) are the following (Guiochon et al., 1994):", "We used a systematic approach to model calibration, as illustrated in Fig. 4.", "In the procedure, targeted micro well experimentation is utilised to estimate the adsorption isotherm parameters, qs and ka,i. Scale down column studies are used to determine the total column porosity, ϵT, and give an initial estimate of the mass transfer, DA, parameters of the system (Fig. 4 Steps 1 and 2).", "Then an iterative procedure is employed, where laboratory scale column runs (7 ml CV, 7.4 cm bed height) of the industrial process using untreated feed material are used to refine parameters in a sequential manner until model predictions exhibit satisfactory agreement with experimental data (illustrated in Fig. 4 Steps 2 and 3, results for the HIC described in detail in Section 4).", "Such a procedure is required when model predictions after the initial calibration effort (Fig. 4 Steps 1 and 2) are not satisfactory, because generating more pure material for further model development is normally prohibitively costly, and using data from the actual industrial process is a superior method from our industrial perspective, as will become clear in the following section.", "Experimental results were used to estimate the values of model parameters using the ‘parameter estimation’ entity in gPROMS based on the SRQPD sequential quadratic programming code.", "Parameter estimation was based on the maximum likelihood formulation, which determines values for the uncertain physical and variance model parameters that maximise the probability that the model will predict the measurement values obtained from the experiments.", "The statistical variance model of constant variance was used in this case (Process Systems Enterprise, 2013).", "In the following section we describe the application of the procedure outlined above to the industrial multicomponent hydrophobic interaction chromatography considered in this work.", "One of the key challenges of developing a mechanistic model of industrial chromatographic processes is the limited availability of purified material, which is required for many tasks in the overall effort to bring a protein therapeutic to market e.g. drug trials, stability studies, toxicology studies, etc.", "We had to generate our own protein solutions for model development experiments from bulk feed material.", "Following initial purification by pseudo affinity capture, the material contained the 6 product forms of interest, as well as a range of product related impurities and host cell proteins (HCP's).", "The product forms were further purified and isolated from impurities over multiple runs on the hydrophobic interaction chromatography considered in this work.", "Multiple runs were required as it was particularly challenging to separate the product isoform BB from closely related product related impurities.", "Fractions were taken every column volume (CV) and analysed by CEX HPLC in order to determine the isoform distribution of the sample.", "Multiple samples with a range of isoform distributions were generated in this way, and later pooled in order to generate material with desired isoform distributions for development experiments.", "Confirmation of removal of product related impurities and HCP's was determined by phenyl RP HPLC.", "We were unable to generate pure samples of each product isoform which prevented determining traditional single component isotherms.", "We therefore required an approach to model development which used multicomponent mixtures of the six product forms.", "This involved (1) measuring isotherms competitively and (2) using an advanced parameter estimation facility to fit the experimental data to a competitive isotherm model.", "The apparent axial dispersion coefficient, DA, and total column porosity, ϵT, were determined from pulse injections of a small unretained molecule using Eq. (2) to Eq. (5).", "The determined total column porosity value, 0.9, was in agreement with previous literature estimations for the same resin (McCue et al., 2007).", "The number of theoretical plates, Np, was determined as 309 with a plate height of 0.0239 cm, resulting in an apparent axial dispersion coefficient, DA, value of 0.0001 cm2/s.", "Batch adsorption in a microwell plate is tedious requiring labour intensive experiments, and can give inaccurate results (Seidel-Morgenstern, 2004).", "However, it can be automated using robotic liquid handling to reduce experimental burden, and is simpler than most alternative methodologies (e.g. perturbation method, dispersed front analysis, peak fitting) which are difficult when applied to multicomponent separations.", "Only frontal analysis is relatively straightforward, but was unfeasible in this work due to high costs and limited availability of required material.", "Our exact methodology for the batch adsorption studies was based upon previous work on high throughput screening of chromatographic separations (Coffman et al., 2008), and is described in the Appendix.", "All batch adsorption experiments were repeated in triplicate, and averages are shown in this paper.", "To ensure that the experimental data used to fit the competitive Langmuir model contained competitive information, we varied the product form distribution in the load material used in the batch adsorption experiments.", "Each graph in Fig. 5 shows adsorption data from microwell experiments conducted at a different load material product form distribution, shown in the ratio AA%:AB%:BB% in the top left hand corner of each graph.", "Note that although the graphs show the bound concentration of the product form as a function of its mobile phase concentration, the mobile phase concentration of the other two product forms are also affecting the bound concentration.", "The effects of competition for binding sites is clear when the graphs in Fig. 5 are compared.", "In graph C, the BB stationary phase concentrations are significantly higher, especially compared to Graph B.", "This is due to the favourable product distribution of the load material resulting in fewer competing components, allowing more BB to bind (graph C load material 20% AA:25% AB:55% BB, graph B load material 28% AA:59% AB:13% BB.", "The estimated isotherm parameter values are shown in Table 1.", "The standard deviations of the estimated parameters are approximately ten percent, indicating there is still some uncertainty around the parameter values.", "The coefficient of determination, r2, for the model fit to experimental data was 0.96, which was found to be sufficient for satisfactory agreement between model predictions and experimental data as shown in Fig. 5, given the inherent uncertainties of the batch adsorption studies.", "The product form distribution in the elution peak, and in samples taken from the column outlet every column volume (CV) during the load and the wash, was measured during experimental small-scale column runs using CEX HPLC.", "The ability of the model to predict these product form distributions was an important and industrially relevant test to validate model accuracy, since achieving a specific percentage distribution of the product isoforms in the elution pool is a key objective of the chromatography.", "This data was also suitable for model calibration should the first iteration of the model give unsatisfactory predictions.", "The first iteration of the model was unable to give satisfactory predictions of the chromatographic process after the initial model calibration, as shown in Fig. 6A.", "This was not unexpected, as although we had spent extra effort ensuring that the isotherm parameters were accurate, we used a lumped mass transfer coefficient and had determined its value using the residence time of a unretained molecule (NaCl) that was significantly smaller than the protein, and thus would be expected to experience faster mass transfer.", "We could have considered using a more complex model (Kaczmarski et al., 2001), and/or used alternative experimental approaches to determining mass transfer parameters more accurately, e.g. conducting multiple pulse injections of the product at non-binding conditions, or using van Deemter plot methodology, etc. (Muller-Spath et al., 2011; Ng et al., 2012).", "However, applying these techniques to this industrial separation was problematic.", "For example, we could not find any non-binding conditions suitable for pulse injections of the product which did not significantly alter the system, and the lack of any pure component material, the highly competitive system and closely related isotherms of the product forms meant that the van Deemter plot method was not practical.", "In addition, we were conscious that there may also have been unidentified phenomenon occurring which could potentially affect these approaches, for example proteins unfolding on the HIC surface.", "Therefore, we took an alternative approach as discussed in the following section.", "We used data from the previously conducted model validation run of the industrial process to refine the model parameters.", "Experimentally determined product distributions in fractions taken every CV were used to estimate the new value of the apparent axial dispersion coefficient, DA, using the ‘parameter estimation’ entity in gPROMS.", "The previously determined value, estimated from the number of theoretical plates of the column, Np, was used as an initial guess.", "Adsorption isotherm parameters, qs and ka,i, were refined manually, guided by the previously determined values, standard deviations and experimental product distributions.", "This method made optimal use of validation data, and meant no further experiments were required other than for further model validation.", "Runs of the industrial process are straightforward to conduct, can be left to run unsupervised with minimal preparation time, and can use feed material that needs no pre treatment and is readily available.", "This part of our approach can be easily integrated with traditional process development where column runs of the industrial process are conducted regularly.", "The drawback of such a procedure is that it can result in sub optimal parameter values.", "Estimating model parameters directly from the industrial process under normal operation was not possible without prior knowledge of suitable initial values, due to the complexity of the feed material and separation considered in this work.", "We found that model predictions were in good agreement with experimental results after one iteration of refining the model parameters using model validation run data, shown in Figs. 6 and 7.", "The figures show the good agreement between the predicted and experimentally determined product form distributions in fractions taken every CV, and in the elution peak.", "The apparent axial dispersion coefficient and the AA equilibrium constant were modified from 0.0001 cm2/s to 0.003 cm2/s, and 5.31 to 3.5, respectively.", "Model validation studies (including the model refinement run) were conducted using qualified scale down columns to provide a rigorous test of model accuracy.", "The flowrate, bed height and mobile phase conditions were kept constant throughout.", "The composition of the load material, total load concentration, load challenge and wash length were varied as described in Table 2.", "Results are presented in Table 3.", "Validation run A was chosen to give a good representation of the isoform distributions experienced in normal load material and the load challenges used during day-to-day operation of the chromatography (Fig. 7A).", "The model gave very accurate predictions of the product form percentages in samples taken during the load and the wash phase, and was able to accurately predict the final products’ monomer subunit ratio in the elution peak.", "The isoform distributions chosen for runs B and C were chosen to provide a challenge for the model, and would rarely be experienced during day-to-day operation.", "It was important to test these artificially created, rare load conditions in order to understand the limitations of the model.", "The model gave good predictions across the extended range of conditions, especially for the case study C (Fig. 7C).", "The difference between model predictions and experimental data for validation run B (Fig. 7B) was due to the very challenging isoform distribution in the load material, in particular that product isoform BB occupied 48% of the load, combined with a high load concentration and load challenge.", "The model slightly overestimated the percentage of AB during the wash length, which was attributed to tailing of BB that was not captured by the model.", "This was not observed during studies with fresh feed material, and thus it is likely that a small amount of product was damaged during the multiple applications, buffer exchanges and concentration steps used to generate the feed material for validation studies.", "Runs D and E were conducted to test model predictions at shorter wash lengths more applicable to producing a product of the desired quality, and thus no samples were taken during the wash.", "We found that the model predictions were again in good agreement with experimental data (predicted component percentages within 3%).", "The development and validation of a predictive mechanistic model of industrial biopharmaceutical multi-component chromatography has been described.", "The equilibrium dispersive model with a competitive Langmuir isotherm was able to successfully predict product quality for an extended range of inlet concentrations, load challenges and inlet product distributions.", "We used targeted small scale experimentation for initial model calibration, spending extra effort on micro well batch adsorption experiments for the estimation of the adsorption isotherm parameters.", "Then an iterative procedure was employed where laboratory scale column runs of the industrial process were used to refine parameters in a sequential manner until model predictions were in satisfactory agreement with the experimental data.", "Final model predictions were within 3% of the final products’ monomer subunit ratio found in the chromatography elution peaks during validation studies.", "The model was also able to accurately predict the product form distribution in samples taken during the wash phase of the chromatographic cycle in validation runs A, B and C (samples were not taken in runs D and E).", "The model can now be used in subsequent studies to explore the effect of load material on product quality.", "The results demonstrate how good understanding of an industrial process can facilitate simpler model development when an exhaustive description is not required, despite considering a chromatographic bioseparation with crude feed material and challenging purification objectives." ]
[ "We present a procedure to deliver models of industrial chromatographic separations.", "Model development occurs sequentially via iterative experimentation/parameter estimation.", "The procedure is applicable even when faced with challenging industrial constraints.", "We consider the purification of a multicomponent protein therapeutic from crude feed.", "Model predictions were in excellent agreement with experimental data." ]
Leaders' sensemaking under crises: Emerging cognitive consensus over time within management teams
When facing a crisis, leaders' sensemaking can take a considerable amount of time due to the need to develop consensus in how to deal with it so that vision formation and sensegiving can take place. However, research into emerging cognitive consensus when leaders deal with a crisis over time is lacking. This is limiting a detailed understanding of how organizations respond to crises. The findings, based on a longitudinal analysis of cognitive maps within three management teams at a single organization, highlight considerable individual differences in cognitive content when starting to make sense of a crisis. Evidence for an emerging viable prescriptive mental model for the future was found, but not so much in the management as a whole. Instead, the findings highlight increasing cognitive consensus based on similarities in objectives and cause-effect beliefs within well-defined management teams over time.
[ "BUSI", "PSYC", "SOCI" ]
[ "Cognitive content", "Cognitive mapping", "Collective leadership", "Mental models" ]
[ "1403", "1407", "3202", "3312" ]
[ "Due to its importance to the viability of organizations, the link between perceptions and interpretations of external change and organizational adaptation to achieve performance outcomes has received considerable research attention (Barr, 1998; Barr, Stimpert, & Huff, 1992; Daft & Weick, 1984; Eden & Ackermann, 2010; Isabella, 1990; Lant, Milliken, & Batra, 1992; Sutcliffe & Huber, 1998; Thomas, Clark, & Gioia, 1993).", "A major reason given for organizational adaptation in the face of a disruptive external change and subsequent crisis is that leaders alter their beliefs to accommodate the changes in the environment, or alternatively, the leaders themselves are changed (Barr, 1998; Daft & Weick, 1984; Janis, 1989).", "Leaders are clearly important when organizations face a crisis and are key when searching for answers to make sense of what is happening (Weick, Sutcliffe, & Obstfeld, 2005).", "Sensemaking under crises is inherently complex because leaders have to think and problem solve in the context of a novel ambiguous situation involving time pressure and stress while interacting with others in management teams (Mumford, Friedrich, Caughron, & Byrne, 2007).", "The key task for leaders in such situations is to develop a mental model, based on their schemas,1 consisting of causal beliefs for understanding and responding to the crisis (Weick, 1995).", "The most significant recent advance in understanding leaders' cognition driving sensemaking under crises has been theoretical.", "Mumford et al. (2007) present a detailed theoretically grounded conceptual model, which includes two particularly important facets, both central to their conceptual model of leader cognition.", "One, is the activation of descriptive mental models which are used to interpret any externally led change.", "It is external change that invariably starts a crisis and individual managers adopting leadership roles, even within the same firm, can interpret the change differently (Jackson & Dutton, 1988).", "Two, is the development of a prescriptive mental model or “…a mental model describing the causes and consequences of performance with respect to the crisis situation at hand” (Mumford et al., 2007, p. 528).", "In other words, leaders develop a mental template for the future, but as Mumford et al. (2007) point out, this development is complex and occurs over time.", "The development of a new prescriptive mental model is critical because the stability of leaders' cognition in the face of a changing external environment has been found to be a contributor to inertia and organizational failure (Barr et al., 1992; Hodgkinson, 1997).", "As an understanding of descriptive mental models and the development of a prescriptive mental model for the future are both cited as key to fully understand leaders' sensemaking under crises, a longitudinal research perspective is required.", "Sensemaking under crises involves cycles of information processing, communication and problem solving, which are likely to be interspersed with action, to make sense and give meaning to events (Smircich & Morgan, 1982; Weick et al., 2005).", "In most settings such a complex longitudinal process invariably involves collective leadership drawing on different experiences and expertise within management teams (Friedrich, Vessey, Schuelke, Ruark, & Mumford, 2009).", "While individuals within management teams will likely interpret change differently, ultimately, consensus needs to be built around an envisioned future (Strange & Mumford, 2002).", "The development of a viable prescriptive mental model provides a basis for vision formation in a crisis (Mumford & Strange, 2002; Strange & Mumford, 2005).", "However, we know little of how the content, or the assumptions and beliefs, contained within leaders' mental models develop and change during the course of a crisis.", "With the exception of an empirical study by Markóczy (2001) there is little direct cognitive empirical evidence to help understand how consensus is developed over time within the mental models of individual managers within the same firm as they grapple with the same change events.", "To help understand how leaders develop a prescriptive mental model for the future when responding to a crisis, we map cognitive diversity and consensus in individuals adopting leadership roles within management teams in the same firm over time.", "As part of a growing stream of research into management cognition (see Hodgkinson & Healey, 2008; Hodgkinson & Sparrow, 2002; Narayanan, Zane, & Kemmerer, 2011; Walsh, 1995 for reviews) scholars have long considered the importance of sensemaking based on interpretive work conducted by the leaders of organizations, such as the CEO or top management team, to make sense of change when it is encountered (Gioia & Chittipeddi, 1991).", "Weick (1995, p. 57) points out that the sensemaking perspective is associated with understanding the cognitive filters that people use and what these filters include and exclude.", "Thus, a hallmark of sensemaking is the simplification of complexity through individuals' mental models so as not to be overwhelmed by data (Daft & Weick, 1984; Walsh, 1988).", "Leaders enact or produce part of the environment they face (Daft & Weick, 1984; Weick, 1995) such as the enactment of a strategic group within their mental models signifying a very limited perception of competition (Porac, Thomas, & Baden‐Fuller, 1989).", "Consequently, the sensemaking perspective highlights that the environments that leaders deal with are, at least in part, socially constructed (Weick, 1995).", "This social construction of environments distinguishes the sensemaking perspective from much of the work on behavioral economics, such as the work which focuses on the cognitive limitations of humans in general operating in objective environments (Hodgkinson & Healey, 2008; Weick, 1995).", "While the social aspects of sensemaking is a theme running through the literature, some researchers put more emphasis on sensemaking as an individual cognitive process (Maitlis & Christianson, 2014).", "Another theme debated within the literature on sensemaking is in connection with its temporal dimension.", "Weick (1995) initially highlighted the retrospective nature of sensemaking, while more recent work raises the possibility of prospective future oriented sensemaking that may be linked to resolving a crisis (Weick et al., 2005).", "Leaders can make presumptions about the future (Weick et al., 2005) and give meaning to others (Smircich & Morgan, 1982) such as through categorizing and labeling a disruptive change as an ‘opportunity’ signifying a positive way to deal with change (Dutton & Jackson, 1987).", "The communication of meaning, such as through the labeling of a disruptive change by trusted leaders is likely to result in more consensus which in turn is more likely to result in organizational action (Dutton & Jackson, 1987; Schwenk, 1984).", "While a strict definition to encompass all crises is problematic, they are often associated with sudden threats to high priority goals and the need to dramatically change prior practice (Hunt, Osborn, & Boal, 2009).", "Crises involve a movement away from stability to established new goals (Lichtenstein & Plowman, 2009) and also imply a time pressured change relative to standard operating procedures (Mumford et al., 2007; Williams, Pillai, Deptula, & Lowe, 2012).", "Sensemaking within an organizational change process is related to understanding and is cognitive in nature (Gioia & Chittipeddi, 1991).", "In the leadership literature, research has highlighted the importance of context specific cognition, which underpins sensemaking and leaders' behavior as well as its influence on outcomes such as performance (Antes & Mumford, 2012; Barrett, Vessey, & Mumford, 2011; Lord & Hall, 2005; Mumford, Connelly, & Gaddis, 2003; Mumford et al., 2007).", "Leaders are critically important in the context of sensemaking under crises.", "They are bombarded with a stream of often conflicting data associated with ill-defined events and possible problems (Dutton & Jackson, 1987).", "It is their key role to make sense of this critical situation and to convey this sense in their role of sensegiving to develop meaning for others (Foldy, Goldman, & Ospina, 2008).", "Leaders are also key in developing a resolution to any crisis.", "They need to project a vision to overcome a crisis based on their prescriptive mental model or mental template for the future (Mumford et al., 2007).", "While the leadership role in sensemaking under crises is critical, leaders are likely to face two main types of problems common in organizations, which add to the difficulty of the task: ambiguity and uncertainty (Weick, 1995).", "Ambiguous facets of crises are likely to result in too many interpretations, while uncertain facets are likely to result in no interpretations as leaders are ignorant of what is happening (Weick, 1995).", "Events incorporating both ambiguity and uncertainty are likely to emerge over time when under a crisis (Mumford et al., 2007).", "Leaders should not act alone when confronting such difficult events.", "Rather they should work in teams because having different perspectives can enrich interpretations (Huber & Lewis, 2010).", "When operating in management teams, leaders are likely to share perceptions and gradually create meaning through communication so there is cross understanding (Huber & Lewis, 2010).", "Cross understanding, through an accurate understanding of the mental models of others within a leadership team, may lead to changes to mental models because some individuals are convinced by and adopt the beliefs of others (Huber & Lewis, 2010).", "Such cross understandings are therefore likely to add to consensus.", "While cross understanding may facilitate more accurate interpretations of events associated with a crisis, errors of judgment are also likely to occur.", "Prior research suggests that leaders put much reliance on case-based prior knowledge and experience when thinking but this can result in errors especially when addressing complex ill-defined problems that are likely to be encountered in crises (Eubanks & Mumford, 2010; Mumford et al., 2007).", "Such errors are also likely to limit leaders' creative thinking required to resolve a crisis (Mumford, Blair, Dailey, Leritz, & Osburn, 2006).", "A number of errors are likely in leaders' cognition, such as those linked to humans' limited information processing capacity (Cyert & March, 1963; Lord & Maher, 1990; Miller, 1956), and others more specific to sensemaking under crises.", "Mumford et al. (2007) discuss that dealing with crises make specific demands on leaders that are likely to result in a number of errors.", "The authors point to errors such as non-recognition of change; errors due to time stress and resource demands; underestimates of the response required due to a form of anchoring bias; and the nature of prior knowledge and the use of specific prototypical cases which may not be appropriate for applying to the current crisis.", "Clearly, to achieve performance in dealing with crises leaders have to reduce such errors.", "One major way they can reduce errors is by involving others with different ways of thinking in collective leadership and decision making.", "When confronting disruptive external change and crises, not all those adopting leadership roles are likely to think the same, even within the same firm.", "As previously observed, differences may be due to errors, such as focal errors, because leaders focus on different information or use different case based prior knowledge.", "Alternatively, some leaders may take more time than others to think through the issues.", "Cognitive diversity and consensus in leaders refers to the differences and similarities in beliefs within their mental models.", "Much of the prior research is directed at strategic consensus or the shared understanding on a common set of priorities (Floyd & Wooldridge, 1992).", "In such studies it is usual to consider consensus in terms of the content of mental models, in particular the ends (objectives, goals) and means (strategies) used to achieve them in top management teams (Kellermanns, Walter, Lechner, & Floyd, 2005).2", "While the study of ends and means are clearly important to understanding sensemaking under crises, Markóczy (2001) suggests that this focus may limit a more holistic investigation.", "Consequently, there is a need to focus on the beliefs associated with the issues most relevant to leaders and how these beliefs are related (Markóczy, 2001).", "In the decision making literature consensus and its implications for performance have been studied considerably but with inconclusive results (see Kellermanns et al., 2005, for a review).", "It is perhaps not surprising then, that prior research is not unequivocal as to the merits of consensus in leaders.", "Research highlights problems when leaders think in similar ways, such as underestimating the full extent of competition (e.g. Porac et al., 1989) so cognitive diversity may help eliminate bias and errors of judgment.", "Cognitive diversity in teams is also likely to increase creativity (Kilduff, Angelmar, & Mehra, 2000; Shin, Kim, Lee, & Bian, 2012) and is said to lead to more extensive discussions about strategic options to enable firms to change (Lant et al., 1992; Miller, Burke, & Glick, 1998).", "Differences in thinking are reported to increase the range of vision so that promising options are not missed (Eden & Ackermann, 2010).", "Such differences are more likely when leadership occurs via large management teams (Amason & Sapienza, 1997), incorporating individuals from different cultural backgrounds (Hitt, Dacin, Tyler, & Park, 1997) and various hierarchical levels (Ireland, Hitt, Bettis, Porras, & Auld, 1987).", "On the other hand, researchers also report positive outcomes for management consensus relating to external change.", "Waiting for consensus to emerge can slow down decision making (Eisenhardt, 1989) but consensus itself has been found to subsequently positively influence the speed of decision making and action (Bourgeois & Eisenhardt, 1988; Dess, 1987; Eisenhardt, 1989).", "Cognitive consensus is also likely to be helpful when undertaking idea evaluation which is a key task for leaders (Mumford, Marks, Connelly, Zaccaro, & Reiter-Palmon, 2000; Mumford et al., 2003).", "It is also postulated to be important for shared commitment within management teams (Floyd & Wooldridge, 1992; Markóczy, 2001; Schwenk, 1986) and therefore important for responding to a crisis.", "While both cognitive diversity and consensus in managers adopting leadership roles are reported to have positive outcomes for firms, there is relatively less research emphasis placed on investigating these at an individual level rather than the firm level of analysis (see the following for exceptions, Hitt et al., 1997; Hodgkinson & Johnson, 1994; Hodgkinson & Maule, 2002; Ireland et al., 1987; Lindell, Melin, Gahmberg, Hellqvist, & Melander, 1998; Markóczy, 2001; Porac et al., 1989; Reger & Huff, 1993).", "We use this individual level of analysis in a single firm to help throw light on leaders' sensemaking over time when dealing with a crisis (Friedrich et al., 2009).", "While there is limited prior empirical research into individual differences in management cognition within the same firm as they deal with a crisis, longitudinal empirical research on stability and change to individual manager's cognition in this context is even more limited.", "Researchers agree, but based on rather limited empirical evidence, that the stability of mental models is a problem when managers face environmental change.", "Empirical evidence for a cognitive explanation for organizational inertia in the face of change has been found in several contexts.", "GrØnhaug and Falkenberg (1989) were amongst the first to investigate changes in strategy when firms experience environmental jolts.", "The authors attributed the lack of change they found to biased management perceptions of their environments and a lack of competencies in dealing with new conditions.", "Other empirical studies have also identified cognitive influences on inertia based on longitudinal documentary evidence (Barr et al., 1992), longitudinal questionnaire evidence (Hodgkinson, 1997) and in-depth cognitive interviews (Reger & Palmer, 1996).", "These studies highlight disastrous consequences for firms when leaders become locked into mental models that are no longer appropriate for new conditions.", "This problem is not easily resolved because mental models have been found to change slowly even when environments change rapidly (Reger & Palmer, 1996).", "In another study by Lindell et al. (1998), based on longitudinal documentary evidence at an individual decision making level, researchers found that a framework of strong stable beliefs, values and assumptions can prevail in an individual over long periods of time (at least over the three-year term of the study).", "This stability, the authors suggest, may be based on those beliefs that are likely to be formed early on in managers' careers.", "However, Lindell et al. (1998) also found continuous mental adaptation, but this was focused on situational factors.", "Evidence suggests that mental models can change due to experience, but once they have been built up over time they remain stable and difficult to change (Barr et al., 1992).", "The study by Markóczy (2001) provides one rare example of more detailed empirical evidence of changes to cognitive consensus over time within individual managers as they grapple with external change.", "The findings highlight that consensus increases over time during strategic change but the locus of consensus was not found within leaders in the top management team.", "The findings also suggest that consensus building occurs due to a larger number of individuals that agree, rather than any alteration to the strength of agreement.", "Such initial findings have led other researchers such as Mumford et al. (2007) to theorize more deeply about leaders' cognition when firms face disruptive change and crises.", "Based on cognitive vision formation theory developed by Mumford and Strange (2002), Mumford et al. (2007) provide a theoretical grounded conceptual model to help explain how leaders address change and resolve crises over time.", "This model postulates that the onset of a crisis would first force leaders to activate descriptive mental models based on case based prior knowledge and experience as they confront ambiguous and novel problems (Eubanks & Mumford, 2010; Mumford et al., 2007).", "In other words, leaders would initially use a mental model based on how their world is currently configured and operates to try to make sense of what is happening.", "As these descriptive mental models are based on prior knowledge and experience individuals will likely produce different interpretations of any external change and its impact on the firm (Mumford et al., 2007).", "Leaders would then develop a prescriptive mental model over time as the basis of a vision formation process for the future to overcome any crisis (Mumford & Strange, 2002; Mumford et al., 2007; Strange & Mumford, 2005).", "Currently, empirical evidence for this theory is rather limited.", "Strange and Mumford (2002) report a study based on documentary evidence which found that it is indeed the content, in terms of goals and causes, that was key when determining a prescriptive mental model as a basis for vision formation in historical charismatic and ideological leaders.", "Strange and Mumford (2005) report a further experimental study which confirms the importance of descriptive mental models based on past experience and the abstraction of key goals and causes for vision formation.", "Both these studies focus on the analysis of visionary statements of leaders rather than their mental models.", "Consequently there is an express need, as Strange and Mumford (2005) report, for more direct longitudinal studies focusing on the changes to mental models which underlie leaders' vision formation in crises.", "Currently, theory implies that leaders near the start of a crisis will draw on different case-based prior knowledge and experience so there is an expectation that considerable cognitive diversity will be present even within leaders within the same firm as they confront a crisis.", "This insight leads to the following working hypothesis:", "Near the start of a disruptive external change and subsequent crisis cognitive diversity will be apparent in managers adopting leadership roles.", "Cognitive diversity will be demonstrated by differences in objectives, means or strategies to achieve objectives and other causal beliefs represented in the mental models of managers adopting leadership roles within the same firm as they address the same crisis.", "Later, as the crisis develops and the impact of the disruptive change becomes clearer, problems are likely to be addressed and start to be resolved.", "When responding to a crisis, theory suggests that cognitive consensus is likely because managers gradually create meaning through communication and agree on a prescriptive mental model for the future.", "Therefore prior research suggests the following working hypothesis:", "When responding to a crisis cognitive consensus will emerge over time in managers adopting leadership roles.", "Cognitive consensus will be demonstrated by increasing similarities in objectives, means or strategies to achieve objectives and other causal beliefs represented in the mental models of managers adopting leadership roles within the same firm as they address the same crisis.", "As part of this developing consensus, some leaders are likely to change their mental models more than others.", "Prior research suggests that this change may be due to a number of factors.", "For example, leaders may initially apply prior case based knowledge but this knowledge may not help in the current crisis.", "There is likely to be errors of judgment initially when confronting ambiguous events.", "These may be resolved by communication with other leaders within the firm and by additional information on important aspects of the disruptive change.", "Consequently, there is an expectation for the following:", "When developing cognitive consensus some leaders will change their mental models more than other leaders.", "Change to mental models will be demonstrated by changes to objectives, means or strategies to achieve objectives and other causal beliefs represented in the mental models of managers adopting leadership roles within the same firm as they address the same crisis.", "These working hypotheses will be investigated and developed in the empirical phase of the research.", "To contribute to the literature on leaders' sensemaking under crises this current study was designed to investigate similarities and differences in beliefs and values at an individual level over time (Bougon, Weick, & Binkhorst, 1977; Markóczy, 1997; Wacker, 1981; Walsh, 1988; Weick, 1979).", "This research agenda did not allow us to use documentary evidence which has been used as a longitudinal data source in some prior studies (e.g. Barr et al., 1992; Nadkarni & Barr, 2008; Nadkarni & Narayanan, 2007).", "Documents such as letters to shareholders assume leaders' consensus and do not highlight individual differences in cognition.", "Instead, multiple data collection techniques were used within single case study method (see Yin, 2014).", "This method allowed the investigation of different leaders' cognition within the same organizational context, including different responses to the same crisis.", "The empirical research was conducted within a single not-for-profit organization anonymously named ‘Health Change UK’ which operates in the health sector in the United Kingdom.", "Cognitive consensus and diversity as well as changes to mental models were investigated within all the leadership teams and included 12 leaders within the organization which has approximately 200 employees.", "The respondents consisted of all 4 top managers (TM) i.e. all of the top management team including the CEO; 2 trustees (TR) including the chair of trustees and the treasurer who both act as non-executive directors on the board; 6 regional leaders operating at middle management level (MM) within the branch network.", "As the purpose of the research is to understand leaders' cognition when facing a crisis, it is essential to provide the detailed research context for the study.", "Therefore, we start by outlining the crisis that ‘Health Change UK’ faced in 2011 at the start of this research.", "‘Health Change UK’, and many other similar providers in health services in the U.K., have sourced large amounts of their funding from various government contracts via taxpayers' money.", "The main government department having overall responsibility for this funding has been the Department of Health within the U.K. Government.", "A large majority of these contracts were commissioned via the National Health Service Primary Care Trusts (NHS PCTs).", "For many years contract commissioners' requirements within Trusts have remained stable and specific contracts have rolled over based on the satisfaction of the work.", "However, due to changes in the external political environment in 2010, the funding and tendering process for these contracts altered significantly based on the plans put forward by a new U.K. Government (Department of Health, 2010).", "The new government introduced major changes to public services.", "The most significant changes related to the proposals to reform the healthcare system.", "Under the plans, general practitioners (doctors in local surgeries at the front line) would be given much more responsibility for spending the budget.", "There were also plans to increase competition so the use of the private sector and third (voluntary) sector was to be encouraged.", "It has been dubbed one of the most radical plans in the history of the health service in the U.K. and has been very controversial.", "Many of the current government's policies are argued to be necessary to combat both the economic crisis of the global recession and also to reduce the nation's large budget deficit.", "In the Comprehensive Spending Review of 2010 it was apparent that efficiency savings would be required from all areas of public funding and also some budgets would face dramatic cuts.", "As a result of these reforms ‘Health Change UK’ were facing some very uncertain times as they were not sure how the funding environment was going to change.", "However, there was more certainty about one major change that was fundamental for the financial viability of ‘Health Change UK’.", "Commissioners of health services were required to ensure that there was competitive tendering on all contracts and that there was a transparency on the performance of service providers.", "They were to ensure that the principle of payment by results (money following service performance) was enshrined in future commissioning of health services.", "Consequently, competition for ‘Health Change UK’ increased dramatically with much larger organizations competing directly with them on tendering for health services.", "Some of these larger organizations had significant economies of scale and introduced lower price competition into the health sector which meant that the continued existence of ‘Health Change UK’ was questioned.", "It was this threat and the subsequent crisis due to the loss of large contracts that the organization was facing at the start of the research.", "To triangulate the data, an interview protocol with different stages was developed to include a variety of data collection techniques to support the investigation of leaders' cognition.", "For the first phase all 12 interviews were conducted over a two-month period by the same researcher using an identical interview protocol in each case.", "The same data collection procedure was followed for the second phase of data collection 18 months later.", "Due to the need to compare leaders' cognition we used sorting technique, common in psychological research (Rosenberg, 1982), as the basis of the standardized procedure for developing cognitive maps outlined by Markóczy and Goldberg (1995).", "The essence of the technique is that a large identical pool of factors is consistently presented to a variety of respondents for them to sort out which are the most important.", "This technique is used to standardize the production of cognitive maps which is vital when they are to be compared and contrasted.", "The pool of factors that represented individuals' beliefs about the organization and its environment were derived from the literature (Buzzell, Gale, & Sultan, 1975; Combe, Rudd, Leeflang, & Greenley, 2012; Hambrick, 1981; Markóczy, 2001; Markóczy & Goldberg, 1995; Miles, 1980; Walsh, 1988) and also adapted through pilot interviews with six employees of the company.", "This pilot was designed to investigate management cognition at all levels of the organization, so managers representative of different organizational levels were included.", "The Business Development Manager who regularly attends board meetings represented the views of the top management team as we wanted to include all individuals for this group in the main study.", "These six individuals were not part of the main study but this small scale evaluation helped test the validity and robustness of the interview protocol and explored different meanings of the factors included in the sorting task.", "The main problem highlighted in this pilot study was the terminology used for the factors in the sorting task.", "They were based on prior research in the profit sector so did not apply completely to the health related not-for-profit case study firm.", "Therefore, the wording of some factors had to be altered to a more commonly accepted language used in this health sector.", "As no further modifications were required 54 factors were promoted to be used in the main study.", "During each interview in the main study all participants were offered the chance to write down any additional factors they believed were important to the success of their organization but no one contributed any further factors.", "All the face-to-face interviews with the managers adopting leadership roles included four stages using multiple data collection techniques.", "All interviews were initiated by using sorting technique as a starting point to the development of causal cognitive maps following the standardized procedure outlined by Markóczy and Goldberg (1995).", "This technique is used to standardize the production of cognitive maps and is also used to reduce interview bias because there is no communication between researcher and respondent during the sorting process (Walsh, 1988).", "Sorting technique is designed to identify each participant's beliefs about important factors for success so a standard aim is introduced in each interview; that of factors ‘important or not important for the success of the organization’.", "After the sorting task each respondent was asked to rank order the ten most important factors for success (rank order 1 for the most important to rank order 10 for the least important) and these ten factors were used to generate cognitive maps in real time during the rest of the interview.", "This approach was taken, as it is possible to verify the accuracy of the cognitive maps produced by the participants during the interviews and it alleviates the need for any post hoc interpretation by the researcher (see Hodgkinson, Maule, & Bown, 2004).", "Production of cognitive maps during the interview also ensures that the elicitation task is meaningful to participants (Hodgkinson & Healey, 2008).", "The cognitive mapping procedure was then followed by an in-depth interview to develop a more detailed understanding.", "Each participant was asked to explain each factor chosen and the links within each cognitive map before laddering was used to investigate antecedents and consequences of a focus on any particular factor (Gutman, 1982; Reynolds & Gutman, 1988).", "Additionally, at the end of the interviews for the second phase, participants were presented with their cognitive maps from phase 1.", "The interviewer then asked the respondents to discuss any similarities and differences over the intervening period.", "From the 24 interviews (both phases) a total of 157 A4 pages of transcriptions were produced.", "Each respondent also completed a questionnaire during the interview process to provide information on their age, gender, job role, location of work, time spent at the company and objectives for the company.", "To investigate similarities and differences in beliefs within the organization, individual differences between pairs of maps were analyzed (see Markóczy & Goldberg, 1995).", "This provided a statistical value between individual maps so each participant's cognitive map was individually compared to the other 11 participants.", "Following Markóczy and Goldberg (1995), if a value of 0 is present then the maps are exactly identical whereas a value of 1 represents a completely different cognitive map.", "A zero value is nearly impossible because this represents identical causal cognitive maps where all 10 factors and all cause–effect beliefs are the same.", "It is only evident on the matrix when compared with the participants' own map.", "The value of 1 can be quite common and occurs several times in the dataset as various participants chose a completely different set of 10 factors to another participant (see Table 1).", "Table 1 presents the data near the start of the crisis.", "There are considerable differences in the factors chosen by the various leaders to achieve success indicating that their objectives are different as are their strategies or ‘means’ to achieve their objectives.", "Of particular interest are the clear differences within the top management team (TM01 − TM04) with high scores for cognitive differences.", "For example, the difference between TM02 and TM03 is 1 (maximum) indicating that two leaders within a small top team have chosen a completely different set of factors as important for success.", "Multidimensional scaling (MDS) was applied to the dataset to provide an initial overview of the whole data.", "The settings were modified using ‘PROXSCAL’ with ‘Proximities = Dissimilarities’, ‘Proximity Transformations = Interval’ and ‘Initial Configuration = Torgerson’.", "The stress values in our study were found to be quite high (S-Stress = 0.14) but expected due to the complexities around dimensionality when analyzing this type of dataset (Markóczy & Goldberg, 1995).", "This technique allowed the data to be presented in two-dimensional space so that the positioning of respondents with respect to each other could be evaluated indicating similarities and differences in their cognitive maps (see Fig. 1).", "Fig. 1 indicates wide differences between the cognitive maps of the leaders.", "Of particular interest is the considerable differences and lack of cognitive consensus within the leadership as a whole and particularly within the top management team and trustees, all of whom sit on the same board of directors.", "From the laddering interview transcripts it was apparent that at the beginning of the crisis the leaders had divergent perspectives on the disruptive external change and its implications for the organization.", "Despite there being a general consensus regarding the nature of the change, which had implications for the way the company needed to operate, sensemaking appeared very idiosyncratic.", "The leaders themselves focused on different aspects of their environments.", "This focus ranged from concentrating on their competitors as in the case of a member of the top team (TM01) to other aspects such as the government, funders (contract commissioners), staff, new markets, strategic alliances, or cost cutting.", "The following quotations highlighted these contrasting perspectives.", "“I think we should look at all our competitors…people that we constantly come up against with tenders and we do need to be looking to see what they're doing and how we can do what they're doing a bit better or more cheaply.”", "TM01", "“I think the change of government has brought about immense changes in the way that government impacts upon services …they've put in place a framework that determines who will do that and how they will do it…government policy in the current environment is integral to everything we do.”", "TM02", "“If we don't get sufficient funding just to keep the service going, then obviously we can't offer anything…suddenly the external world has hit us…how do we motivate staff so they don't just think that we're pushing them to do targets, targets, targets; actually we're also looking after them.”", "TM03", "“I think trustees sometimes feel very frightened about moving into new areas of work…I understand their reluctance, but for me it feels crucial for us to be able to thrive in a marketplace that's becoming more and more difficult.”", "TM04", "“Sometimes you're going to see benefits in partnering with somebody else, sometimes as senior partner, sometimes as junior partner…it's probably less risky to do that — to recognize your own limitations and to work alongside somebody else.”", "TR01", "“The fact that the world is changing around us all the time.", "The funding is uncertain…when people are under threat they ‘hunker down’ and sort of get set in their ways kind of thing, whereas organizationally we have constantly to be ready to change.”", "TR02", "“It's how you then structure an organization, isn't it?", "If you've got to do cost-cutting, then that has an effect, doesn't it?", "What do you cut?", "Is it staffing and how do you deliver that and how does that affect morale for example?", "And I think that's quite a complex one to deal with really.”", "MM01", "“Last year I've seen so much change…than in the whole of my working profession… the support from head office to translate what funders want is really critical… The way that one communicates really has an effect on whether staff want to go along with what's changing or not.”", "MM03", "In summary, the data indicate that in phase 1 of the interviews considerable cognitive diversity existed in the leadership as a whole and even within each leadership team.", "Considerable differences were found in the cognitive maps of the leaders highlighting alternative focus of issues of importance as well as different objectives and strategies or means to achieve them.", "The findings support Hypothesis 1.", "Next, we wanted to assess any changes to individuals' mental models as the crisis progressed.", "Therefore, we analyzed the cognitive maps based on the factors contained in the cognitive maps and the causal relationships between them during phases 1 and 2.", "To provide a more detailed analysis, the standard method of calculating in-degrees (or the number of links leading into a factor) and out-degrees (or the number of links leading out of a factor) for all factors in all the cognitive maps was used (see Bougon et al., 1977).", "Calculating the in-degrees of the different factors helped identify the objectives or end states and also the ‘means’ or strategies respondents believed the best way to achieve these objectives.", "Additionally, the frequency of chosen concepts and the accumulated rank order was collated.", "To keep a simplified scoring system a conventional ordinal ranking model was used (Cook & Kress, 1985) but without any intensity of preference (so 1st = 10pts to 10th = 1 pt).3", "The etiographic representations and accumulated rank order scores for phases 1 and 2 are represented in Table 2.", "Table 2 indicates the changes in objectives in the leadership as a whole between phases 1 and 2 of the data collection.", "The following objectives have become much more important to the leadership as a whole as the crisis has progressed: ‘Service quality’, ‘Motivation of staff’, ‘Relationships with partners’ and ‘Innovative services’.", "‘Service quality’ in particular is considerably more important in phase 2 moving from an in-degree score of 43 and a rank order score of 44 in phase 1 to 125 and 90 respectively in phase 2 (see Table 2).", "These increases indicate that ‘Service quality’ and its improvement is seen much more widely by the leadership as the most important objective for the organization.", "This increasing consensus towards a focus on improving service quality and the importance of staff motivation is reflective of trial and error learning based on losing contracts and tendering for new contracts.", "Considerable change, such as improving the tendering process was required to subsequently resolve the financial crisis.", "Tendering was improved by collating data on past successes, such as on patient improvement and demonstrating efficiency savings, and quoting this data in new tender documents.", "Table 2 also indicates other factors have moved up the agenda in the leadership as a whole such as the factors representing the increased importance on analysis of data such as ‘Detailed information/data on customers’ and ‘Detailed analysis of company finances’.", "Other factors also increased in importance as the crisis progressed such as ‘Developing staff’ and ‘Employee flexibility’ that impact on improving service quality.", "The focus on attempting to resolve the organizational crisis following the disruptive change is also evident in other data.", "In phase 1 only three leaders chose ‘Responsibility to commissioners’ (the fund holders) as an important factor.", "However, by phase 2 this stakeholder is discussed much more widely.", "Additionally, by phase 2, ‘Competition’ is discussed just as often but it has moved from a dialogue of the unknown to the known with specific competitors named in discussions.", "A major theme that came out of the in-depth laddering interviews in phase 2 was the increased chaos produced within the organization by losing contracts and the subsequent learning that occurred.", "In accordance with the sensemaking literature these discussions were framed retrospectively (Weick, 1995).", "The following quotations from leaders highlight this retrospective sensemaking.", "“XXX (Service A — 55% of turnover) was tanking seriously (experiencing significant problems) and…people were really unhappy whereas I think now we've been through the pain of the restructure…losing XXX (Service B — 21% of turnover), losing XXX (Service C — 6% of turnover), big wake up calls for the staff.”", "TM01", "“I think that 12 months ago ‘Health Change UK’ was a different organization.", "It was slightly myopic and didn't have much of a sense of what its competitors were up to and we lost contracts…I think now we've got a good appreciation of what our competitors do and how they compete.”", "TM02", "“I was still feeling the ricochet of the fallout from losing the XXX (Service B) contract actually and we were in a bit of a state of anxiety around that…last year has seen us win new contracts…it's allayed those anxieties.”", "TM03", "“In the world that we live in currently with competitive tendering if we're not good value we won't get the tender....if our price differential is too great we won't even get anywhere close…So for me knowing what competitors' prices are and knowing what our prices are is absolutely crucial.”", "TM04", "“I think we have learnt that the amount of change that we've got means we need more resource in the sense of in head office, in the sort of senior (top) management group…So where we were previously doing the same thing in lots of different places you could have a fairly light touch management.”", "TR01", "“We lost XXX (Service B), we got things wrong in XXX (Service C).", "We've had to spread our boundaries…a year ago we weren't in XXX (Service D — 2% of turnover), we weren't in XXX (Service E — 4% of turnover), we weren't in XXX (Service F — 19% of turnover)…the committee (board of directors) has certainly become much more aware of it's a new world.”", "TR02", "“The sense I've got is we're more aware of, as it were, fragility.", "I think we're far more in a competitive market.", "I think that's had a rocky start on various occasions, but we've won some large contracts and lost some large contracts as well.", "I think we've learnt pretty rapidly.”", "MM01", "“Learning from mistakes, learning from our losses.", "XXX (Service B) was the other big thing…this time last year that people were recovering from and then we had the XXX (Service A) change…XXX (Service B), XXX (Service A) and XXX (Service C), that's a huge learning curve in one year.”", "MM03", "In summary, the crisis at the organization caused by a reduction in funding and changes to competitive tendering meant that ‘Health Change UK’ was questioning its financial viability.", "The commissioners supplying the contracts were seen as increasingly important so the leadership was finding out what was needed to be successful in a new competitive landscape.", "By phase 2 of the data collection the leadership had learned about its new competitors and had taken steps to improve its service quality.", "They had also become much more effective at competitive tendering.", "Consequently, the organization had retained some contracts and added some new ones.", "The sensemaking and learning that occurred over time resulted in increased cognitive consensus by phase 2.", "We used another form of analysis to compare the leadership as a whole over the two phases.", "The analysis listed in Table 3 demonstrates the number of mapped factors and the number of links between the factors within each cognitive map.", "Additionally, the link density for mapped nodes shows the number of links divided by the number of mapped factors (see Eden, Ackermann, & Cropper, 1992).", "Similarly, the absolute link strength density for mapped nodes exhibits the sum of all link strengths divided by the number of mapped factors (see Langfield-Smith & Wirth, 1992).", "Map Density for Mapped Nodes — The number of links divided by the theoretical number of maximum links between mapped factors (number of mapped factors ∗ number of mapped factors − 1) (see Hart, 1976).", "These scores are important to investigate issues such as cognitive complexity and focus within cognitive maps.", "Table 3 indicates a reduction in cognitive complexity as cognitive consensus emerged.", "Of particular significance is the reduction in the number of links between factors important for success and the reduction in link strength.", "The reduction in these two measures suggests that the leadership as a whole became much more focused on fewer cause–effect relationships when managing their organization in the later phase of sensemaking when attempting to resolve the crisis.", "In this second phase the leaders became less focused on external issues and more focused on internal issues as their response to the crisis took shape.", "Additionally, the movement towards cognitive consensus over the course of the crisis is further supported in Table 4.", "Table 4 presents the data of comparing the similarities and differences in individual cognitive maps (Markóczy & Goldberg, 1995) 18 months later.", "While there are still some high scores for cognitive differences there is also some movement to cognitive consensus.", "For example, the difference between TM02 and TM03 has moved from maximum distance value of 1.00 in phase 1 to a value of 0.541 in phase 2 indicating a considerable movement to cognitive consensus and agreement on some factors for achieving success.", "In diagrammatic terms the dimensionality (distance ratio) has narrowed in the cognitive maps of the leadership as a whole as can be seen in Fig. 2.", "In summary, there was an expectation, but based on limited prior longitudinal cognitive research, that we would find a narrowing in the dimensionality in common space maps indicating more cognitive consensus due to sensemaking over time as the crisis progressed (Figs. 1 and 2 taken together highlight this to an extent).", "The findings, therefore, support Hypothesis 2 to some extent.", "However, while consensus increased slightly in the leadership as a whole, much more consensus was evident within the three leadership teams as the responses to the crisis developed.", "This finding is contrary to Markóczy (2001) who found that there was no increased consensus within the top management team when studying the development of cognitive consensus within individual firms as they responded to change.", "We found an increase in the similarities of objectives and strategies (or means) to achieve them as well as other cause–effect beliefs in the cognitive maps of the leaders within each well-defined leadership team.", "Cognitive diversity was reduced within the top management team, within trustees (non-executive directors) and within middle managers (regional leaders).", "As our findings run contrary to prior research we discuss the possible explanations for our findings and their implications in the discussion section below.", "We have already discussed some of the changes to the cognitive maps of the leaders over time (18 months), such as a movement to cognitive consensus on the main objectives (see Table 2 and discussion).", "However, we have not compared each complete cognitive map for the leaders from phases 1 and 2.", "Table 5 provides the values for each individual's map when each map is compared between phase 1 and phase 2 using the same MDS technique.", "The data indicate that large changes have occurred to the cognitive maps of nearly all the leaders in the organization.", "Only two middle managers (regional leaders MM01 and MM05) show low scores for cognitive differences and therefore only the cognitive maps of these two leaders were relatively stable between phases 1 and 2.", "The findings, therefore, support Hypothesis 3.", "Before outlining the conclusions and discussing the possible fruitful areas for future research arising from the present study we first discuss the limitations.", "The findings are based on a single in-depth case study when leaders confront a single crisis.", "Therefore, the study offers little in the way of generalization.", "A limitation of case study research is that it allows the findings to be used for theory building or discussed in relation to theory only (analytical generalization).", "This limitation, however, is balanced by the advantage that case study method can provide a deep understanding of leaders' cognition under crisis in a naturalistic setting.", "As the context is so important for understanding leaders' cognition under crises we feel that case study research has a lot to offer this emerging domain.", "The use of causal cognitive mapping is a technique with known limitations.", "Hodgkinson and Sparrow (2002), for example, report that the type of errors to be expected is related to the technique used to generate the maps.", "The hand drawn mapping technique used in this study as a modification to the standard method outlined by Markóczy and Goldberg (1995) has advantages and limitations.", "The main advantage is that the respondents draw the maps themselves in real time which is likely to be more accurate than post hoc drawing by the researcher.", "The main limitation is that the hand drawn method puts particular demands on the respondents' recall memory and is therefore likely to provide some inaccuracies in the representation of individuals' beliefs.", "It is difficult to determine whether all the changes to cognitive maps represent learning how to respond to the crisis as the basis of vision formation or whether changes to maps highlight initial errors of judgment.", "We suggest that future research could tackle this issue by additional data collection using an experimental research phase.", "Experimental research methods are well developed in management cognition research and these methods could be used in conjunction with the data collection techniques used in this study, such as cognitive mapping, to explore the influence of errors in more detail.", "Limited changes to cognitive maps could highlight a time delay which is a form of error identified as contributing to inertia in prior research (Barr et al., 1992).", "The longitudinal research reported in this current study included two phases of data collection separated by 18 months.", "There are some advantages to this research design, such as allowing enough time for changes to take place but without experiencing a significant number of respondents leaving the organization.", "However, we note that further data collection phases would have been beneficial.", "Our findings point to the slow development in cognitive consensus towards a prescriptive mental model for the future.", "Therefore, additional data collection phases would likely have contributed to additional insights into this slow development.", "We note that additional phases of data collection could also have contributed to further understanding of how much cognitive consensus is likely within the leadership as a whole when responding to a crisis.", "We did not focus on the communication of vision in statements from leaders to followers.", "Analysis of documentary evidence and attending staff meetings, in conjunction with cognitive mapping of both leaders and followers, may help in understanding the articulation of a prescriptive mental model for the future in more detail.", "Currently, we do not know how many of the ‘new’ objectives and cause–effect beliefs were articulated to followers and in what form.", "Given the cognitive diversity between the leadership as a whole, perhaps the CEO articulated a single vision based on one way forward (their own) to resolve the crisis, or possibly delayed articulation due to this lack of consensus.", "Modifying the research design to include additional data would help clarify the links between leaders' mental models and the articulation of a vision to resolve a crisis.", "When considering the main contributions emerging from our empirical study we highlight the following.", "First, prior longitudinal research on leaders' cognition largely assumes consensus because evidence is drawn from organizational level documentary evidence (e.g. Barr et al., 1992).", "This approach has several weaknesses.", "It underplays the importance of sensemaking in individuals and ignores individual differences that can occur within teams of leaders.", "An understanding of cognitive diversity and consensus within the leadership is important for a deep understanding of organizational responses to disruptive change and crises.", "Given the findings, assuming consensus in leaders within firms may be regarded as an assumption too far.", "Second, the findings point to less cognitive diversity and more cognitive consensus within leaders as sensemaking occurs over time.", "Evidence for an emerging viable prescriptive mental model for the future was found but in a way not expected from a review of prior theory (Mumford et al., 2007) or empirical findings (Markóczy, 2001).", "Cognitive consensus occurred within well-defined leadership teams much more than within the leadership as a whole.", "The findings point to emerging cognitive sub-cultures of leadership when facing a crisis based on converging beliefs within leadership teams but different beliefs between leadership teams even in a single organization.", "The findings therefore lead to an additional hypothesis to be investigated by further research:", "When responding to a crisis, cognitive consensus around a viable prescriptive mental model for the future will initially emerge within well-defined leadership teams.", "Third, we contribute data and analysis to compare with the theoretically grounded conceptual model presented by Mumford et al. (2007) for investigating leaders' cognition under conditions of organizational crisis and change.", "Our data did not attempt to test or address the complete conceptual model but the data allows some theoretical generalization by comparing our data with theory.", "Our findings focus on two parts of the conceptual model only; the activation of descriptive mental models and the development of a viable prescriptive mental model for the future.", "When we compare our findings to the conceptual model presented by Mumford et al. (2007) of particular significance is the considerable cognitive diversity within leaders as a whole when developing a viable prescriptive mental model for the future.", "Given the time lapse since the crisis first became apparent (well over 2 years before phase 2 of data collection) there were still considerable differences in beliefs in how to achieve success within the new competitive landscape.", "Considering that a number of successful changes had been implemented to deal with the crisis before stage 2 of the data collection, we find this lack of consensus surprising.", "The data implies that consensus over a viable prescriptive mental model occurs initially within teams of leaders but is problematic between teams and there is little cognitive consensus when considering the leadership as a whole.", "Another possible explanation for our findings is that our data needs to take an even longer-term perspective on the development of a prescriptive mental model within the leadership as a whole.", "When comparing the data with the discussion of the conceptual model presented by Mumford et al. (2007), we confirm the importance of experiential and tacit knowledge to problem solving when making sense of new conditions and the development of a viable prescriptive mental model for the future.", "Of particular importance to the leaders in our case study organization was learning about new competitors, and the services they were offering, as well as the new reality of competitive tendering.", "Learning how contract commissioners (fund holders) evaluated tenders in respect to competitors was key to this latter issue.", "Experiential knowledge, based on both successes and failures in tendering for contracts, led to an improved tendering process.", "These improvements enabled the organization to obtain more new contracts and become more financially viable.", "An improved success rate in competitive tendering resulted in four new contracts equating to 30.8% of turnover following the original contract losses of 27.2% of turnover.", "Fourth, the findings also confirm that it was not the identification of the crisis or change that was problematic for the leaders but learning how to operate through trial and error in a new competitive landscape (see Barr et al., 1992).", "The two important issues of note are the increased significance of service quality and the importance of staff motivation.", "The focus on these two issues is consistent with some of the problems faced by the organization as a result of increased low price competition after government changes.", "The organization had to improve its service delivery to justify its higher prices.", "Fifth, the findings also confirm that studying the locus of consensus is particularly important when understanding sensemaking and an organizational response to change (Markóczy, 2001).", "Consensus is particularly important in leadership teams (Yammarino, Mumford, Connelly, & Dionne, 2010).", "In contrast to Markóczy (2001), however, we found emerging consensus in all leadership teams within the organization as the response to the crisis developed.", "This finding can be explained by the focus on a specific crisis in the current study.", "Markóczy (2001) found consensus within a specific interest group that benefited from change, but when in crisis the implications for any specific interest group is likely to be unclear.", "When responding to more benign and gradual change, an interest group is more likely to develop because they can see the benefits of change more clearly and this might explain the different findings in this case.", "To add to deeper understanding future research should analyze the interaction within leadership teams to help understand more fully how cognitive consensus is formed.", "Interaction within the leadership teams as well as with external stakeholders could influence individuals to change their mental models.", "Communication between team members, both formally and informally, is likely to facilitate cross understanding and this is a possible explanation for our findings (see Huber & Lewis, 2010).", "We did not investigate communication between leaders and the influence of dominant leaders on others so this may be a fruitful field of research for the future.", "Further studies could also look into the role of followers (other employees) in this context because of the differences found between leadership teams.", "It would be interesting to see if followers recognized the differences in the mental models within different leadership teams as this may influence how they responded.", "Given our findings, an interesting question to investigate would be: did followers agree with the analysis and future objectives put forward to resolve the crisis by one leadership team more than another?", "Another possible fruitful field of research is into the influence of emerging consensus on individual performance, team performance, and/or organizational performance.", "This research option is particularly important due to the cognitive differences found within individuals and leadership teams in our study.", "Finally, future research could be directed at the antecedents to any change and lack of change in the mental models of leaders as they respond to a crisis.", "Mumford and Strange (2002) and Strange and Mumford (2002) suggest an explanation for a lack of change to mental models based on the style of leadership.", "Ideological leaders may be more likely to be consistent in their thinking even when responding to a crisis so future research could explore this possible effect." ]
[]
The integrated stress response system in cardiovascular disease
The integrated stress response system represents an ancillary, extremely conserved signalling pathway present in virtually all eukaryotic cells, which plays an important part in the pathophysiology of several disorders such as cancer and neurodegeneration. However, its role in the cardiovascular system remains largely elusive. Hence, this review aims to acknowledge recent findings regarding the action of the eIF2α kinases in the cardiovascular system and their role in the pathophysiology of related disorders.
[ "PHAR" ]
[]
[ "3002", "3004" ]
[ "The integrated stress response (ISR) system is one of the most important signalling pathways involved in the cellular adaptation to stress, and it is presumed to exist in all eukaryotic cells [1].", "It consists of four serine/threonine kinases, namely general control nonderepressible (GCN)2, heme-regulated inhibitor (HRI), PKR-like endoplasmic reticulum kinase (PERK) and protein kinase double-stranded RNA-dependent (PKR), that, when activated, owing to either extrinsic or intrinsic stressors, phosphorylate the α subunit of eukaryotic translation initiation factor 2 (eIF2α) at Ser51, leading to an immediate cellular gene expression reprogramming to overcome the imposed stress and restore normal cell function [2].", "Each eIF2α kinase senses and responds to distinct cellular stresses, and some overlap of their activities does exist [3].", "These kinases share great homology, albeit some differences in their regulatory domains, and, upon a stressful stimulus, they need to dimerize and undergo autophosphorylation to become fully functional and to phosphorylate eIF2α [4].", "The phosphorylation of eIF2α results in the inhibition of eIF2α-GTP recycling, which is necessary for the initiation of mRNA translation, reducing overall translation, while selectively favouring the translation of proteins implicated in stress recovery, such as activating transcriptional factor (ATF)4 [5].", "This key factor can induce the expression of several genes involved in the regulation of apoptosis, metabolism, redox balance, amino acid (AA) biosynthesis and transport, by forming homo- and/or hetero-dimers that bind to specific DNA regions designated as the C/EBP-ATF response element (CARE) [4].", "According to the nature of the stimulus that triggered eIF2α phosphorylation, as well as its intensity and duration, the gene reprogramming can help to promote cell survival or induce cell death [1].", "Proper termination of the ISR signalling is essential, because it enables cells to reactivate protein synthesis and return to normal function.", "eIF2α dephosphorylation is mediated by the association of protein phosphatase (PP)1 and its catalytic subunit PP1c to either growth arrest and DNA-damage-inducible protein (GADD34), which is induced downstream of ISR signalling, or to constitutive repressor of eIF2α phosphorylation (CReP), which exists at basal levels even under unstressed conditions and helps to maintain low levels of phosphorylated eIF2α [4] (Fig. 1).", "Since the discovery of these kinases and their implication in cellular stress adaptation and fate-decision, they have been increasingly studied in the context of several disorders, such as respiratory [6], metabolic [7], tumoral [8] and neurodegenerative diseases [9].", "However, their role in the cardiovascular system remains largely unexplored.", "The scope of this review is therefore not to detail the structure and mechanics of the eIF2α kinases, extensively reviewed elsewhere [2], but rather to overview and explore the current knowledge on eIF2α kinases in the cardiovascular function and pathophysiology.", "GCN2, also called eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4), is a protein kinase that was initially discovered as being responsible for the detection and resolution of AA starvation in yeast [10].", "However, it is currently acknowledged that GCN2 is also capable of detecting and answering to several other types of stimuli, such as hypoxia [11], DNA damage [12], ultraviolet radiation [13], among others [1], although the mechanisms by which they are able to activate GCN2 remain unknown.", "It has been speculated that other stresses, aside from AA unavailability, might end up disturbing the AA pool of the cell, eventually leading to GCN2 activation through its canonical pathway [4].", "During periods of AA deprivation, the cellular levels of uncharged transfer RNAs (tRNAs) increase, and GCN2 can sense and bind to these tRNAs through its histidyl-tRNA synthetase (HisRS)-like and C-terminal domains, undergoing autophosphorylation at Thr898/Thr903 leading to the activation of its protein kinase catalytic domain and subsequent phosphorylation of eIF2α [10].", "Recently, GCN2 was also found to be present in the cell nucleus where it interacts with small RNA transcripts.", "GCN2 was able to modulate polymerase-III-mediated RNA transcription in the nucleolus to repress the excessive production of small RNA during AA shortage situations, inhibiting excessive protein translation [14].", "HRI, also termed eIF2α kinase 1 (EIF2AK1), is a protein mainly expressed in erythrocytes, unlike the other eIF2α kinases that are widely expressed, that can detect low heme (an iron-coupled co-factor present in haemoglobin) levels and can promote the survival of erythroid precursors.", "HRI is also able to coordinate erythropoiesis and the production of globin chains according to heme availability [2], and it can regulate macrophage maturation and iron homeostasis, although its exact role in this setting remains elusive [15].", "HRI possesses two heme-binding domains and, upon periods of heme deprivation, they dissociate from HRI, enabling its autophosphorylation and activation [16], resulting in eIF2α phosphorylation and protein synthesis inhibition.", "HRI is thus able to reduce globin aggregate formation, thereby protecting erythrocytes from its toxicity [4].", "PERK or eIF2α kinase 3 (EIF2AK3), in contrast to the other eIF2α kinases, is a transmembrane protein present in the endoplasmic reticulum (ER) that is able to sense and promote adaptation in situations of ER stress, typically caused by accumulation of unfolded or misfolded proteins in the ER lumen [4].", "PERK normally interacts with (and is kept latent by) a chaperone protein called 78 kDa glucose-regulated protein (GRP78).", "ER stress triggers the dissociation between GRP78 and PERK, allowing dimerization of the latter and subsequent autophosphorylation [2].", "When activated, PERK phosphorylates eIF2α, thus inhibiting the synthesis of new peptides and reducing their entry into the ER lumen [17].", "PERK is also a sensor involved in the unfolded protein response (UPR) system, which is sensitive to protein-folding alterations and ER stress [4].", "PKR, also called eIF2α kinase 2 (EIF2AK2), is a protein kinase that responds to viral infection by sensing double-stranded RNA, further blocking the translation of viral mRNAs and inducing cell apoptosis.", "This kinase can also be activated in a dsRNA-independent manner by other stimuli, such as oxidative and ER stress, or bacterial infection, through unknown mechanisms [4].", "Cardiovascular diseases remain a leading cause of death worldwide, and therefore research for further therapies is more important than ever [18].", "Major advances in the cardiovascular area are in part dependent on the discovery of new signalling pathways that contribute to the development of these pathologies.", "Increasing numbers of studies have been targeting the role of ISR in the stressed cardiovascular system, however its complex actions need to be further dissected.", "Given its important role in AA homeostasis, GCN2 has been widely studied in the setting of dietary restriction and associated benefits, and whether these either are or are not mediated by GCN2.", "Dietary restriction is not only associated with extended lifespan in numerous organisms but also with increased resistance to different forms of stress [19].", "In the context of cardiovascular diseases, dietary restriction promotes antiapoptotic and anti-inflammatory effects in the myocardium of murine models after ischemia/reperfusion (I/R) injury [20,21].", "In hypertensive patients [22] and mice [23], caloric restriction can improve endothelial-dependent vasodilation through maintenance of nitric oxide (NO) availability, while preventing cardiac-overload-induced hypertrophy [24].", "Although other signalling pathways have been implicated in these beneficial effects, such as mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK), GCN2 represents an important mediator for AA homeostasis [25,26], working in concert with mTOR and AMPK to regulate energy and protein metabolism according to nutritional demands [27] (Fig. 2).", "A link between chemotherapy exposure, especially alkylating agents, and pulmonary veno-occlusive disease (PVOD) development in humans has been established [28,29].", "Subsequently, an experimental animal model of PVOD induced by the chemotherapeutic agent mitomycin C was created [30].", "In addition to developing high pulmonary arterial pressures, these animals also displayed pulmonary vascular lesions mimicking the human pathology.", "Interestingly, the lungs of these animals showed decreased GCN2 expression that was proportional to the dose of mitomycin and to the severity of pulmonary hypertension (PH) [30].", "Moreover, humans with recessive biallelic mutations in the GCN2 gene were found to develop hereditary PVOD [31], a rare and venous-focused cause of PH [32].", "All mutations, either in a homozygous or compound-heterozygous state, disrupted the function of the gene.", "Still, the cause–effect relationship between mutations in the GCN2 gene and the development of PVOD remains to be identified [31].", "Considering that hypoxia is an important trigger for the development of PH [33], and that GCN2 and PERK are subsequently activated in response to hypoxia [11], one might hypothesise that one of the missing links between GCN2 and PH development could involve hypoxia-inducible factor (HIF)-1α regulation, a central mediator in response to hypoxia and pulmonary vascular remodelling [34].", "In fact, Liu et al. hypothesised that low levels of phosphorylated eIF2α trigger cell apoptosis by activating p53 and inhibiting cell cycle arrest, whereas high levels of phosphorylated eIF2α favour adaptation by promoting G1 arrest, revealing that different outcomes can arise upon ISR activation according to the intensity of the stress [11].", "Apart from activating HIF-1α, hypoxia can also activate PERK/eIF2α signalling to inhibit global protein synthesis and reduce energy consumption [35].", "However, Papadakis et al. showed that PKR was able to suppress HIF-1α transcription independently of eIF2α phosphorylation by impairing signal transducer and activator of transcription (STAT)3 activity through the activation of the tyrosine phosphatase TC-PTP.", "Inhibition of STAT3 is sufficient to decrease HIF-1α transcription and dampen the expression of the targeted genes of HIF-1α, inhibiting the accumulation of HIF-1α in hypoxic cells [36] (Fig. 3).", "ER stress is implicated in the progression of systemic [37] and pulmonary [38] hypertension.", "In the former, not only PERK but also activating transcription factor (ATF)6 and inositol-requiring enzyme (IRE)1 (sensors of the UPR) contribute to the abnormal vascular function in spontaneously hypertensive rats [37].", "By contrast, only ATF6 and associated downstream targets (and not PERK directly) are involved in PH [39,40].", "However, recent findings also position PERK and IRE1 in the picture, suggesting that all the three axes of the UPR are involved in the pathogenesis of PH, presumably through the modulation of cell proliferation and inflammation [38].", "The levels of endothelin (ET)-1, another key mediator in vascular homeostasis, are normally elevated in several cardiovascular diseases, such as chronic heart failure (HF) [41] and PH [42].", "Recently, ATF4 was pointed to as a novel regulator of ET-1 transcription in human microvascular endothelial cells.", "ATF4-dependent induction of ET-1 is triggered by PERK and PKR activation, which subsequently stimulate the eIF2α/ATF4 signalling pathway.", "ATF4 and c-JUN form complexes that interact with the activator protein (AP)1 response element to initiate ET-1 transcription [43].", "At high concentrations, ET-1 is not only a potent vasoconstrictor but also a stimulator of inflammation and fibrosis, which might contribute to disease progression [44].", "ER stress has also been implicated in myocyte apoptosis in the failing heart.", "After a few days, mice subjected to transverse aortic constriction showed a significant increase in cardiac ER chaperones that was accompanied by an increase in myocyte apoptosis.", "These effects were mediated by CHOP-, JNK- and caspase-12-dependent signalling pathways [45].", "Recently, AGGF1 (an angiogenic factor with a G-patch domain and a forkhead-associated domain 1) was found to counteract the negative effects of ER stress through the activation of a noncanonical pathway involving downstream activation of the p-eIF2α/ATF4 axis, while concomitantly inhibiting the expression of sXBP1 and CHOP, its main apoptotic inducers [46].", "Cardiovascular disorders are often associated with comorbidities such as diabetes, anaemia, obesity, renal dysfunction and local or systemic inflammation [47].", "In this context, Jung and colleagues described that the GCN2/eIF2α/ATF4 signalling pathway was able to increase the levels of fibroblast growth factor (FGF)21 in the skeletal muscle cells of diet-induced HF rats.", "FGF21 is known to have antidiabetic, antiobesity and anti-inflammatory effects in animals with HF and associated diseases [48], but in this study the interference with the GCN2 pathway resulted in reduced levels of FGF21, wearying its beneficial effects [49].", "In an experimental rat model of diabetic cardiomyopathy, one of the most common complications of diabetes, PERK was identified as mediating the myocardial susceptibility to arrhythmias.", "This prejudicious effect seems related to the fact that PERK interacts directly with calcineurin to regulate ryanodine receptor (RyR)2 opening, thus leading to an abnormal increase in intracellular calcium and impairing the normal electrical functioning of the heart [50] (Fig. 4).", "Recent studies showed that PERK is responsible for reactive oxygen species (ROS)- [51] and hypoxia/reoxygenation-triggered [52] ER stress-induced cardiomyocyte apoptosis, because cardiac cells deficient in PERK showed greater protection against cell death.", "Furthermore, in animal models subjected to myocardial I/R injury, downregulation of the PERK/eIF2α/ATF4 signalling pathway correlates with cardioprotective features, such as antiapoptotic and antioxidative effects as well as decreased infarct size and improved cardiac function [53,54].", "Iron deficiency affects one-third of people worldwide, and has been notably linked with coronary heart disease and atherosclerosis [55].", "Iron is a very important constituent of heme and the levels of the latter are in part regulated by HRI.", "Indeed, Liu et al. showed that HRI confers cellular adaptation upon iron deficiency, through activation of an adequate gene expression and maintenance of Gata-binding protein (Gata)1/friend of Gata1 (Fog1) – key regulators of erythropoiesis [16] (Fig. 5).", "High levels of NO are able to induce eIF2α phosphorylation, possibly via HRI activation, although the precise mechanism by which NO activates HRI remains partially unknown [56].", "Indeed, NO appears to be able to bind directly to the N-terminal heme-binding domain of HRI and to initiate its catalytic activity.", "NO is a key mediator in several physiological processes and extensively studied in the cardiovascular setting.", "Its reduced availability is a central abnormality in several cardiovascular disorders [57].", "Therefore, even though HRI is mainly expressed in erythrocytes, the connection established between NO and HRI activation warrants further research, specifically the possible effects of the HRI kinase on the cardiovascular system.", "Further research is needed to assess the cardiovascular implications of HRI activation, keeping in mind that HRI is not expressed in the heart [58] but could indirectly exert beneficial or harmful effects on the cardiovascular system.", "Several reports have already shown the effects of ISR knock-down in rodents and the effects on the cardiovascular system upon a stressful stimulus.", "Under normal conditions, knockout (KO) mice for the ISR genes (individually) are fertile and display a normal development.", "All of them present a normal cardiac structure and function under normal conditions [58–61].", "When submitted to transverse aortic constriction, GCN2−/− mice are less prone to ventricular dysfunction, myocardial apoptosis and fibrosis, as well as to lung congestion, inflammation and remodelling, when compared with wild-type mice [60].", "Likewise, PKR−/− mice show preserved left ventricular contractility along with reduced myocardial fibrosis and pulmonary congestion when submitted to pressure overload, when compared with wild type.", "Although the hypertrophy level was similar between KO and wild-type mice, the former also showed decreased inflammation and reduced expression of proapoptotic factors [59].", "By contrast, cardiac-specific PERK KO mice show impaired systolic function, increased myocardial fibrosis and apoptosis, as well as lung remodelling (increased lung weight) when submitted to transverse aortic constriction and when compared with wild-type mice [61].", "Contrarily to GCN2 and PKR, the absence of PERK is deleterious in the overloaded heart.", "HRI−/− mice display a normal development, except for mild haematological abnormalities such as macrocytosis (increased erythrocyte volume) and hyperchromia (increased haemoglobin concentration).", "Surprisingly, in these mice, iron deficiency induces a hyperchromic and normocytic anaemia, leading to cardiomegaly [58], instead of following the normal adaptive response seen in mammals, which is a decreased red blood cell (RBC) volume and haemoglobin content – in other words a microcytic and hypochromic anaemia.", "Taking all these studies into account, one can acknowledge that dysregulation of ISR signalling can lead to pathologic consequences at the cardiovascular level.", "Hence, individual pharmacological modulation of the eIF2α kinases, as well as eIF2α itself, might represent a promising therapeutic approach to cardiovascular diseases.", "Several drugs have been described to act either by activating or inhibiting the eIF2α kinases, therefore controlling the eIF2α phosphorylation state or by acting on the proteins that dephosphorylate eIF2α.", "To discriminate between the specific consequences of eIF2α phosphorylation and ISR gene activation from other stress pathways, Lu et al. created an artificial PERK, in which the stress-sensing domain was exchanged by a modified FKBP domain (Fv2E).", "Fv2E-PERK does not respond to stress but is rather receptive to a small synthetic compound called AP20187.", "Because its activation is drug-induced only, one can dissect the specific effects of eIF2α phosphorylation from other signalling pathways that are also induced upon stress [62].", "This very valuable tool should be extended to the other eIF2α kinases to clarify the effects of ISR components.", "GCN2 can be stimulated by the action of histidinol, halofuginone, arginine deiminase and asparaginase [4], although only the first two have been described in the cardiovascular setting.", "Histidinol, a precursor of the histidine biosynthetic pathway, in co-treatment with doxorubicin, is able to boost its antitumoral effect in mice, while not increasing the associated cardiotoxicity and lethality [63].", "In a rat model of balloon-injured carotid arteries, local administration of halofuginone, an alkaloid obtained from Dichroa febrifuga, promotes an adaptive remodelling and selectively prevents neointima growth without inhibiting the reconstitution of the endothelial layer [64].", "In vitro, Nagler et al. showed that vascular endothelial cells (ECs) are less susceptible to the inhibitory effect of halofuginone than vascular smooth muscle cells (SMCs).", "Indeed, by inhibiting the synthesis of collagen type α1, halofuginone can prevent the alteration of the SMC phenotype from a contractile to a synthetic one, which is promoted by this type of collagen [65].", "In the cardiac setting, in an animal model of virus-induced myocarditis, halofuginone treatment was able to suppress cardiac inflammation by decreasing T helper 17 cells and inhibiting the transforming growth factor (TGF)-β1 signalling pathway [66].", "When considering PERK, because several diseases like cancer and neurodegeneration have been associated with its overexpression, effort has been made to find new inhibitors rather than activators [67].", "One of the first PERK-specific inhibitors that was discovered was GSK2606414 [68] which successfully inhibited PERK activation in vitro and in vivo in a human tumour xenograft model.", "In an attempt to improve its pharmacokinetics by diminishing its lipophilicity, another inhibitor designated as GSK2656157 was developed [69].", "In several studies in the field of oncology [68,69] and neurodegeneration [70], PERK inhibition showed beneficial effects, such as tumour growth arrest, sensitisation to chemotherapy and inhibition of tau phosphorylation [67].", "Other PERK inhibitors have been developed by other companies but limited data are available and further research is needed to assess the properties of these molecules [67].", "So far, only one specific activator has been discovered and characterised: CCT020312, and, in contrast to most of the studies mentioned above, activation of PERK showed potential benefits in tumoral and neurodegenerative pathologies [67].", "Besides these studies and discrepancies, none of these compounds has yet been studied in the setting of cardiovascular diseases, where one can expect that modulation of PERK might be helpful, for example in situations of post myocardial infarction or hypertension, owing to the ability of PERK to control cellular proliferation and apoptosis as well as the inflammatory response [37,54].", "Regarding the inhibitors of eIF2α dephosphorylation, salubrinal, an inhibitor of PP1, can inhibit eIF2α dephosphorylation by impairing the formation of the GADD34–PP1 and CReP–PP1 complexes, therefore promoting prolonged downstream signalling [4].", "In an animal model of pressure-overload-induced cardiac hypertrophy, salubrinal treatment could attenuate cardiac hypertrophy, fibrosis and apoptosis with associated improvement of overall cardiac function.", "These beneficial effects seemed to be primarily linked to a cytoprotective effect against ER stress [71].", "Salubrinal treatment also protects cardiomyocytes against apoptosis during myocardial infarction (MI).", "Administration of salubrinal before MI injury was able to enhance eIF2α phosphorylation and decrease the infarction site [72].", "In an in vitro model of doxorubicin-induced cardiac toxicity [73] and tunicamycin- and hypoxia-induced apoptosis [74], salubrinal protected primary cultured rat cardiomyocytes against apoptosis and decreased ROS production in a p-eIF2α-dependent manner.", "In the vascular setting, salubrinal administration attenuated the development of PH in rats exposed to chronic hypoxia.", "The treated group showed decreased mean pulmonary artery pressure (mPAP), right ventricular systolic pressure (RVSP), right ventricular and vascular remodelling, as well as decreased levels of ET-1 and oxidative stress [75].", "It has also been suggested that activation of UPR signalling is involved in early PH, by contributing to an exacerbated inflammation and vascular remodelling.", "Indeed, in a rat model of severe PH induced by monocrotaline exposure, salubrinal treatment was able to attenuate well-established PH by decreasing macrophage recruitment and thus reducing the inflammatory environment [40].", "Although these studies show a beneficial role for salubrinal in several backgrounds, attention must be paid to the adverse effects that might occur owing to continuous ISR stimulation and improper termination of this pathway, making it impossible for cells to restore their normal function.", "Guanabenz, an α2-adrenergic receptor agonist and GADD34–PP1c inhibitor, can grant protection against tunicamycin-induced ER stress in rat cardiac myocytes.", "Guanabenz treatment was able to antagonise the effect of tunicamycin on the expression of ER stress markers such as IRE1α, GRP78 and CHOP [CCAAT-enhancer-binding protein (C/EBP) homologous protein], while mildly prolonging eIF2α phosphorylation [76].", "Further studies are needed to better understand the dual role of guanabenz on the inhibition of signalling while maintaining eIF2α activation.", "Owing to its role on the α2-adrenergic receptors and possible adverse effects on the cardiovascular system as a result of its activation, there is a need to find specific GADD34 inhibitors.", "Sephin1, a guanabenz derivative, specifically binds to GADD34 but not to CReP, therefore enabling prolonged eIF2α phosphorylation only in stressful situations.", "As a consequence, sephin1 can attenuate the expression of stress genes, as long as eIF2α remains phosphorylated and the protein translation remains inhibited [77].", "Conversely, Crespillo-Casado et al. found no effect for sephin1 in in vitro enzymatic assays dependent on the formation of the binary complex GADD34-PP1, nor any interference with the p-eIF2α dephosphorylation state in cells.", "This study alerts to the possibility that other compounds might exist that selectively target p-eIF2α dephosphorylation [78].", "Mice prone to developing medulloblastoma (Ptch1+/−) were crossed with GADD34−/− mice to obtain heterozygous mice for the GADD34 gene.", "Although none of the mutations (GADD34+/− or GADD34−/−) had any effect on medulloblastoma cells in the adult mice, the young GADD34+/− mice displayed an increased number of hyperplastic lesions and increased incidence of medulloblastoma.", "By contrast, young GADD34−/− mice showed reduced lesions owing to an increase in pre-malignant cell apoptosis, therefore reducing overall medulloblastoma incidence [79].", "This study shows the dual role that the ISR signalling can assume according to the intensity of activation.", "The question remains whether the modulation of ISR through GADD34 inhibition would be beneficial in proliferative disorders that affect the cardiovascular system.", "Although not yet explored in the cardiovascular setting, nelfinavir, a protease inhibitor used in the treatment of HIV patients, has been shown to be able to decrease HIF-1α/vascular endothelial growth factor (VEGF) expression in normoxic and hypoxic conditions.", "Through HIF-1α dampening, nelfinavir can also decrease VEGF transcription (VEGF promotor is regulated by HIF-1α) and therefore attenuate angiogenesis [80], which could be an important feature in vascular diseases and in MI.", "Nelfinavir downregulates CReP levels leading to the maintenance of the phosphorylation state of eIF2α and to a perpetuation of ISR signalling downstream [4].", "Inhibitors of ISR signalling have also been used and proved to have some therapeutic effects.", "Some studies have described the beneficial role of indirubin-3′-monoxime in the inhibition of vascular SMC abnormal proliferation and migration.", "Indirubin-3′-monoxime is a potent glycogen synthase kinase (GSK)-3β inhibitor that was also shown to inhibit GCN2, although not specifically [4].", "In a femoral artery cuff model [81], local application of indirubin-3′-monoxime inhibited DNA synthesis in platelet-derived growth factor (PDGF)-activated SMCs and reduced neointima formation.", "These effects seemed to be mediated by STAT3 inhibition.", "In another study, primary rat aortic SMCs submitted to indirubin-3′-monoxime treatment also showed a reduced proliferative capacity that was associated with an impairment of glycolysis via inhibition of STAT3/hexokinase (HK2) signalling [82].", "Further studies are required to elucidate whether part of these effects occur as a result of eIF2α signalling dampening or not.", "PKR inhibition through 2-aminopurine, an analogue of guanine and adenine, was able to protect mice neonatal cardiomyocytes against H2O2-induced injury by attenuating apoptosis and inflammation [83].", "Many other molecules have been described to inhibit the eIF2α kinases [4] but so far only a few of them have been tested or applied to cardiovascular diseases.", "In brief, the ISR signalling network is crucially involved in the cellular ability to adapt to a wide range of stressful stimuli that can be imposed on cardiac and vascular cells, either in normal or pathological conditions.", "Inhibition of the ISR system might result in an impaired cellular response to stress, whereas its activation leads to an immediate cellular gene expression reprogramming to overcome the imposed stress and restore normal cell function.", "By contrast, overstimulation of this pathway can also lead to deleterious effects, such as cell death.", "Further studies are needed to better understand the mechanisms that lead to the activation of these eIF2α kinases, specifically in the cardiovascular setting, and the subsequent effects of such activation.", "It is also important to bear in mind that these concerns need to be addressed specifically for each eIF2α kinase, as a unit and as a group, because some overlap between their activities exist.", "Strategies aimed at either suppressing or enhancing ISR signalling need to be tested and understood in the cardiovascular setting, in vitro and in vivo, so ISR-based therapies can be applied in future clinical practice.", "The authors have no conflicts of interest to declare." ]
[ "ISR signalling is a conservative pathway focused on cellular stress response.", "Activation of ISR can be a double-edge sword depending on the underlying conditions.", "The eIF2α kinases are important in cardiac and vascular homeostasis.", "The ISR system might open new therapeutic windows for cardiovascular diseases." ]
Cost and Outcome of Behavioural Activation versus Cognitive Behavioural Therapy for Depression (COBRA): a randomised, controlled, non-inferiority trial
Background Depression is a common, debilitating, and costly disorder. Many patients request psychological therapy, but the best-evidenced therapy—cognitive behavioural therapy (CBT)—is complex and costly. A simpler therapy—behavioural activation (BA)—might be as effective and cheaper than is CBT. We aimed to establish the clinical efficacy and cost-effectiveness of BA compared with CBT for adults with depression. Methods In this randomised, controlled, non-inferiority trial, we recruited adults aged 18 years or older meeting Diagnostic and Statistical Manual of Mental Disorders IV criteria for major depressive disorder from primary care and psychological therapy services in Devon, Durham, and Leeds (UK). We excluded people who were receiving psychological therapy, were alcohol or drug dependent, were acutely suicidal or had attempted suicide in the previous 2 months, or were cognitively impaired, or who had bipolar disorder or psychosis or psychotic symptoms. We randomly assigned participants (1:1) remotely using computer-generated allocation (minimisation used; stratified by depression severity [Patient Health Questionnaire 9 (PHQ-9) score of <19 vs ≥19], antidepressant use, and recruitment site) to BA from junior mental health workers or CBT from psychological therapists. Randomisation done at the Peninsula Clinical Trials Unit was concealed from investigators. Treatment was given open label, but outcome assessors were masked. The primary outcome was depression symptoms according to the PHQ-9 at 12 months. We analysed all those who were randomly allocated and had complete data (modified intention to treat [mITT]) and also all those who were randomly allocated, had complete data, and received at least eight treatment sessions (per protocol [PP]). We analysed safety in the mITT population. The non-inferiority margin was 1·9 PHQ-9 points. This trial is registered with the ISCRTN registry, number ISRCTN27473954. Findings Between Sept 26, 2012, and April 3, 2014, we randomly allocated 221 (50%) participants to BA and 219 (50%) to CBT. 175 (79%) participants were assessable for the primary outcome in the mITT population in the BA group compared with 189 (86%) in the CBT group, whereas 135 (61%) were assessable in the PP population in the BA group compared with 151 (69%) in the CBT group. BA was non-inferior to CBT (mITT: CBT 8·4 PHQ-9 points [SD 7·5], BA 8·4 PHQ-9 points [7·0], mean difference 0·1 PHQ-9 points [95% CI −1·3 to 1·5], p=0·89; PP: CBT 7·9 PHQ-9 points [7·3]; BA 7·8 [6·5], mean difference 0·0 PHQ-9 points [–1·5 to 1·6], p=0·99). Two (1%) non-trial-related deaths (one [1%] multidrug toxicity in the BA group and one [1%] cancer in the CBT group) and 15 depression-related, but not treatment-related, serious adverse events (three in the BA group and 12 in the CBT group) occurred in three [2%] participants in the BA group (two [1%] patients who overdosed and one [1%] who self-harmed) and eight (4%) participants in the CBT group (seven [4%] who overdosed and one [1%] who self-harmed). Interpretation We found that BA, a simpler psychological treatment than CBT, can be delivered by junior mental health workers with less intensive and costly training, with no lesser effect than CBT. Effective psychological therapy for depression can be delivered without the need for costly and highly trained professionals. Funding National Institute for Health Research.
[ "MEDI" ]
[]
[ "2700" ]
[ "Clinical depression is a common and debilitating mental health disorder, being the second largest cause of global disability.1", "Globally, the effect of depression on aggregate economic output is predicted to be US$5·36 trillion between 2011 and 2030.2", "Reduction of these substantial costs is a key objective for low-income, middle-income, and high-income countries alike.", "Antidepressant medication and cognitive behavioural therapy (CBT) have the most clinical evidence.", "However, although antidepressant medications are cheap, their use is limited by side-effects, poor patient adherence, and discontinuation relapse risk.", "CBT is as effective as are antidepressants3 and provides long-term protection against relapse, but it is complex and its effectiveness is dependent on the skills of psychological therapists, who are expensive to train and employ.", "For low-income and middle-income countries in particular, the need for an extensive professional infrastructure of such therapists limits access to CBT.", "Evidence before this study", "Authors of published systematic reviews, including a Cochrane review, have commented on the limitations of existing evidence for the effectiveness of behavioural activation (BA) for depression compared with cognitive behavioural therapy (CBT) and the scarcity of cost-effectiveness data, with the existing evidence insufficiently robust to establish comparability.", "Authors of the Cochrane review called for studies that improve the quality of evidence.", "Our pretrial evidence took published review findings from the UK National Institute for Health and Care Excellence (NICE), who reported no difference in treatment outcome between BA and CBT immediately after treatment (Hedges' g 0·139 [95% CI −0·400 to 0·122]; p=0·296) and subsequent follow-up (0·135 [−0·456 to 0·186]; p=0·409).", "The authors of NICE's review regarded the existing international evidence as insufficient to recommend BA for first-line treatment in clinical guidelines for depression.", "Added value of this study", "This trial addresses these research recommendations and is, to our knowledge, the only high-quality, fully powered non-inferiority and cost-effectiveness study addressing both the effects and costs of BA compared with CBT for depression.", "When we combine the data from our study with data from other international studies in the meta-analysis done by NICE, our data reduce the 95% CIs around the effect size for depression symptoms immediately after treatment (Hedges' g 0·054 [95% CI −0·214 to 0·107]; p=0·514) and at follow-up (0·059 [−0·234 to 0·115]; p=0·503) and unequivocally show both non-inferiority of BA compared with CBT and that BA is more cost-effective than is CBT against commonly applied decision maker willingness-to-pay thresholds.", "Implications of all the available evidence", "Junior mental health workers with no professional training in psychological therapies can deliver behavioural activation, a simple psychological treatment, with no lesser effect than CBT has and at less cost.", "Effective psychological therapy for depression can be delivered without the need for costly and highly trained professionals.", "Globally, health services require effective, easily implemented, and cost-effective psychological treatments for depression that can be delivered by less specialist health workers than are needed at present to close a treatment gap that can be as much as 80–90% in some low-income countries.4", "One potential alternative, behavioural activation (BA), is a simple psychological treatment for depression.", "It might be easy and quick to train junior mental health workers (MHWs) in BA who have no professional training in psychological therapies.5", "However, this method is only appropriate if BA delivered in this way is as effective as and more cost-effective than is CBT.", "Although BA compares favourably with CBT in systematic reviews,6,7 the existing evidence is insufficiently robust to establish comparability.8", "Authors of a Cochrane review7 called for more quality studies than have been done so far and the UK National Institute for Health and Care Excellence (NICE) regarded the international evidence as insufficient to recommend BA for first-line treatment in clinical guidelines,8 instead recommending a large non-inferiority study: “to establish whether behavioural activation is an effective alternative to CBT”.8", "Given these recommendations, we hypothesised that BA is non-inferior to CBT for depression treatment response in adults with depression and that BA is cost-effective compared with CBT.", "We found that BA for depression is not inferior to CBT in terms of reduction of depression symptoms and is more cost-effective than is CBT against commonly applied decision maker willingness to pay thresholds.", "We observed our results using both mITT and PP analyses, using a conservative non-inferiority margin.", "Our economic analyses were driven by the lower costs of the MHWs who delivered BA compared with the more experienced psychological therapists who routinely deliver CBT.", "Our study results therefore substantiate the hypothesis that BA is as effective as is CBT and that its simplicity renders BA suitable for delivery by junior MHWs with no professional training in psychological therapies.5", "This trial is the largest trial of BA to date and is one of the largest trials of psychological treatments for depression.", "We followed up participants for 18 months and our economic analysis is one of few in this field.", "Therapists and MHWs working in three different routine UK care settings delivered treatment, providing evidence of potential generalisability.", "We assessed therapy quality using independent raters and ensured that treatment in both arms was delivered to the standard recommended guidelines.", "Our levels of attrition and outcome loss to follow-up were low at 12 months and 18 months, similar to other trials in this area, but are still a limitation.", "Although participants in the per-protocol population attended similar numbers of sessions to those in other CBT trials,15 35% of participants chose to not even attend a minimal number of sessions, a problem well known to routine psychological therapy services.", "This pragmatic trial done in routine environments means that we were unable to quantify or control for the contribution of antidepressant medicines to outcomes.", "However, most participants who were taking medication had been doing so for a considerable time before entering the trial, making it unlikely that our results were driven by pharmacological treatment.", "Given the nature of the intervention and comparator, we could not mask patients or the mental health workers or therapists who were delivering the interventions to treatment allocation, but we used self-reported outcome measures and robust outcome assessor-masking procedures to reduce researcher unmasking to less than 5%.", "Missing data for the primary outcome measure was substantial.", "However, our between-group inferences were robust to data imputation.", "Our findings could have substantial implications for the scalability of psychological treatment for depression internationally4 given the greater availability and ease with which a BA workforce could be trained than could a CBT workforce.", "For many years, CBT has been the foremost psychological therapy recommended by therapists, researchers, and policy makers.", "Our results challenge this dominance.", "Although more work needs to be done than has been done so far to find ways to effectively treat the 20–23% of patients whose depression was unchanged by BA or CBT, our findings suggest that BA should be a front-line treatment for depression, with substantial potential to improve reach and access to psychological therapy globally.", "Our results in both groups compare favourably with a meta-analysis3 of the effects of CBT that estimate proportions of patients with remissions of around 50%.", "Our cost-effectiveness analyses show the high probability that BA is cost-effective and affordable compared with CBT at standard willingness to pay thresholds.", "Our most striking finding is that BA leads to similar clinical outcomes for patients with depression, but at a financial saving to clinical providers of 21% compared with the costs of provision of CBT, with no compensatory use of other health-care services by patients.", "Driving these savings is the fact that BA can be delivered by inexperienced MHWs with no professional training in psychological therapies, with no lesser effect than that of more highly trained and experienced psychological therapists giving patients CBT.", "Although many obstacles exist to successful dissemination in addition to training of MHWs, our findings suggest that health services globally could reduce the need for costly professional training and infrastructure, reduce waiting times, and increase access to psychological therapies.4", "Our findings have substantial implications given the increasing global pressure for cost containment across health systems in high-income countries and the need to develop accessible, scalable interventions in low-income and middle-income countries.", "Such countries might choose to investigate the training and employment of junior workers over expensive groups of psychological professionals.", "Our results, therefore, offer hope to many societies, cultures, and communities worldwide, rich and poor, struggling with the effect of depression on the health of their people and economies.", "In this randomised, controlled, open-label, non-inferiority trial (the Cost and Outcome of Behavioural Activation versus Cognitive Behaviour Therapy for Depression [COBRA] trial), we recruited participants from primary care and psychological therapy services in Devon, Durham, and Leeds (UK).", "Eligible participants were adults aged 18 years or older who met diagnostic criteria for major depressive disorder assessed by researchers using a standard clinical interview (Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition [SCID]9).", "We excluded people at interview who were receiving psychological therapy, were alcohol or drug dependent, were acutely suicidal or had attempted suicide in the previous 2 months, or were cognitively impaired, or who had bipolar disorder or psychosis or psychotic symptoms.", "We recruited participants by searching the electronic case records of general practices and psychological therapy services for patients with depression, identifying potential participants from depression classification codes.", "Practices or services contacted patients to seek permission for researcher contact.", "The research team interviewed those that responded, provided detailed information on the study, took informed written consent, and assessed people for eligibility.", "The UK South West Research Ethics Committee gave national approval for the study (NRES/07/H1208/60).", "The protocol has been published previously.10", "After eligibility was established, consent agreed, and baseline data collected, we randomly allocated participants (1:1) to BA or CBT using computer-generated allocation, stratified by depression severity according to the Patient Health Questionnaire 9 (PHQ-9)11 (<19 vs ≥19), antidepressant use (taking antidepressants or not), and recruitment site (Devon, Durham, or Leeds).", "A computer-based system allocated the first 20 participants to each group on a truly random basis.", "For subsequent participants, allocation was minimised to maximise the likelihood of balance in stratification variables across the two study groups.", "The registered Peninsula Clinical Trials Unit (Plymouth University, Plymouth, UK) allocated participants remotely after baseline data entry to ensure allocation concealment.", "Treatment was given open label, but outcome assessors were masked to participants' allocations.", "Concealment was ensured by use of an externally administered password-protected trial website with retention of a stochastic element to the minimisation algorithm.", "We recorded instances when outcome assessors were unmasked during interviews if participants informed them of their allocation.", "We developed our clinical protocols in line with published treatment protocols,12,13 including those from our own trials,14,15 advice from international collaborators, and NICE recommendations8 for duration and frequency of BA and CBT.", "Full-time National Health Service (NHS) MHWs and therapists worked half of their working week for COBRA (with the other half worked as normal) and followed written manuals to deliver a maximum of 20 sessions over 16 weeks, with the option of four additional booster sessions if the patients wanted them.8", "Treatment included core and supplementary techniques appropriate to the BA or CBT protocol to be used as clinically indicated; for example, behavioural or cognitive strategies for management of anxiety.", "All core components of both treatments were delivered by session eight, which we considered to represent a minimally sufficient dose of therapy (appendix).", "Sessions were face to face, lasting for 60 min.", "BA and CBT experts on the trial team trained MHWs and therapists for 5 days in either BA or CBT.", "MHWs and therapists were assessed for competence at the end of training with use of standardised quality criteria instruments consistent with the relevant treatment: either the Quality of Behavioral Activation Scale (Dimidjian S, University of Colorado, personal communication) or the Revised Cognitive Therapy Scale for CBT.16", "Further training was given if competency was not demonstrated.", "MHWs and therapists received 60 min of clinical supervision fortnightly from NHS psychological therapists clinically experienced in BA or CBT, overseen by trial team experts.", "Junior MHWs—graduates trained to deliver guided self-help interventions, but with neither professional mental health qualifications nor formal training in psychological therapies—delivered an individually tailored programme re-engaging participants with positive environmental stimuli and developing depression management strategies.", "Participants were encouraged to increase their contact with individually specified positive situations and reduce their avoidance of other situations.", "Specific BA techniques included identification of depressed behaviours, analysis of the triggers and consequences of depressed behaviours, monitoring of activities, development of alternative goal-orientated behaviours, scheduling of activities, and development of alternative behavioural responses to rumination.", "Professional or equivalently qualified psychotherapists, accredited as CBT therapists with the British Association of Behavioural and Cognitive Psychotherapy, with a postgraduate diploma in CBT, delivered a personalised treatment programme based on an assessment of how participants' beliefs lead to emotional distress and ineffectual coping.", "Participants used cognitive and behavioural exercises to specifically test the accuracy of those beliefs by identifying and modifying negative thoughts and beliefs that give rise to them.", "Specific techniques included participants monitoring moods and activities, planning of exercises to test negative beliefs, and thought records to identify and examine the accuracy of negative automatic thoughts and underlying beliefs.", "We did follow-up assessments 6 months, 12 months, and 18 months after randomisation.", "We assessed the quality of and adherence to treatment using audiotapes and written records of therapy sessions.", "Independent experts in both treatments rated a random (with use of a computer-generated random number sequence) sample of tapes, stratified by therapist, therapy session, and intervention, for competence using the Revised Cognitive Therapy Scale16 for CBT (range 0–72) and the Quality of Behavioral Activation Scale (Dimidjian S, University of Colorado, personal communication) for BA (range 0–96).", "All therapists recorded the specific therapeutic techniques that they had used for each session on a checklist.", "The primary outcome was self-reported depression severity (PHQ-9 score11) at 12 months.", "Secondary outcomes were PHQ-9 score at 6 months and 18 months and Diagnostic and Statistical Manual of Mental Disorders IV major depressive and anxiety disorder status and number of depression-free days between follow-ups (SCID),9 anxiety (Generalized Anxiety Disorder 7),17 and health-related quality of life (36-Item Short Form Survey)18 at 6 months, 12 months, and 18 months.", "For adverse events, we recorded deaths from whatever cause and all self-harm and suicide attempts.", "The independent Data Management Committee reviewed all adverse events and made relevant trial conduct recommendations.", "Previous research has suggested that non-inferiority margins should be half of the mean controlled effect size from historical trials.19", "Accordingly, we estimated the non-inferiority margin for the primary outcome using meta-analysis data from trials of BA14 for which BA was superior to controls by a mean of 0·7 SD units (95% CI 0·39–1) or 3·8 PHQ-9 score units (2·1–5·4).", "Therefore, our non-inferiority margin was 1·9 PHQ-9 points (ie, 0·5 × 3·8).", "We inflated our sample size by 20% for participant follow-up attrition.", "We planned to recruit 220 participants per arm to detect a between-group non-inferiority margin of 1·9 PHQ-9 points with a one-sided 2·5% α.", "Furthermore, although findings from a trial20 of CBT have shown little effect of outcome clustering by therapists, the presence of a small therapist clustering effect (ie, an intracluster correlation coefficient of 0·01) would still provide the same power.", "We did all analyses using a statistical analysis plan prepared in the first 6 months of the trial, agreed with the Trial Management Group, Trial Steering Committee, and Data Management Committee.", "We assessed between-group equivalence of baseline characteristics and outcomes descriptively and did a descriptive analysis of baseline characteristics by recruitment method.", "We compared observed primary and secondary outcomes between groups 12 months after randomisation using linear regression models adjusted for baseline outcome values and stratification variables.", "We did modified intention-to-treat (mITT) and per-protocol (PP) analyses, as security of inference depends on both PP and intention-to-treat analyses showing non-inferiority.21", "PP analysis provides some protection for any theoretical increase in the risk of type I error (erroneously concluding non-inferiority).", "Our mITT population comprises all patients according to and included in random allocation with complete data.", "We defined the PP population as participants meeting the mITT definition and receiving at least eight treatment sessions (representing a minimally sufficient dose of therapy).", "We analysed safety in the mITT population.", "We did sensitivity analyses for our primary outcome and for different definitions of PP (eight, 12, 16, and 20 treatment sessions) to check security of inference of non-inferiority.", "We accepted non-inferiority of BA to CBT (in a 0·025 level test) if the lower bound of the two-sided 95% CI (equivalent to the upper bound of one-sided 97·5% CI) was within the non-inferiority margin of −1·9 PHQ-9 points.", "We checked for non-equivalence of the primary outcome at all follow-up points using the same approach.", "We did secondary analyses to compare groups at follow-up across 6 months, 12 months, and 18 months using hierarchical linear regression.", "To ease clinical interpretation, we calculated proportions of recovery (participants with PHQ-9 scores of ≤9) and response (50% reduction from baseline PHQ-9 scores).", "We ran sensitivity analyses to assess the likely effect of missing data using multiple imputation models.", "We did imputation by treatment group using chained equations to create 20 complete datasets under the assumption that data were missing at random.22", "Imputation models included covariates as defined for the primary analysis model and auxiliary variables that were predictive of outcomes.", "After analysis, we combined the effect estimates from the imputed datasets using Rubin's rule.23", "For economic analyses, we took the UK NHS and personal social services perspective consistent with the NICE reference case,24 also examining a wide societal perspective, adding productivity losses due to time off work in a sensitivity analysis.", "We collected participants' use of BA and CBT from clinical records, with additional resource information (eg, training, supervision, and other non-face-to-face activities) from therapists and trainers.", "We used the Adult Service Use Schedule to measure other health and social care services used, including psychotropic medications.", "We measured productivity losses using the absenteeism and presenteeism questions from the Health and Work Performance Questionnaire.25", "We calculated effectiveness in terms of quality-adjusted life-years (QALYs) using the EuroQol-5D-3L measure of health-related quality of life.26", "We assigned health states from the EuroQol-5D-3L measure a utility score using responses from a representative sample of adults in the UK.27", "We calculated QALYs as the area under the curve defined by the utility values at baseline and each follow-up, assuming that utility score changes over time followed a linear path.", "We compared the costs and cost-effectiveness of treatments at 18 months to capture the economic effect of events like relapse with unit costs from the 2013–14 financial year.28,29", "We discounted costs and QALYs in year 2 at 3·5%.24", "We used complete case analysis with missing data explored in a sensitivity analysis using multiple imputation with chained equations.", "We calculated the cost of each treatment using a microcosting (bottom-up) approach.30", "We based MHW costs on NHS Agenda for Change salary band five (salary range £21 909–28 462; US$31 662–41 130; €27 726–35 993) for BA and band seven (£31 383–41 373; US$45 350–59 786; €39 738–52 388) for CBT therapists and included employer National Insurance and pension contributions plus capital, administrative, and managerial costs.", "We calculated cost per h using standard working time assumptions,31 weighted to account for time spent on non-patient-facing activities.", "We applied nationally applicable unit costs for other health and social care services.", "We assessed cost-effectiveness in terms of QALYs using the net benefit approach.32", "We analysed differences in mean cost per participant at 18 months using parametric t tests, with the validity of results confirmed using bias-corrected, non-parametric bootstrapping.33", "We calculated incremental cost-effectiveness ratios and constructed cost-effectiveness planes using 1000 bootstrapped resamples from regression models of total cost and outcome by treatment group.", "We used these bootstrapped replications to calculate the probability that each of the treatments is the optimal choice for different values a decision maker is willing to pay for a unit improvement in outcome, representing uncertainty around the cost and effectiveness estimates, with cost-effectiveness acceptability curves illustrating the probability that BA is cost-effective compared with CBT, dependent on willingness to pay per QALY.34", "We controlled for stratification variables and baseline values of the variables of interest, truncating data to exclude influential outliers—ie, cases with total costs in the 99th percentile that make a significant difference to the results.", "We did all analyses using Stata version 14.1.", "This trial is registered with the ISCRTN registry, number ISRCTN27473954.", "The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.", "DAR, RST, FCW, SB, SR, and BB had full access to all the data in the study and DAR had final responsibility for the decision to submit for publication.", "Between Sept 26, 2012, and April 3, 2014, we recruited 440 participants, randomly allocating 221 (50%) to the BA group and 219 (50%) to the CBT group (figure 1).", "175 (79%) participants were assessable for the primary outcome in the mITT population in the BA group compared with 189 (86%) in the CBT group, whereas 135 (61%) were assessable in the PP population in the BA group compared with 151 (69%) in the CBT group.", "We noted no evidence of a difference in patient characteristics between recruitment methods (appendix).", "Patient-level and trial-level characteristics at baseline were well balanced between groups (table 1).", "PHQ-9 score at baseline was negatively skewed, with a high proportion of participants scoring towards the upper end of the distribution (data not shown), but scores were similar between groups (table 2).", "Ten MHWs provided BA (median 22 participants each [IQR 19–25]) and 12 therapists provided CBT (21 [13–23]).", "MHWs had a mean of 18 months mental health experience (SD 11) and CBT therapists had a mean of 22 months post-CBT qualification (24).", "We removed one CBT therapist from the trial in the early stages who did not meet acceptable competency.", "Participants received a mean of 11·5 BA sessions (7·8) or 12·5 CBT sessions (7·8).", "305 participants (69%) completed the PP number of at least eight sessions (BA 147 [67%] patients, mean 16·1 sessions [SD 5·3]; CBT 158 [72%] patients, mean 16·4 sessions [5·4]); participants completing less than eight sessions (135 [31%]; BA 74 [33%], CBT 61 [28%]) completed a mean of 2·5 BA sessions (SD 1·9) or 2·6 CBT sessions (2·1).", "MHWs and therapists met acceptable competency standards: mean Quality of Behavioral Activation Scale BA competence was 55 (7·5) and mean Revised Cognitive Therapy Scale for CBT competence was 37·9 (10·9).", "We found no evidence of inferiority of PHQ-9 score at 12 months in either the mITT (CBT 8·4 PHQ-9 points [SD 7·5]; BA 8·4 PHQ-9 points [7·0]; mean difference 0·1 PHQ-9 points [95% CI −1·3 to 1·5]; p=0·89) or PP (CBT 7·9 PHQ-9 points [7·3]; BA 7·8 [6·5]; mean difference 0·0 [–1·5 to 1·6]; p=0·99) populations (table 2).", "The non-inferiority of BA to CBT was accepted for both the mITT and PP populations as the lower bound of the 95% CI (one-sided 97·5% CI) of the between-group mean difference lies within the non-inferiority margin of −1·9 PHQ-9 points (appendix).", "Although we initially planned to include therapist as a random-effects variable, given the low levels of observed clustering, we parsimoniously fitted our models without therapist as a variable.", "We checked for no inference difference with and without inclusion of a random-effects therapist term.", "We ruled out superiority of CBT to BA as the lower bound of the 95% CI included zero for the mITT and PP populations.", "The inference of non-inferiority was robust to sensitivity analysis across different PP definitions.", "We found no evidence of a significant between-group treatment interaction across the mITT or PP populations with the primary outcome at 12 months as stratified by depression severity, antidepressants use, and recruitment site (appendix).", "We found that BA was not different from CBT in anxiety (Generalized Anxiety Disorder 7), depression status, and depression-free days and anxiety diagnoses (SCID) for either the mITT or PP populations using observed or imputed data at 12 months (table 2).", "Because of substantial missing 36-Item Short Form Survey data at baseline, we analysed these data adjusted for stratification variables only.", "We found no difference in numbers of participants with at least one anxiety diagnosis: BA 43 (28%) of 153; CBT 43 (27%) of 161 (mITT population; χ2 0·08; p=0·78).", "Between 61% and 70% of mITT and PP participants in both groups met criteria for recovery from depression or response to treatment at 12 months, with no differences in the proportions of patients in each group who recovered or responded (table 3).", "Using observed data for all outcomes, we found no evidence of a difference between the CBT and BA groups over the period of the trial, as indicated by a non-significant time-by-treatment effect interaction, for both the mITT and PP populations (appendix).", "We found a small, negligible clustering of primary and secondary outcome scores at follow-up across therapists overall and within BA and CBT groups (intracluster correlation coefficient ≤0·04).", "Two (1%) non-trial-related deaths (one [1%] multidrug toxicity in the BA group and one [1%] cancer in the CBT group) and 15 depression-related, but not treatment-related, serious adverse events (three in the BA group and 12 in the CBT group) occurred in three [2%] participants in the BA group (two [1%] patients who overdosed and one [1%] who self-harmed) and eight (4%) participants in the CBT group (seven [4%] who overdosed and one [1%] who self-harmed).", "Of the 440 participants recruited, 76 (17%) had missing primary outcome data at 12 month follow-up.", "The proportion of missing PHQ-9 data was higher in the BA than in the CBT group (46 [21%] vs 30 [14%]; odds ratio 1·6 [95% CI 1·0–2·7]; p=0·05).", "Imputation of data for primary and secondary outcomes at 12 months showed that in accordance with the observed data analysis, no difference existed between groups (tables 2, 3), supporting our conclusion of non-inferiority.", "The odds of missing PHQ-9 data were higher for patients with increased baseline severity of depression (PHQ ≥19, odds ratio 1·6 [95% CI 1·0–2·6]; p=0·05) and increasing age (in years) was associated with lower odds of missing PHQ-9 data (odds ratio 0·97 [0·96–0·99]; p=0·01).", "We found no evidence of an association between missingness and any other baseline characteristic (data not shown).", "Outcome assessors reported having been unmasked for 16 (4%) participants (five [2%] in the BA group and 11 [5%] in the CBT group; due to participants informing assessors of their treatment allocation).", "For economic analyses, at 18 months, full service use data was available for 159 (90%) of 176 participants in the BA group and 168 (93%) of 180 participants in the CBT group.", "We found a significant difference in mean intervention costs between the two groups, but no differences in other categories of cost or in total cost (table 4).", "Mean health state utility scores according to EuroQoL-5D-3L were slightly higher in the BA group than in the CBT group across the entire follow-up period, with resultant QALYs also higher for BA, but the QALY difference was not significant.", "Costs were lower and QALY outcomes better in the BA group than in the CBT group, generating an incremental cost-effectiveness ratio of –£6865.", "The scatterplot of bootstrapped cost and effectiveness pairs for BA versus CBT illustrates dominance of BA over CBT, with the point estimate and two-thirds of scatter points falling in the southeast quadrant of the cost-effectiveness plane, where BA replications are cheaper and more effective than are CBT ones (figure 2).", "The cost-effectiveness acceptability curve (appendix) showing the probability of BA being cost-effective compared with CBT does not fall below 75% and is closer to 80% at NICE-preferred willingness24 to pay £20 000–30 000 per QALY.", "In all sensitivity analyses, including complementary therapies and productivity losses, as well as analyses taking narrow intervention and mental health service perspectives, BA was significantly less costly than was CBT, so BA continues to have a higher probability of being cost-effective than does CBT at the NICE threshold (appendix).24", "Imputation of missing data increased the difference in total cost (BA £1841·67; CBT £2282·40; difference –£440·73 [95% CI −1007·71 to 126·26]; p=0·13), but reduced the difference in QALYs (BA 1·22; CBT 1·19; difference 0·03 [–0·06 to 0·11]; p=0·55), increasing the incremental cost-effectiveness ratio to –£16 951.", "The cost-effectiveness acceptability curve for the missing data analysis again supported the likelihood that BA is cost-effective compared with CBT (appendix)." ]
[]
Changes in plant species richness and productivity in response to decreased nitrogen inputs in grassland in southern England
Biomass production and plant species diversity in grassland in southern England was monitored before and after a change from conventional to organic farming. Our 18-year study, part of the UK's Environmental Change Network long-term monitoring programme, showed that the cessation of artificial fertiliser use on grassland after conversion to organic farming resulted in a decrease in biomass production and an increase in plant species richness. Grassland productivity decreased immediately after fertiliser application ceased, and after two years the annual total biomass production had fallen by over 50%. In the subsequent decade, total annual grassland productivity did not change significantly, and yields reached 31-66% of the levels recorded pre-management change. Plant species richness that had remained stable during the first 5 years of our study under conventional farming, increased by 300% over the following 13 years under organic farm management. We suggest that the change in productivity is due to the altered composition of species within the plots. In the first few years after the change in farming practice, high yielding, nitrogen-loving plants were outcompeted by lower yielding grasses and forbs, and these species remained in the plots in the following years. This study shows that grassland can be converted from an environment lacking in plant species diversity to a relatively species-rich pasture within 10-15 years, simply by stopping or suspending nitrogen additions. We demonstrate that the trade-off for increasing species richness is a decrease in productivity. Grassland in the UK is often not only managed from a conservation perspective, but to also produce a profitable yield. By considering the species composition and encouraging specific beneficial species such as legumes, it may be possible to improve biomass productivity and reduce the trade-off.
[ "AGRI", "DECI", "ENVI" ]
[ "Biomass production", "Grassland", "Long-term monitoring", "Organic farming", "Plant species richness", "UK Environmental Change Network" ]
[ "1105", "1800", "2303" ]
[ "In 1939 the British parliament removed covenants protecting old grasslands from ploughing restrictions (Duffey et al., 1974).", "This, coupled with a desire for self-sufficiency, accentuated by the onset of the Second World War, marked the start of intensive agriculture in the United Kingdom.", "This intensification substantially increased between the 1940s, when two thirds of Britain's food was imported (Lloyd and Wibberley, 1977), and the 1980s when overproduction and subsequent detrimental effects on the environment were causing concern.", "By 1984 production of cereals was 10 million tonnes more than the population of the UK could utilise (Marren, 2002).", "Increased mechanisation and inorganic fertiliser use, as well as new strains of pasture species, including ryegrass and clover, created highly productive but species-poor swards.", "In response to the loss of wildlife and degradation of landscape associated with this agricultural intensification (Ratcliffe, 1984; Hopkins et al., 2000), agri-environment schemes (AES) were introduced into the UK.", "Over the past 20 years three types of schemes have been introduced: Environmentally Sensitive Areas (ESAs), Countryside Stewardship (CSS) and Environmental Stewardship (ESS).", "The uptake of these schemes has substantially increased in the last 10 years: in 2005, 13% of the UK (Defra, 2007) was covered by AES, and by 2009 this had reached 66%, in excess of 6 million ha.", "In 2013 the area of land enrolled in entry level AES had risen to 7.4 million ha, compared to just under 2 million ha in 2005 (Defra, 2014).", "Little is known about how the change from intensive to extensive farming methods affects land and biodiversity.", "Taylor and Morecroft (2009) followed and described plant and insect diversity trends at our study site in southern England for five years after the farmland was converted from conventional to organic agriculture.", "Those authors showed that above-ground biomass production in the pasture decreased rapidly after the management change, and that plant species richness had increased within two years.", "Although other authors have compared the effect of organic farming on plant species biodiversity and biomass production with that of conventional farming as paired comparisons, they do not detail – or have not assessed – the direct effect of the change in one area over time (Hole et al., 2005).", "There is some disagreement about the effect of nitrogen addition in agricultural systems.", "It has been shown that long-term nitrogen inputs increase standing crop biomass and decrease plant species richness, shifting plant composition to a few dominant species (Bakelaar and Odum, 1978; Tilman, 1987; Hunneke et al., 1990; Inouye and Tilman, 1995).", "However, other studies have shown that low species richness resulted in low above-ground biomass (Hector et al., 1999; Palmborg et al., 2005), although a recent re-analysis of one of these experiments has demonstrated that species richness and composition are likely to be of similar importance for productivity (Hector et al., 2011).", "In this study, which forms part of the Environmental Change Network (ECN), a long-term monitoring programme started in 1992, we followed changes in vegetation in permanent pasture on the Wytham estate in Oxfordshire, UK, prior to and following the adoption of agri-environment measures under an ESA agreement.", "Building on the work of Taylor and Morecroft (2009), we assessed continued impacts on above-ground productivity and plant species richness for eight years beyond the original study, using data collected as part of additional ECN vegetation surveys in other parts of the study area.", "All experimental plots were situated on the Wytham estate (51.77 N, 1.34 W), as described by Taylor and Morecroft (2009).", "The estate, owned by Oxford University, is situated five km northwest of Oxford, and currently consists of approximately 670 ha of organic farmland, leased to FAI Farms Ltd, and 390 ha of woodland.", "The woodland consists of a variety of types, including ancient semi-natural woodland, secondary woodland and plantations.", "Until September 2001 the farmland was managed as a commercial mixed farm.", "In 2002 organic management was adopted, and by 2005 the farm had obtained organic status certification.", "The experimental plots in this study were of two types: grazing ‘exclusion plots’ in permanent pasture and ‘vegetation plots’ in grassland within the woods.", "Ten exclusion plots were first set up on Lower Seeds permanent pasture (British grid reference: SP46720847) in 1996 to prevent grazing from livestock or wild herbivores (Fig. 1).", "Each exclusion plot measured 1.5 m × 2.5 m, was positioned randomly within a subplot (750 m2) of a single main plot of 7500 m2 and was covered with a wire mesh cage.", "The plots remained in the same position throughout the year and were relocated annually in March.", "Six 10 m × 10 m vegetation plots were established between 1994 and 1998 in grassland within Wytham woods and in the surrounding farmland on the Wytham estate.", "Within these plots, ten 40 cm × 40 cm quadrats were randomly located and permanently marked with aluminium pegs, thus allowing the exact plots to be revisited annually.", "Three of the six grassland plots, U1 (SP45710812), U2 (SP460008309), and U3 (SP46540762) were left ‘unmanaged’, other than for sporadic grazing and the ensuing natural fertilisation by sheep (Fig. 1).", "The remaining three plots, M1 (SP46710923), M2 (SP46570839) and M3 (SP46620770) were classified as ‘managed’ plots.", "They were all grazed by sheep and/or cattle, and were fertilised with nitrogen compounds prior to 2002; the nitrogen applications stopped in 2001.", "Between 1993 and 2000 (we do not have farm records for 2001) nitrogen was applied to the exclusion plots and the managed plots at a mean rate of 225 kg ha−1 year−1.", "In addition, plot M3 was managed as historic grassland under the Higher Level Stewardship (HLS; Natural England, 2013) scheme.", "The pasture in the exclusion plots was cut four times during each year in May, July, September, and October, using a power scythe (Alpina, Global Garden Products Italy S.p.A. Veneto, Italy).", "A 10 cm strip was cut around the edge of each plot, raked off and discarded.", "The remaining uncut area was measured and the vegetation was then cut, collected and bagged for further processing and biomass calculation.", "The mesh cage was replaced in the exact spot it had been removed from (two opposite corners had been marked with aluminium tent pegs) and firmly pegged down to prevent accidental movement by grazing livestock.", "The vegetation removed from each plot was weighed and dried at 25 °C until the weight had stabilised and no more water was being lost.", "If the cut was large (>1 kg), a subsample of 100 g was used.", "Biomass was calculated and expressed as g m−2 year−1.", "Between June and August, the six vegetation plots were surveyed annually as part of the ECN vegetation survey.", "Prior to the July cut the exclusion plots were also surveyed.", "The presence of all plants (graminoids, forbs, woody species and mosses) rooted in the soil within the small quadrats and in the exclusion plots were recorded.", "Species names follow Stace (2010) for vascular plants and Smith (2004) for bryophytes.", "Data analysis was carried out in R version 3.2.2 (R Core Team, 2015).", "All analyses addressed the central hypothesis of whether a measured outcome (i.e. biomass, species richness or species frequency, depending on the experiment and model) exhibited a change significantly different from zero across the temporal shift from conventional to organic farming, here represented by the cessation of nitrogen fertilisation.", "The optimal structures of the individual models for each experiment and outcome type are discussed below; standard approaches to the selection of fixed effects, random effects, temporal auto-correlation and error structure distribution were used following Zuur et al. (2009).", "Biomass was log-transformed prior to inclusion in linear mixed models.", "Season of harvest and a categorical indicator for nitrogen application (i.e. during the period of annual nitrogen applications vs. the post-application period) were included as the covariates of interest.", "The inclusion of a random intercept term for each exclusion cage was preferred over a simple linear model (likelihood ratio test statistic = 3.91, P = 0.048), although the variance associated with the random intercept term was very low (0.005).", "Auto-correlation function (ACF) residual plots indicated the presence of strong temporal correlation.", "The inclusion of an auto-regressive moving average (ARMA) (1,0) error structure (Zuur et al., 2009) considerably reduced Akaike's Information Criterion (AIC) over a model that did not account for auto-correlation (1204.83 vs. 1228.44), and was included in the final model.", "The resulting linear mixed model was fitted using restricted estimate maximum likelihood using the R package ‘nlme’ (Pinheiro et al., 2015).", "A generalised estimating equation (GEE; Højsgaard et al., 2006; Zuur et al., 2009) was used to assess the significance of the change in species richness across the temporal change in nitrogen application.", "The use of a GEE with a Poisson error structure improved the quantile–quantile (QQ) diagnostic plots of model residuals over a Gaussian linear model, and allowed for the inclusion of temporal auto-correlation (an auto-regressive series of order one was used; Zuur et al., 2009).", "Unlike the biomass model (Section 2.4.1 above), a random intercept for exclusion plot was not used; this was because model comparisons using AIC found little difference between simple and mixed models (with random intercept: 977.43; without: 975.43), and because of the fact that accounting for a Poisson error structure, temporal auto-correlation and random effects in a single model remains highly challenging (Bolker, 2015).", "Fisher's Exact Test was also used to compare the incidence of species pre- and post-farming management change; P values from these tests were adjusted for multiple comparisons using the false-discovery rate method of Benjamini and Hochberg (1995).", "The significance of the effect of plot type and the presence or absence of nitrogen applications on species richness were assessed using a linear mixed model.", "AIC comparisons indicated that a model structure including nested random intercepts for quadrats within plots and a temporal auto-correlation structure (ARMA(1,0), as above) were both required.", "To achieve this, species richness was square-root transformed to enable the use of a linear mixed model (using the R package ‘nlme’; Pinheiro et al., 2015).", "Comparison of QQ plots of linear model residuals using square-root transformed species richness with generalised linear models with a Poisson error structure on untransformed data showed similar improvements over linear models applied directly to count data.", "Similarly, Poisson generalised linear mixed models had much higher AIC values than linear mixed models accounting for temporal autocorrelation but using square-root transformed data (5073.04 vs. 1077.45); this suggested that accounting for temporal auto-correlation and spatial structure (the random effects) using transformed count data was preferred over modelling untransformed data where only one of the auto-correlation or random effects structures could be accounted for (Bolker, 2015).", "Fisher's Exact Test was again used to compare the incidence of species pre- and post-farming management change, as described above (Section 2.4.2).", "An analysis of deviance of the final linear mixed model showed significant effects of season, nitrogen application and their interaction (Table 3).", "After nitrogen applications ceased in 2001 there was a rapid decline in biomass production (Fig. 2).", "This can be mostly attributed to the decrease in production in spring and early summer; autumn cut biomass showed no clear variation over years (Fig. 3).", "Seasonal variation, however, was dependent on the addition of nitrogen, with the autumnal decline in biomass being greater during the period of the applications (Table 3; Fig. SI1).", "In spring, productivity peaked in 1999 (527 g m−2) and in summer productivity was greatest in 1997 (556 g m−2).", "In the first year after the cessation of nitrogen fertilisation, spring biomass production decreased by 70%, and between 2002 and 2014 levels then fluctuated between 30 and 50% of the pre-2002 productivity.", "In summer, there was a lag in the response to the change in management: in 2002 biomass production was 523 g m−2, 97% of the maximum production pre 2002, but by 2005 production had decreased to a low of 170 g m−2.", "Since 2010, and in contrast to the spring production, the summer yields have steadily increased, reaching 400 g m−2.", "As expected, productivity was lower in autumn than in spring and summer.", "Annual monitoring of the vegetation in the exclusion plots in the permanent pasture showed that stopping the application of nitrogen fertilisers resulted in an increase in plant species diversity (Fig. 4; Fig. SI2).", "The mean number of species per exclusion plot rose from 4.2 before the change to organic farming to 8.7 after.", "In 2014 there was a mean of 15.8 species in each exclusion plot, with the maximum number of species detected in any one exclusion plot being 20.", "The mean number of species identified in each year between 1996 and 2001 was 10.3 and rose to 13.8 species between 2002 and 2006.", "Between 2007 and 2014, a mean of 24.9 species were detected.", "The greatest number of species was recorded in 2014, when 33 plant species were identified.", "An ANOVA of the final GEE model demonstrated a highly significant effect of a change in farming management on species richness (χ2 = 19.2, d.f. = 1, P < 0.0001).", "One effect of the cessation of nitrogen fertilisation was an alteration of the ratio of forbs to graminoids.", "Up to and including 2001, graminoids were most frequent in four out of six years, but after 2001 this was the case in only four out of 13 years.", "Comparison of the plant species detected between 1994–2001 and 2002–2014 (Table 2) showed that there were significant increases for Agrostis stolonifera (P < 0.001) Arrhenatherum elatius (P < 0.001), Brachythecium rutabulum (P < 0.05), Bromus hordeaceus (P < 0.001), Cerastium fontanum (P < 0.001), Cynosurus cristatus (P < 0.01), Festuca rubra (P < 0.01), Geranium dissectum (P < 0.001), Medicago lupilina (P < 0.001), Phleum bertolonii (P < 0.001), Poa trivialis (P < 0.001), Ranunculus repens (P < 0.001), Taraxacum officinale (P < 0.001) and Trifolium repens (P < 0.001).", "Not all species benefitted from the change in farming practice; Alopecurus pratensis (P < 0.05), Cirsium arvense (P < 0.01), Poa annua (P < 0.001), Poa pratensis (P < 0.05) and Veronica agrestis (P < 0.001) all suffered a significant decrease.", "The unmanaged plots supported a more diverse range of plant species than the exclusion plots or the managed plots throughout the survey period (Table 1).", "A mean of 78.5 species was recorded in each plot between 1994 and 2014 and a mean of 53.3 species was identified in more than one year.", "In most years the number of forb species detected was greater than the number of graminoid species detected (Fig. 5), although the differences were largely insignificant.", "Comparison of each species detected prior to 2002 with records between 2002 and 2014 showed that 24 species increased significantly (Table 2) and 20 species decreased significantly.", "Of those that increased, 14 were forbs, five were graminoids, four were mosses and there was one woody species.", "For the forbs, the most significant increases in detection were for Agrimonia eupatoria, present in none of the quadrats prior to 2002 but 49% after (P < 0.01), Cirsium eriophorum (6.7–18.7%, P < 0.001), Clinopodium vulgare (0–12.8%, P < 0.001), Convolvulus arvensis (10–24%, P < 0.001), Galium verum (9–19.5%, P < 0.01), Linum catharticum (0–6%, P < 0.001) and Veronica chamaedrys (8–23.6%, P < 0.001).", "Of the four mosses that increased in detection, the greatest changes were for B. rutabulum (20–34.1%, P < 0.01) and Kindbergia praelonga (8.1–19.7%, P < 0.001).", "The detection of Fraxinus excelsior also increased significantly (4.7–26.4%, P < 0.001).", "Of the graminoid species that decreased, the most significant were Luzula campestris (21.9–10.8%, P < 0.01) and P. pratensis (15.2–4.6%, P < 0.001).", "Other species that decreased significantly were Aphanes arvensis (9–0.5%, P < 0.001), C. fontanum (31.9–15.9%, P < 0.001), Galium aparine (12.9–1.8%, P < 0.001), G. dissectum (21.4–7%, P < 0.001), Sherardia arvensis (10.9–2%, P < 0.001), Rumex acetosa (30.5–16.1%, P < 0.001), and Veronica arvensis (4.3–0%, P < 0.001).", "Species richness in the managed vegetation plots within the Wytham estate was lower than in the unmanaged plots: a mean of 35.3 species was recorded in the plots between 1994 and 2014 (M1) or 1998 and 2014 (M2 and M3).", "Of these, a mean of 21.7 species were detected in more than one year.", "In the managed plots the majority of significant changes in species presence were increases in detection post-2001.", "Only four species significantly decreased in detection after this date: one forb, A. arvensis (2.7–0%, P < 0.001), and three graminoids: Lolium perenne (100–85.6%, P < 0.001), P. annua (37.3–5.1%, P < 0.001) and P. pratensis (12–0.7%, P < 0.001).", "In comparison, the detection of 31 species increased after the change from conventional to organic farming, 17 significantly so.", "Four species were not present until after 2001: B. hordeaceus (0–5.9%, P < 0.001), P. bertolonii (0–8.9%, P < 0.001), R. repens (0–26.2%, P < 0.01) and T. repens (0–25.6%, P < 0.001).", "The other species for which the change was significant were all graminoids and they increased between 6% and 41%.", "The greatest increase occurred in Holcus lanatus (22.7–63.3%, P < 0.001).", "For the other graminoids, the increase was smaller, but still significant, Agrostis capillaris (2–33.3%, P < 0.001), A. stolonifera (26.7–50%, P <0.001), Dactylis glomerata (10.7–42.8%, P < 0.001), Phleum pratense (0.7–8.7%, P < 0.001), and P. trivialis (8.7–28.7%, P < 0.001).", "Not every plot contained all the species found throughout the survey, a reflection of the different soil and habitat types of the Wytham estate.", "We observed an increase in species diversity over the period of the survey in all managed plots and two out of three unmanaged plots (Table 1).", "An analysis of deviance of the final linear mixed model results is presented in Table 4.", "For the unmanaged plots, only small changes in the mean number of species detected pre- and post-management change were observed (Table 1).", "Changes in the managed plots and the exclusion cages in permanent pasture were more marked, with clear increases in average species richness (Table 1; Figs. SI2 and SI3).", "The detection of species in the functional groups graminoids, forbs, mosses and woody species followed different patterns for different management systems.", "In the managed plots and exclusion cages graminoids were most common, but in the unmanaged plots the incidence of forbs and graminoids was similar, with forbs being more abundant in most years (Fig. 5).", "Woody species and mosses were more abundant in the unmanaged vegetation plots (Fig. 5) than in the managed plots.", "There was a gradual increase in the mean number of graminoids detected in the exclusion plots with 3.5 species in 1996 and 7.8 species in 2014 (Fig. 5a) and a similar steady increase in the managed plots, ranging from 1.9 in 1994 to 5.1 in 2014 (Fig. 5b).", "Forb species were absent or present at a low rate until the mid-2000s.", "In the exclusion plots less than two forb species per exclusion plot were detected until 2007, when there was a mean of 4 species per exclusion plot, and this continued to increase to 2014 when 6.8 species were detected.", "Similarly, forbs were almost absent from the managed plots until 2009 when a mean of 1.2 species per quadrat was detected.", "Until then less than one species per quadrat had been recorded.", "There were 35 plant species present in at least one of the managed or unmanaged plots or the exclusion plots and eight species (seven graminoids and one moss) were found in all plots.", "Of these eight species, four showed little or no difference frequency between the plot types, and four showed similarities between the managed plots and exclusion plots, but differences when compared with the unmanaged plots.", "The presence of some species was affected by farming practice.", "P. annua was abundant in the managed plots and exclusion plots up to 2001 (between 90% and 100%, in the managed plots), but after the change to organic farming was implemented, the frequency of P. annua decreased to 50% (2004) and continued to decrease to 30% by 2014.", "Detection in the exclusion plots followed a similar pattern.", "In the unmanaged plots, however, P. annua was present in less than 20% of quadrats in all years between 1994 and 2014 and there was no change prior to and post 2001.", "The frequency of B. hordeaceus was also affected by the farming management change; this grass was detected in the unmanaged plots before 2002, but not in the exclusion plots or managed plots (except for one record in 1997).", "The presence of B. hordeaceus in unmanaged plots decreased after 2002, but increased in the managed plots and in the exclusion plots.", "Similarly, there were increases in the managed plots and exclusion cages for H. lanatus after 2002, but these increases were also seen in the unmanaged plots.", "Prior to 2002, H. lanatus was not detected in the exclusion cages, and was only detected in less than 10% of the managed plots in 1994.", "This study is one of a few long-term experiments demonstrating large shifts in plant community composition and grassland productivity following changes in nitrogen application and deposition, some of which have been reviewed by Dise and Stevens (2005) and Phoenix et al. (2012).", "In this study, high productivity before the move from a conventional to an organic system of farming corresponded to low species richness.", "After the change, the productivity of the pasture remained low although the species richness rapidly increased.", "However, in the last two years of this study productivity began to increase.", "In the UK, studies at Wardlow Hay Cop in Derbyshire showed that adding nitrogen contributed to the loss of species richness by decreasing forb cover and resulting in increases in graminoids (Morecroft et al., 1994; Carroll et al., 2003).", "Prior to 2002, the dominant species in the exclusion plots at Wytham were fast growing pasture grasses: L. perenne, a palatable and nutritious grass (van Wijk et al., 1993), P. annua, A. stolonifera and D. glomerata.", "Although most of these productive grasses were still recorded in the exclusion plots after the change to organic farming, the composition of the plots had changed to include a greater number of forbs and low-yielding grass species.", "Species that increased in frequency included C. cristatus, which has a low yield due to the high stem to leaf ratio; those that would not withstand heavy grazing e.g. A. elatius; and those regarded as grass weeds with low yield or which are typically unpalatable to stock, e.g. P. trivialis and H. lanatus.", "Our results suggest that the composition of species is more important for determining productivity than simply the number of species present.", "Marquard et al. (2009) found that in a six year biodiversity experiment in Germany, the increase in biomass was positively correlated with species richness, and that doubling the number of species increased community biomass by 91 g m2.", "They also showed that some groups of species had different effects on productivity, particularly legumes, which had a positive effect on community biomass.", "At Wytham, one legume species, T. repens, was present in only one plot prior to 2001 (in 1997 and 1999).", "However, the frequency of many legumes has increased since 2008 (L. corniculatus, M. lupilina, Trifolium dubium, T. repens, Trifolium pratense, Vicia sativa and Vicia tetrasperma) with all but V. tetrasperma being detected in 2014.", "In the German study, grasses had only a small effect on community biomass, and Marquard et al. (2009) suggested that these graminoids were being outcompeted by tall forbs and legumes.", "However, our study shows that when nitrogen inputs were high, and grasses more frequent than forbs, mosses and woody species, the productivity was also high.", "If legumes generally make significant contributions to biomass production (Marquard et al., 2009), this may explain why we observed a 100% increase in spring productivity in our exclusion plots in 2014 compared with 2005.", "Marquard et al. (2009) also suggested that tall herbs made a significant contribution to productivity; however, in our studies we recorded few tall herbs in our plots.", "Other studies have also shown that the composition of the grassland plant community can affect above-ground biomass, and that increased species richness can result in increased biomass productivity (Tilman et al., 1996, 2001; Hector et al., 1999; Bullock et al., 2001), and provides a reasonable explanation for the recent yield increases at Wytham.", "A meta-analysis of 44 biomass and species composition experiments showed that mixtures of species produce on average 1.7 times more biomass than species monocultures (Cardinale et al., 2007).", "These results suggest that the precise composition of a community, and how the species with it interact and compete with each other for available resources, is also important.", "Cardinale et al. (2007) also found that as the duration of the study increased, so did the probability that species mixtures would yield more than their most productive species alone.", "This may be another reason why the productivity of our exclusion plots increased in the last two years of the study as species richness increased.", "It may, therefore, take several more years before maximum productivity in our permanent pasture is reached, especially if the diversity of species is increasing.", "All of our managed plots showed an increase in species richness after 2001, albeit to different extents depending on the location of the plot, but in the unmanaged plots there was no such identifiable change in species richness.", "After the cessation of nitrogen inputs, it took two years for species richness to increase in the managed plots, and seven years for the average number of species detected in the plots to double.", "In contrast, after five years of adding nitrogen fertiliser to a sandy grassland in North-eastern China, Shi et al. (2014) found that three years post-fertilisation was long enough for species richness to begin to show signs of recovery.", "Rundlöf et al. (2010) showed that, in Sweden, organic farming resulted in a significantly greater abundance of forbs than in conventional farming.", "Other farm surveys have shown that increasing the rate of nitrogen fertiliser increases the proportion of Lolium spp. at the expense of Agrostis spp. (Jones and Hayes, 1999; Hopkins et al., 1990), white clover (T. repens) and forbs (Hopkins et al., 1990).", "As mentioned above, we showed that clover and other legumes benefitted greatly from the cessation of nitrogen inputs.", "The nitrogen-loving grasses were still detected in a high percentage of the plots, but we did not measure percentage cover so we cannot comment on their abundance within exclusion plots.", "In the USA, Foster and Gross (1998) found that escalating nitrogen inputs increased the dominance of one native grass species (Andropogon gerardi), thus preventing establishment of subordinate forb species and reducing overall species diversity.", "At Wytham, we also observed a change in species composition: some nitrogen loving species such as P. annua disappeared from the exclusion plots, and there was an increase in generalist grasses such as Agrostis spp. and Phleum spp.", "Mountford et al. (1996) observed that at Tadham Moor (Somerset, England) the effects of fertiliser inputs were reversible: low levels (25 kg ha−1) of fertiliser resulted in increased H. lanatus and L. perenne, and significantly reduced species diversity within six years.", "However, once nitrogen additions had stopped, H. lanatus and L. perenne declined in the plots and their place was taken by A. capillaris and C. cristatus.", "Hopkins et al. (2000) corroborated these findings, also suggesting that these botanical changes due to increased nitrogen were to some extent temporary if nitrogen additions ceased.", "Whilst this was the case for the managed plots at Wytham, we found that H. lanatus did not appear in the exclusion plots until the nitrogen applications had stopped, and that H. lanatus then increased rather than decreased.", "In a study in North America examining the effect of cessation of nitrogen fertilisation on species re-colonisation of grassland over a 30 year period, Isbell et al. (2013) reported that, subsequent to 10 years of high nitrogen input, it took 20 years to return for plots to return to control levels of species diversity.", "These studies suggest that the species diversity in our plots may continue to rise for several more years.", "In a study in the Netherlands, Smits et al. (2008) found that maximum species richness was reached 10–15 years after the cessation of fertiliser application and that, after 20 years, species richness then began to decrease.", "These authors attributed this decrease to two reasons: either plant species had reacted to the changing levels in the available nutrients, or atmospheric nitrogen deposition and soil eutrophication had altered the levels of available nitrogen.", "Mountford et al. (1996) estimated that it would take 9 years for grassland that had received nitrogen inputs of 200 kg ha−1 to revert back to its plant species composition prior to fertilisation.", "In our study, all managed plots, including the exclusion plots, were subjected to over 200 kg ha−1 nitrogen each year between 1992 and 2001.", "Over the subsequent 13 years, the species diversity in the managed plots (again including the exclusion plots) increased.", "In the exclusion plots, by 2014, there were more species present than in the unmanaged plots, but in the other managed plots species diversity was considerably lower.", "In our study, 20 years of vegetation monitoring has provided valuable and rare data on the long-term effect of the cessation of nitrogen inputs on grassland.", "We have demonstrated that moving to an organic farm management system is beneficial to plant species diversity in permanent grassland, resulting in a greater number of both grasses and forbs without introducing seed mixtures.", "Although not investigated in this study, it is likely that the increase in plant species diversity will also impact other organisms, especially invertebrates (Duffey et al., 1974).", "We showed that biomass production initially decreased after artificial fertiliser inputs had ended, but that as species diversity subsequently increased, biomass also began to show signs of recovery, possibly due to the increase in plants such as legumes that not only add to the quality of the herbage but can also increase soil fertility (Brockwell et al., 1995).", "By not re-sowing the grassland and using minimal management, i.e. occasional grazing and/or cutting, grassland can recolonise with species local to the area.", "This work suggests that in the medium term, biodiversity can be improved without having to spend time and money improving soil quality, or habitat features, or investing effort to sow imported seed, although it should be noted that this may depend on the history and landscape-context of the grassland in question.", "Increasing biodiversity by this approach acknowledges that there may be a temporary reduction in grassland productivity.", "Grassland monitoring at Wytham as part of the UK ECN programme will continue, and we hope ultimately to be able to determine when maximum species diversity is reached, and whether biomass productivity will eventually reach levels recorded before the conversion to organic farming." ]
[]
Low back pain and its relationship with sitting behaviour among sedentary office workers
The relationships between sedentary lifestyle, sitting behaviour, and low back pain (LBP) remain controversial. In this study, we investigated the relationship between back pain and occupational sitting habits in 64 call-centre employees. A textile pressure mat was used to evaluate and parameterise sitting behaviour over a total of 400 h, while pain questionnaires evaluated acute and chronic LBP. Seventy-five percent of the participants reported some level of either chronic or acute back pain. Individuals with chronic LBP demonstrated a possible trend (t-test not significant) towards more static sitting behaviour compared to their pain-free counterparts. Furthermore, a greater association was found between sitting behaviour and chronic LBP than for acute pain/disability, which is plausibly due to a greater awareness of pain-free sitting positions in individuals with chronic pain compared to those affected by acute pain.
[ "ENGI", "HEAL", "SOCI" ]
[ "Dynamic sitting", "Low back pain", "Office chair", "Pressure distribution", "Sitting behaviour" ]
[ "2201", "2213", "3307", "3612" ]
[ "Today, sedentary lifestyle has become omnipresent, as an increasing number of individuals spend extended periods in a seated position at work as well as during leisure time (Jans et al., 2007; Saidj et al., 2015; Hadgraft et al., 2015).", "Simultaneously, the prevalence of low back pain (LBP) has increased among office workers in general (Ayanniyi et al., 2010; Collins and O'Sullivan, 2015).", "Specifically, call-centre employees have recently become the focus of attention in this field as they spend up to 95% of their total work time in a seated position (Toomingas et al., 2012), but their jobs are also recognised for potentially high levels of stress, especially when dealing with difficult or aggressive customers (Johnson et al., 2005; Oh et al., 2017).", "Since high job-related stress is additionally thought to be related to musculoskeletal disorders of the lower back (Sprigg et al., 2007), it is therefore unsurprising that a higher proportion of call-centre workers report musculoskeletal symptoms than other professional office users (Norman et al., 2004).", "Since LBP represents the third leading cause of self-perceived disability due to various diseases (Vos et al., 2016) and indicates a major economic burden to society (Wieser et al., 2011; Nöllenheidt and Brenscheidt, 2016), identifying risk factors, especially within the office environment, appears to be of high importance for implementing suitable prevention programs.", "While it might be expected that LBP and sedentary office work are highly related, the literature offers only little evidence.", "On the one hand, recent studies report that seated working periods of longer than 7 h per day significantly increase the risk of LBP (Odds Ratio = 1.89) (Cho et al., 2012; Subramanian and Arun, 2017).", "On the other hand, several systematic reviews have failed to prove that sitting duration on its own is linked to the onset of LBP and found no significant association between sitting itself and the risk of LBP in office workers (Chen et al., 2009; Lis et al., 2007; da Costa and Vieira, 2009; Bakker et al., 2009; Kwon et al., 2011; Hartvigsen et al., 2000).", "This lack of evidence is assumed to mainly result from the multifactorial nature of LBP, as well as from possible methodological weaknesses, including unreliable subjective measurement instruments, low measurement durations, and low number of subjects in the scientific literature (Kwon et al., 2011; Hartvigsen et al., 2000), which complicate the establishment of any causal relationships (Hoy et al., 2010).", "Even though associations between sitting duration and LBP seem to be controversial, other aspects of sitting behaviour might have critical links to LBP among office workers.", "Here, Womersley and May (2006) reported that individuals with pain sat uninterrupted for longer periods and showed a more flexed and relaxed sitting posture than pain-free individuals, suggesting that individual sitting habits may be related to LBP, even if the causal links are unclear.", "Despite several investigations into “perfect” sitting positions in terms of the “optimal” spinal curvature during sitting (Waongenngarm et al., 2015; O'Sullivan et al., 2012a; Pynt et al., 2001; Baumgartner et al., 2012; Zemp et al., 2013), broad consensus is lacking, suggesting that the “correct” sitting position might be subject-specific (Claus et al., 2016).", "Moreover, Claus et al. (2009) proposed that any sustained sitting posture could result in fatigue, discomfort and pain, suggesting that a “good” posture could still be detrimental if it persists uninterrupted for extended periods (Coenen et al., 2017).", "As a result, postural variability as well as regular small movements are plausibly beneficial for the prevention of LBP (Davis and Kotowski, 2014; Vergara and Page, 2002; Pynt et al., 2001; Srinivasan and Mathiassen, 2012; Aarås et al., 2000).", "Dynamic sitting behaviour is thought to provide beneficial biological and physiological effects, since postural variations can reduce spinal loads (Davis and Kotowski, 2014) and spinal shrinkage (van Deursen et al., 2000), prevent muscle fatigue through alternating motor unit activation (van Dieën et al., 2001), and inhibit damage to the posterior aspect of the annulus pulposus by means of low magnitude dynamic movements (Callaghan and McGill, 2001).", "Moreover, Straker and Mathiassen (2009) indicated that short periods of inactivity can already cause local changes regarding biomechanical, physiological and neurological capability.", "It therefore appears reasonable that less dynamic sitting habits may result in discomfort and pain, especially in the lower back.", "In previous studies, different measurement technologies such as video analysis (Womersley and May, 2006), accelerometers (Ryan et al., 2011), optoelectronic motion analysis (Dunk and Callaghan, 2005), force sensors (Yamada et al., 2009; Zemp et al., 2016b) and pressure distribution sensors (Zemp et al., 2016a) have all been used to assess sitting behaviour.", "Here, pressure distribution sensors offer a relatively cheap measurement approach that neither disturbs nor affects the subject during measurement, and allows high accuracy for classifying individual sitting behaviour and positions (Zemp et al., 2016a; Kamiya et al., 2008).", "Additionally, pressure mats are easily attachable and therefore offer a practical solution for analysing the sitting behaviour of participants on their own chair.", "A previously conducted pilot study (N = 20) demonstrated tendencies towards a more static sitting behaviour in participants with mild LBP (Zemp et al., 2016a).", "Towards gaining a deeper understanding of the relationships between LBP and occupational sitting habits, the goal of this study was to build on the previous pilot data and establish whether call-centre employees with LBP express different sitting behaviour patterns from those without LBP.", "Since the working task is known to strongly impact on sitting behaviour (van Dieën et al., 2001; Dunk and Callaghan, 2005; Ellegast et al., 2012; Groenesteijn et al., 2012; Grooten et al., 2017), this study selected a large number of participants that worked in an environment with highly standardised working tasks.", "Furthermore, in order to maintain real-world validity, no additional work assignments, or sitting/movement instructions were provided to the participants during the measurement period.", "Therefore, seventy office workers from a professional call-centre company located in Dresden and Leipzig (Germany) were recruited.", "Participants were required to speak German and were excluded if they were pregnant, took glucocorticoids, or were currently undergoing medical treatment for other physical complaints besides back pain.", "All participants provided written informed consent prior to participation in this study, which was conceptualised and performed in accordance with the principals of the declaration of Helsinki and was approved by the ethics committees of the University Potsdam, Germany (no. 42/2014) and confirmed by the ethics commission of ETH Zürich, Switzerland.", "After measurement completion, all participants received 15 Euros compensation and were provided with their individual study results.", "The call-centre environment offers a contemporary office work setting with regard to job assignments and physical organisation.", "Our selected call-centre specifically dealt with difficult and challenging customer situations, and it was therefore assumed that participants were exposed to a considerable mental stress burden (Johnson et al., 2005; Oh et al., 2017).", "The employees' work tasks were highly standardised, comprising typing at a computer and calling clients using a head-set, with nearly all duties undertaken in a sitting position.", "Since the company's work policy required a change of workplace every 3 h, it was not possible to ergonomically adjust the office desk and computer set-up to the individual requirements and preferences.", "This study was conducted within 2 weeks at two different worksites of the call-centre company and each participant was assessed during one complete working shift.", "In order to investigate the true relationships between daily sitting behaviour and LBP, it was essential that the measurements were based on each subjects' real-world performance in the natural office environment, with participant's each using their own office chair, undertaking their own daily office tasks.", "The company provided three different office chair models, which all allowed adaptation of seat height and depth, as well as the option to fix the backrest at a certain angle or allow dynamic reclination.", "After measurement system set-up, data was collected for the entire working shift, including breaks.", "At the end of the working day, calibration measurements for the classification of the different sitting positions were performed.", "Similarly to the preceding pilot study (Zemp et al., 2016a), participants were asked to sit four times in seven predefined sitting positions: upright (P1), reclined (P2), forward inclined (P3), laterally tilted right/left (P4/P5), crossed legs right over left/left over right (P6/P7).", "Afterwards the participants were asked to fill out the questionnaire (section 2.5).", "In order to assess sitting behaviour, spatio-temporal changes in the distribution of pressure across the participants' sitting interface were monitored by means of the textile pressure mat “sensomative science” (sensomative GmbH, Rothenburg, Switzerland) consisting of a 196 (14 × 14) sensor matrix with a size of 45 cm × 45 cm (www.sensomative.com).", "The pressure data were recorded at 1.5 Hz with a resolution of 8 bits and a maximum pressure limit of 60 kPa.", "Using Bluetooth Low Energy, the data were transferred from the textile mat to a connected mobile phone (Nexus 5X, Google, LG, Seoul, Korea) where the data were stored in the corresponding mobile phone application.", "In order to prevent the mat from sliding, the system was laterally fixed with two textile straps and belt loops (Fig. 1).", "Due to the pressure mats’ thin and flexible nature, participants were not able to feel its presence.", "In order to gather information about short- and long-term pain status, including corresponding functional limitations, as well as sociodemographic information, the following standardised questionnaires were used:", "To assess pain intensity and pain related functional limitations in the previous three months, participants were requested to complete the Chronic Pain Grade (CPG) questionnaire (Von Korff et al., 1992), which is divided into two subscales: (1) Korff characteristic pain intensity (CPI) and (2) Korff disability (DISS).", "Each CPG item ranged from 0 (“no pain/impairment”) to 10 (“worst possible pain”/“I wasn‘t able to do anything”).", "For data analysis, items of each subscale were presented on a scale ranging from 0 to 100.", "Missing or inconsistent data were treated according to the CPG recommendations.", "The Brief Pain Inventory (BPI) (Radbruch et al., 1999) was used to estimate subjects’ acute LBP within the previous 24 h. Similar to the CPG questionnaire, the BPI is subdivided into two subscales: (1) pain severity (BPISeverity) and (2) pain-related interference (BPIInterfere) of daily functions.", "Answering possibilities for the BPI ranged from 0 (“no pain”/“no interference”) to 10 (“pain as bad as you can imagine”/“interferes completely”) (Daut et al., 1983).", "The BPI also included a body chart to illustrate each participant's pain area(s), which allowed confirmation (or otherwise) of pain in the lower back region.", "Missing or inconsistent data were treated according to the BPI recommendations.", "Data processing and analysis was performed similarly to the pilot study of Zemp et al. (2016a), which is only briefly described below:", "The four pain variables (CPI, DISS, BPISeverity, BPIInterfere) were used to allocate participants into either subgroup A: no pain; no functional disability, or into subgroup B: with pain; with functional disability.", "Thereby, all participants with scores of 0 were allocated to subgroup A and all participants with scores greater than 0 were assigned to subgroup B.", "Raw pressure data were analysed using MATLAB (R2017a MathWorks Inc., Natick, USA).", "The random forest classification approach was applied to determine the sitting position of each subject at any instant during the entire working day (Zemp et al., 2016a, 2016b).", "The calibration measurements of all participants were used to create one general random forest classifier.", "Here, all pressure values of every calibration measurement were normalised to the maximal value of the 196 sensors, and an ensemble of 500 decision trees was used while all other parameters were kept at MATLAB's default levels.", "In order to quantify the reliability of the sitting position classifier within this study, a leave-one-out (LOO) cross-validation was performed.", "Here, the calibration measurements of all participants except one was used as training data and the remaining measurement was used for validation.", "The classified sitting positions were then identified as correct or incorrect.", "This procedure was repeated for every calibration measurement in order to quantify the overall classification accuracy.", "This study included 70 call-centre employees, from which six participants (8.6%) were excluded due to participation withdrawal (N = 2) or incomplete data sets of the measured pressure distribution (N = 4), resulting in a study sample of 64 participants (43 ± 13 years; 78 kg ± 21 kg; 170 cm ± 10 cm; 40 females).", "Furthermore, two subjects could not be included for analysis of the CPI and BPISeverity due to inconsistent and/or missing entries resulting in a total of 62 participants for the CPI and BPISeverity, and a total of 64 participants for the DISS and BPIInterfere grouping variable.", "The two subscales of the CPG and BPI questionnaires showed good internal consistencies with Cronbach's alpha values of 0.92 (CPI), 0.92 (DISS), 0.94 (BPISeverity) and 0.92 (BPIInterfere).", "Overall, the majority of participants reported some level of either chronic or acute back pain (N = 48, 75%), with an average low to medium pain intensity (CPI = 39.49 ± 20.01; BPISeverity = 2.13 ± 1.73) and related disability (DISS = 28.75 ± 19.06; BPIInterfere = 2.22 ± 1.69).", "Moreover, the findings indicated a large variability within all four pain groupings (Table 1).", "Since most of the participants preferred to work with a fixed backrest, the positions “upright” (P1) and “reclined” (P2) were considered as one and the same position.", "The random forest classifier demonstrated an overall classification accuracy of 90% (Table 2) ranging from 70% up to 100% for the different participants.", "The p-values and Cohen's effect size of the two-tailed independent t-tests for SitBePar and the four grouping variables indicated that the relationship between sitting behaviour and chronic pain grouping variables (CPI: p = 0.052, d = 0.579; DISS: p = 0.076, d = 0.471) was higher than the relationship between SitBePar and acute pain conditions (BPISeverity: p = 0.625, d = 0.120; BPIInterfere: p = 0.253, d = 0.291).", "Participants experiencing chronic LBP showed a lower overall percentage of transient periods (25.69 ± 11.69%) compared to pain-free participants (35.23 ± 14.55%) indicating a moderate effect (p = 0.011, d = 0.723).", "Similarly, a moderate effect (p = 0.036, d = 0.544) was observed between participants who felt disabled due to chronic LBP (DISS) (25.64 ± 12.28%) and corresponding non-disabled counterparts (32.75 ± 13.81%) (Figs. 2 and 3; Appendix: Table A1).", "Mean values of almost all four sitting behaviour parameters for the four pain groupings indicated that participants with pain and pain related disability demonstrated a rather static sitting behaviour compared to their pain-free counterparts (Appendix: Table A1).", "This study aimed to analyse the relationships between sitting behaviour and LBP by investigating the sitting habits of call-centre workers whose assignments were undertaken in an almost continuous sitting position.", "We found a small association between general sitting behaviour and participants reporting chronic LBP and/or pain related functional disability.", "These observations over extended periods were consistent with reported sitting activity over short periods (1hr), where subjects also exhibited more frequent postural shifts than chronic LBP workers (Akkarakittichoke and Janwantanakul, 2017).", "The lack of a stronger relationship between LBP and sitting behaviour was most likely due to the highly multifactorial causality of LBP, including socio-psychological and physiological factors (Hoy et al., 2010).", "Another reason could be the complex and largely individual sitting habits, which are known to vary considerably among office workers (Goossens et al., 2012; Zemp et al., 2016a), thereby producing inhomogeneity in sitting behaviour.", "Since almost all analysed sitting behaviour parameters for all pain groupings showed more dynamic activity in pain free participants, this study indicates a possible trend (0.011 < p < 0.453) towards a more static sitting behaviour among the majority of participants perceiving pain and/or suffering from pain related disability.", "These results are in line with several studies showing that participants with LBP or lumbar discomfort exhibit a more static sitting behaviour by demonstrating less micro-movements and longer periods of uninterrupted sitting (Zemp et al., 2016a; O'Keeffe et al., 2013; O'Sullivan et al., 2012b; Vergara and Page, 2002; Womersley and May, 2006).", "A reasonable explanation for this observation could be the so-called “fear-avoidance behaviour” (Vlaeyen and Linton, 2000) meaning that, for instance, regular movements or positional alternation are reduced or avoided due to fear of experiencing pain (Vlaeyen et al., 2016).", "In this study, a greater association between sitting behaviour and LBP was found for participants in the chronic LBP and/or related disability grouping than for those with acute pain/disability.", "It is therefore plausible that participants with chronic pain have a higher level of awareness to pain-free sitting positions and pain provoking movements compared to individuals affected by acute pain.", "Such an habitual awareness could result in fewer transitions between sitting positions as well as a reduction in small movements, indicating a type of avoidance learning based on their pain history (Krypotos, 2015).", "A similar phenomenon was reported by Panhale et al. (2016) who found a strong correlation between fear-avoidance belief and activity limitation in patients with chronic LBP; hence reflecting an underestimated impact of fear avoidance beliefs on the patient‘s behaviour.", "Overall, transient periods were lower in participants with chronic LBP than pain free participants, indicating reduced movement throughout their working day.", "Since less frequent postural shifts have previously been observed among subjects with chronic LBP compared to healthy participants (Akkarakittichoke and Janwantanakul, 2017), it is likely that dominant drivers of sitting behaviour may exist that are related to chronic LBP.", "Although these results do not allow any conclusions nor definite statements to be drawn regarding a possible causal relationship or adaptational process among individuals with chronic LBP, a more static sitting behaviour is generally known to have physiological and biological consequences.", "Sustained pressure under the buttocks due to prolonged, uninterrupted sitting could be reduced by varying posture (Søndergaard et al., 2010; Vergara and Page, 2002; Zemp et al., 2015, 2016c, 2019) by means of e.g. regular pelvis rotations (van Geffen et al., 2008).", "Moreover, since continuous compression on an intervertebral disc can result in reduced disc nutrition (Kingma et al., 2000; Pynt et al., 2001) frequent postural movements are also recommended through lordosis and kyphosis.", "In this manner, sufficient metabolic balance of various musculoskeletal structures can be supported, including a reduction of ischaemic effects due to prolonged static sitting (Reenalda et al., 2009; Todd et al., 2007).", "O'Sullivan et al. (2013) defined dynamic sitting as “increased motion in sitting, which is facilitated by the use of specific chairs or equipment”.", "However, recent studies have shown that dynamic chair equipment is not sufficient to affect muscle activation, postures and core kinematics (Ellegast et al., 2012; O'Sullivan et al., 2013; Grooten et al., 2017; Kingma and van Dieën, 2009).", "Therefore, it can be concluded that dynamic sitting should be actively stimulated, which can then be supported by dynamic chair mechanisms, indicating the requirement for a redefinition of dynamic sitting (Pynt, 2015).", "Such a redefinition, however, would require valid information in terms of movement patterns and positional changes that reflect a normal physiological sitting behaviour, which has not yet been fully established.", "In order to enhance a more dynamic sitting behaviour among office employees, technical devices supporting dynamic sitting have recently been discussed.", "Several studies (Haller et al., 2011; Goossens et al., 2012; Davis and Kotowski, 2014) have demonstrated devices capable of monitoring behaviour and providing feedback for avoiding discomfort and musculoskeletal disorders at an early stage, with the aim to change sitting patterns among office workers.", "However, Roossien et al. (2017) reported that tactile feedback integrated into an office chair was unable to change sitting behaviour, suggesting that such feedback is insufficient for reducing musculoskeletal discomfort of office workers on a sustained basis.", "Their study used feedback signals to improve sitting duration in an “optimal supported posture”, instead of facilitating regular small movements, for instance.", "Therefore, tactile input combined with visual features may potentially promote more beneficial sitting habits than either alone (Straker et al., 2013).", "The textile pressure mat “sensomative science“ used in the presented study, includes a corresponding smart phone application providing visual feedback of the current pressure distribution while sitting.", "Given further development in the application, for instance by enabling regular tactile or visual feedback to the user (smart phone or watch) to initiate small movements, could have potential for more dynamic sitting behaviour in sedentary office workers.", "In the present study only one working shift per employee was analysed, which might not comprehensively reflect the complete patterns of sitting behaviour in each individual.", "Our study aimed to achieve a general impression of sitting behaviour characteristics among a sedentary population of office workers.", "Since call-centre work is predominantly repetitive and inactive in nature (Thorp et al., 2012; Toomingas et al., 2012; Straker et al., 2013), call-centre employees were chosen as the focus group of our study.", "Indeed, observations during the ongoing measurements indicated that 95% of the working shift was indeed seated, except for scheduled breaks of 15–30 min within a working day – data that is highly consistent with the observations of Toomingas et al. (2012).", "This regular schedule indicated that our participants' work was not only highly sedentary, but also largely equivalent among all workers, which reduced confounding effects.", "It is also important to consider that, contrary to ergonomic guidelines, office tables and chairs were not ergonomically adjusted to each individual since the company's structure required regular workplace rotations within the office.", "To ensure ecological validity, the researchers did not intervene with the provided chair/table placement or rotation schedule.", "Nevertheless, the experimental setup including chair instrumentation, providing study information as well as the presence of the investigators could have altered the working routine.", "From the results of our study, it seems that levels of LBP in call centre employees are only partially linked to sitting behaviour itself, and that the multifactorial nature of LBP is therefore possibly more associated with sedentary lifestyle or other factors such as job tenure, daily working hours, general fitness, customer reaction, and psychological stress etc.", "When examining sitting behaviour and its relationship with LBP, future studies should therefore consider these confounding variables, as well as different levels of pain intensity on a more continual scale.", "Since our study mainly analysed participants exhibiting on average low to medium back pain as well as pain related disability, such a detailed classification was not included and should therefore be addressed in future studies.", "An alternative classification of individuals with non-specific LBP can define subgroups similar to those presented by O'Sullivan (2005), distinguishing between mechanically and non-mechanically triggered pain.", "In this way, different pain drivers can be considered when analysing the relationship between sitting behaviour and LBP.", "Due to the highly sedentary working conditions, call-centre employees are at particular risk of developing musculoskeletal disorders.", "As a result, these population-related characteristics need to be carefully considered when generalising these findings to the majority of office workers.", "With scientific discussion to date leading to unclear relationships between sedentary lifestyle, sitting behaviour, and low back pain, we have provided standardised (real-world) conditions to investigate whether sitting behaviour and LBP are inherently linked.", "Our results show a possible trend towards more static sitting behaviour among call-centre workers with chronic LBP pain and pain related disability.", "A greater association was found between sitting behaviour and chronic LBP than for acute pain/disability, which was a possible result of the fact that participants with chronic pain have a higher level of awareness to pain-free sitting positions and pain provoking movements compared to individuals affected by acute pain." ]
[ "Sitting behaviour was analysed in 64 office employees using textile pressure mats.", "The overall classification accuracy for the different sitting positions was 90%.", "Subjects with chronic low back pain tended to show a more static sitting behaviour." ]
Hepatocyte nuclear factor 4α regulates megalin expression in proximal tubular cells
Hepatocyte nuclear factor 4α (HNF4α) is a member of the nuclear receptor superfamily and upregulates expression of many genes in the liver, pancreas, small intestine, and colon. HNF4α is also highly expressed in proximal tubular epithelial cells (PTECs) in kidney. PTECs reabsorb various substances through transporters, ion channels, and receptors, but the target genes for HNF4α in PTECs have not been investigated in detail. In the present study, we aimed to identify novel HNF4α target genes that are highly expressed in PTECs. Expression of many solute carrier transporter genes was upregulated by HNF4α in human PTEC-derived HK-2 cells. Notably, expression of megalin (LRP2), an endocytic receptor of various molecules involved in development and progression of chronic kidney disease (CKD), was strongly induced by HNF4α and the transactivation potential of the megalin promoter was dependent on HNF4α expression. Moreover, HNF4α was found to directly bind to an HNF4α binding site near the transcription start site in the megalin gene. These results indicate that HNF4α plays an important role in maintaining reabsorption and metabolism in PTECs by positive regulation of several solute carrier transporter and megalin genes at the transcriptional level.
[ "BIOC" ]
[ "Hepatocyte nuclear factor 4α", "Kidney", "Megalin", "Proximal tubule" ]
[ "1303", "1304" ]
[ "Hepatocyte nuclear factor 4α (HNF4α), an orphan member of nuclear receptor superfamily, is highly expressed in epithelial cells in liver, pancreas, small intestine, colon, and kidney, and HNF4α is essential for maintenance of the homeostasis in these tissues [1,2].", "HNF4α binds to approximately 40% of the promoter region of the genes expressed in human hepatocytes and pancreatic islets [3], indicating that HNF4α is a strong regulator in the liver and pancreatic.", "Since Hnf4a null-mice results in embryonic lethality [4], tissue-specific Hnf4a-null mice were generated in hepatocytes, pancreatic β cells, and intestinal epithelial cells.", "Based on many studies using liver-specific Hnf4a-null mice, hepatic HNF4α was found to be a central regulator for hepatocyte differentiation and function though direct regulation of many liver-specific genes [5–9].", "In addition, pancreatic β cell-specific Hnf4a-null mice impaired glucose-stimulated insulin secretion [10].", "Mutations in HNF4α gene related to maturity-onset diabetes of the young 1 (MODY1) [11], suggesting that pancreatic β cell-specific Hnf4a-null mice exhibited the similar phenotype as MODY1.", "Also, intestinal epithelial cell-specific Hnf4a-null mice were liable to get inflammatory bowel disease (IBD) [12].", "Thus, HNF4α plays an essential role in maintenance of specific function in these tissues, but kidney-specific Hnf4a-null mice have not generated yet.", "In kidney, HNF4α is highly expressed in proximal tubular epithelial cells (PTECs) [13,14].", "The proximal tubule is a part of the nephron of the kidney, and the main function of the proximal tubule is reabsorption of many substances such as water, glucose, potassium and sodium ions, phosphate, amino acids, and proteins filtrated in glomerulus.", "PTECs play an important role in renal injure and repair in acute kidney injury (AKI) and chronic kidney disease (CKD) progression [15–17].", "Many transporters such as solute carrier (SLC) transporters and ATP-binding cassette transporters are expressed in the proximal tubule to reabsorb and excrete biomolecules.", "For example, sodium glucose cotransporter 2 (SGLT2), as known SLC5A2, is expressed in the proximal tubule, and Sglt2-deficient mice exhibit glycosuria and decreased glucose reabsorption [18].", "Thus, many SGLT2 inhibitors are on the market for type 2 diabetic medications.", "Moreover, several studies were reported that HNF4α regulates expression of the transporters.", "Expression of Slc6a19 gene, the neutral amino acid transporter in enterocytes, is transactivated by HNF4α and HNF1α [19].", "HNF4α was also shown to bind to the promoter regions of Slc22a1, Slc22a6, and Slc22a8 genes in rat kidney [20], indicating that HNF4α could play a central regulator of the transporters in the proximal tubule.", "However, the detailed regulation of the proximal tubule-enriched genes including the transporters by HNF4α remains poorly unexplained.", "In the present study, we investigated to identify novel HNF4α target genes that are highly expressed in PTECs by overexpression of HNF4α in human HK-2 cells that express many characteristics of PTECs [21].", "We found that expression of many transporters was induced by HNF4α, but expression of megalin (known as LRP2), a receptor for a large number of ligands, was strongly induced by HNF4α.", "Promoter activity of megalin gene was dependent on an HNF4α binding site and HNF4α expression, and HNF4α directly bound to the HNF4α binding site.", "Because megalin is involved in CKD development and progression, these finding may contribute to a detailed understanding of CKD development and progression through direct regulation of megalin expression by HNF4α.", "HK-2 and HEK293T cells were cultured at 37 °C in Dulbecco's modified Eagle's medium (Wako) containing 10% fetal bovine serum (HyClone) and 100 units/ml penicillin/streptomycin (Thermo Fisher Scientific).", "Full-length of human HNF4α cDNA was cloned into EcoRI and BamHI sites of pCMViR [22] and pEBMulti-Hygro vectors (Wako).", "pCMViR/HNF4α or empty plasmids were transfected into HK-2 cells cells using Fugene HD (Roche) and HEK293T cells using polyethyleneimine Max (Polyscience) as transfection reagents.", "After 48 h, the cells were harvested with Isogen II (Wako).", "Total RNA was extracted from the Isogen II solution and was transcribed using ReverTraAce qPCR RT Master Mix with gDNA Remover (TOYOBO).", "cDNA was used for Real-time PCR using Power SYBR Green Master Mix (Thermo Fisher Scientific) with the specific primers on a LightCycler 480 system II (Roche).", "Levels of mRNA expression were normalized relative to TATA-binding protein (Tbp) mRNA as an internal control using ∆∆Ct method.", "Sequences for the primers are shown in Supplementary Table 1.", "The -3996, -3135, -2071, -806, -539, -104/+200 fragments from the transcription start site of the human megalin promoter were amplified with genomic DNA from A549 cells by PCR and cloned into the luciferase reporter vector, pGL4.11 (Promega).", "Mutations were introduced into the HNF4α binding sites in the megalin promoter by inverted PCR based site-directed using the primers, 5′-agggttgcagggggcgggccgggcg-3′ and 5′-cagcgcggggaggagtgggcactcgaa-3′.", "The induced mutations at -1/+4 in the megalin promoter are indicated as bold and underlined.", "Wild-type, or mutated megalin promoters were cloned into pGL4.11, and pGL4.74 encoding Renilla luciferase as an internal control were co-transfected into HEK293T cells in the presence or absence of HNF4α expression plasmid.", "After 48 h, promoter activities were measured using Dual-Glo Luciferase Assay System (Promega).", "Whole cell lysates from cultured cell lines were also prepared as described previously [8].", "The samples were diluted with Laemmli sample buffer, incubated at 65 °C for 15 min, fractionated by 10% SDS-polyacrylamide gel electrophoresis.", "The gels were transfered onto a PVDF membrane (GE healthcare).", "The membrane was incubated for 1 h with PBS containing 0.1% Tween 20% and 5% skim milk, and then incubated for 1 h with anti-HNF4α (Perceus Proteomics) and anti-γ-tubulin (Sigma-Aldrich) antibodies.", "After washing, the membrane was incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology), and the reaction product was visualized using Western Lightning Ultra (PerkinElmer).", "Electrophoretic mobility shift analysis was carried out using LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific) and nuclear extracts from HNF4α-transfected cells.", "The following double-stranded probes were used (mutations are indicated as bold and underlined); the HNF4α binding site at -13/+17 in the human megalin promoter (wild-type (WT); 5′-ctccccgcgctgcaaagtgcagggggcggg-3′ and 5′-cccgccccctgcactttgcagcgcggggag-3′, mutant (Mut); 5′-ctccccgcgctgagggttgcagggggcggg-3’ and 5’- cccgccccctgcaaccctcagcgcggggag-3’) and the HNF4α binding site at -203/-192 in the mouse ornithine transcarbamylase (Otc) promoter as a positive control (5′-gttaggcttaaagttcaagtg-3′ and 5′-cacttgaactttaagcctaac-3′) [7].", "Nuclear extracts (3 μg) and the 5′-biotin labeled probe of the megalin promoter (WT) were added and the reaction mixture incubated on ice for 10 min.", "For competition experiments, a 50-fold excess of the unlabeled megalin (Mut) or Otc probes was added to the reaction mixture and the mixture was incubated on ice for 10 min prior to the addition of the 5′-biotin labeled probe.", "For supershift analysis, 1 μg of anti-HNF4α or anti-PPARβ antibodies (Santa Cruz Biotechnology) was added to the reaction mixture, and the mixture was incubated on ice for 10 min after the addition of the 5′-biotin labeled probe.", "DNA-protein complexes were fractionated by 7% PAGE, and blotted onto a Hybond-N+ membrane (GE healthcare).", "After washing, DNA-protein complexes were visualized using detection module in the kit.", "HNF4α-transfected HEK293T cells were fixed in 1% formaldehyde for 10 min at room temperature and chromatin immunoprecipitation was carried out using SimpleChIP Plus Enzymatic IP kit (Cell Signaling Technology) and anti-HNF4α antibody (Santa Cruz Biotechnology).", "Purified DNA was amplified by real-time PCR using ΔΔCt method.", "Enrichment of the HNF4α binding site was normalized to the input samples and expressed as fold-enrichment as compared to the control normal IgG antibody.", "Nucleotide sequences of the primers are as follows: megalin gene with HNF4α binding site (5′-ggggttcagtaatcggaaga-3′ and 5′-gtgacaggacagcgaggtg-3′), megalin gene without HNF4α binding site (5′-ggggttcagtaatcggaaga-3′ and 5′-gtgacaggacagcgaggtg-3′), OTC gene with HNF4α binding site (5′-aaatgaggaggccaggcaa-3′ and 5′-ggttagagatactgcagggca-3′), and OTC gene without HNF4α binding site (5′-tggcaataccacactgtttagt-3′ and 5′-ctgaaccacaaggaccccaa-3′).", "All values are expressed as the mean ± standard derivation (S.D.).", "HNF4α upregulates many genes in liver, pancreas, small intestine, and colon [3,6–10,12].", "PTECs also highly express HNF4α, but involvement in PTEC function by HNF4α has not been investigated in detail.", "Thus, we analyzed to identify novel HNF4α target genes that are highly expressed in PTECs.", "By overexpression of HNF4α in human PTEC-derived HK-2 cells, we investigated whether HNF4α has a potential to induce the expression of proximal tubule-enriched genes.", "When HNF4α expression vector was transfected into HK-2 cells, HNF4α mRNA and protein was strongly induced (Fig. 1A and B).", "Then, we compared expression of 82 genes including transporters, ion channels, and receptors that are highly expressed in the proximal tubules.", "Of these, expression of 78 genes was induced 0.5–1.5-fold, or no expression by HNF4α (Supplementary Table 2).", "Expression of 4 genes including SLC4A1, 7A7, 16A4, and megalin was increased more than 1.5-fold by HNF4α (Fig. 1C).", "Similar results were observed in HEK293T cells (Fig. 1D-F).", "In rat kidney, HNF4α was shown to bind to Slc22a1, Slc22a6, and Slc22a8 promoters [20], but the expression of these genes was not induced by HNF4α, or no expression in HK-2 cells (Supplementary Table 2).", "Of these, a noteworthy result was that expression of megalin was markedly induced more than 5- and 15-fold by HNF4α in HK-2 and HEK293T cells, respectively (Fig. 1C and F).", "Megalin plays an important role in reabsorption of albumin and low molecular weight proteins by endocytosis [23].", "Interestingly, because megalin is invovled in obesity/metabolic syndrome-related CKD [16], we investigated whether HNF4α directly transactivates the expression of megalin.", "By the JASPAR, an open access database for transcription factor binding sites, five HNF4α binding sites with high score were expected in the human megalin promoter region from -4000 to +200 from the transcription start site.", "Thus, promoter analysis was performed to determine whether HNF4α can transactivate the megalin promoter (Fig. 2A).", "The mouse ornithine carbamylase (Otc) gene containing functional two HNF4α binding sites was strongly transcativated by HNF4α [7].", "The megalin promoters of -3996/+200 fragment containing five HNF4α binding sites and -3135 and -2071/+200 fragments containing two HNF4α binding sites at -1525/-1511 and -6/+ 9 were transactivated by approximately 15–20 fold by HNF4α (Fig. 2A).", "The -806/+200 fragment containing only one HNF4α binding site at -6/+9 was still transactivated by HNF4α, but the promoter activity was decreased by approximately 5-fold, indicating that two regions at -1525/-1511 and -6/+9 might be essential for transactivation of the megalin gene by HNF4α.", "Thus, mutations were introduced into these HNF4α binding sites, resulting that the promoter activity of the mutant at -6/+9 (MT1) was decreased by approximately 80%, but the mutant at -1525/-1511 (MT2) had no significant difference in the promoter activity compared with wild-type promoter (Fig. 2B).", "These data indicate that the region at -6/+9 of the megalin promoter is a critical cis-element for transactivation by HNF4α.", "The binding site at -6/+ 9 of the megalin gene was highly conserved among species (Fig. 3A).", "To confirm the direct binding of HNF4α at -6/+9 in the megalin promoter, electrophoresis mobility shift analysis was performed (Fig. 3B).", "Nuclear extracts from HNF4α-transfected HEK293T bound to biotin-labeled probe including the -6/+9 fragment of the megalin promoter (lane 2, the lower open arrow).", "This complex band was eliminated by the addition of excesses of unlabeled probe containing an HNF4α binding site in the mouse Otc promoter (lane 3) and the unlabeled megalin probe containing the -6/+9 fragment (lane 4), but not the unlabeled megalin probe whose mutations were introduced into the HNF4α binding site at the -6/+9 region (lane 5).", "The complex was supershifted by anti-HNF4α antibody, not by anti-PPARβ antibody (lanes 6 and 7, the upper closed arrow), indicating that HNF4α directly binds to the megalin promoter.", "Furthermore, chromatin immunoprecipitation analysis using HNF4α-transfected HEK293T cells showed that HNF4α indeed bound to the HNF4α binding sites of the OTC promoter as the positive control (Fig. 3C).", "HNF4α strongly bound to the megalin promoter region, suggesting that HNF4α directly and physiologically binds to the promoter region of the megalin gene.", "HNF4α is a master regulator in liver, and many function and the target genes of HNF4α have been identified [7–9].", "Similarly, function of HNF4α in pancreatic β-cells and intestinal epithelial cells was investigated [10,12].", "HNF4α also highly expresses in PTECs in kidney, but function of HNF4α in the PTECs has been poorly investigated due to lack of proximal tubule-specific Hnf4a-null mice.", "Thus, we aimed to identify novel HNF4α target genes that are highly expressed in the PTECs by HNF4α overexpression system.", "Expression of 3 SLC transporters for chloride/bicarbonate (SLC4A1) [24], cationic amino acids/neutral amino acids (SLC7A7) [25], and orphan monocarboxylate (SLC16A4) [26], was upregulated more than 1.5-fold by HNF4α in human PTEC-derived HK-2 cells, these upregulated SLC transporters may be the novel target genes for HNF4α in the PTECs.", "HNF4α binds to Slc22a1, Slc22a6, and Slc22a8 promoters in rat kidney [20], but the expression of these genes was not induced by HNF4α, or no expression in HK-2 cells.", "Thus, HNF4α only binds to these promoters, and transactivation of these genes may not be induced in human PTECs.", "Interestingly, expression of megalin was strongly induced by HNF4α.", "Megalin was first discovered as the pathological antigen of Heymann nephritis in the rat proximal tubule and cloned as gp330/megalin that is a large glycoprotein belonging to LDL receptor superfamily [27,28].", "Subsequently, megalin was found to be a receptor for a large number of ligands such as calcium ion, retinol-binding proteins, vitamin D-binding proteins, albumin, transthyretin, and liver-type fatty acid binding protein [29].", "Whole body megalin-null mice indeed result in low molecular weight proteinuria of serum carrier proteins such as retinol-binding proteins and vitamin D-binding proteins, and the similar symptoms occur in Fanconi syndrome [30].", "Kidney-specific megalin-deficient mice also exhibit decreased plasma vitamin D, probably due to decreased uptake of vitamin D-binding protein/25-OH vitamin D3 complex, and result in hypocalcemia and severe osteopathy, suggesting that megalin is a critical receptor for calcium homeostasis and bone metabolism [31].", "Mutations in megalin gene were found in families with Donnai-Barrow syndrome and facio-oculo-acoustico-renal syndrome, revealing that megalin is an important regulator of many compounds in the bloodstream [32].", "Interestingly, PTECs in high-fat diet fed wild-type mice, but not kidney-specific megalin-null mice, showed autolysosomal dysfunction with autophagy impairment, indicating that megalin might be associated with CKD development and progression [33].", "Although transcriptional regulation of megalin have not been investigated in detail, it was reported that peroxisome proliferator-activated receptor α (PPARα) and γ (PPARγ) positively regulates the expression of megalin through the binding sites in the promoter region [34].", "However, expression of megalin was only about 2-fold induced by the ligands for PPARs, indicating that other transcription factors synergistically regulate expression of megalin gene with PPARα/γ.", "Because HNF4α induced megalin expression about 15-fold, HNF4α may be an important factor to transativate the megalin gene in PTECs.", "Several studies have reported that HNF4α and PPARα could share the binding sites in the promoter regions [35].", "For example, HNF4α and PPARα bind to the same sequence in acyl-CoA thioesterase 1 (Acot1) promoter [36].", "In the report, expression of Acot1 was upregulated by treatment of ligand for PPARα, but the expression of Acot1 is repressed in the presence of HNF4α.", "Conversely, PPARα was shown to bind to multiple binding sites in the megalin promoter [34], but no transactivation by HNF4α was observed through these binding sites in this study.", "HNF4α bound to the other region including the transcription start site and transactivated the expression of megalin, indicating that HNF4α and PPARα may positively regulate the expression of megalin though different binding sites in PTECs.", "Because saturated and unsaturated free fatty acids and their derivatives are endogenous ligands for PPARα [37] and megalin uptakes albumin-bound fatty acids into PTECs, PPARα might be transactivated by these fatty acids uptaken through megalin.", "Because of expression of PPARα is positively regulated by HNF4α [38], HNF4α may play a dual role in megalin expression through direct regulation of megalin itself and indirect regulation of megalin by PPARα.", "In conclusion, HNF4α was found to upregulate many genes including transporters and megalin that are highly expressed in PTECs.", "Of these, HNF4α strongly upregulated megalin expression through an HNF4α binding site in the promoter region.", "Because dysfunction of transporters and megalin causes many diseases including CKD, HNF4α may play a critical role in maintaining the function of PTECs.", "This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant–in-Aid for Scientific Research, No. 25460490).", "The authors declare no conflicts of interest associated with this manuscript." ]
[ "HNF4α upregulates expression of several SLC transporters that are highly expressed in proximal tubular epithelial cells.", "HNF4α upregulates expression of magalin, multifunctional endocytic receptor in human proximal tubular epithelial cells.", "Transactivation of megalin gene is dependent on HNF4α expression and an HNF4α binding site in the megalin promoter." ]
Data supporting the activation of autophagy genes in the diabetic heart
This data article contains full list of autophagy related genes that are altered in diabetic heart. This article also shows data from in vitro cultured cardiomyocytes that are exposed the high glucose treatment to simulate hyperglycemic state in vitro. The interpretation of these data and further extensive insights into the regulation of SG biogenesis by AMPK can be found in "Type-2 diabetes increases autophagy in the human heart through promotion of Beclin-1 mediated pathway" (Munasinghe et al., in press) [1].
[ "MULT" ]
[]
[ "1000" ]
[ "RT profiler array showed marked activation of several autophagy related genes with beclin-1 being markedly increased compared to other genes (Table 1).", "Importantly, exposure of adult cardiomyocytes to high glucose markedly increased the level of beclin-1 within 24 h with a peak increase at 48 h (Fig. 1A).", "Importantly, the caspase activation which indicates cell death followed the beclin-1 activation (Fig. 1B).", "Male obese leptin-receptor mutant BKS.Cg-+Leprdb/+Leprdb/OlaHsd mice (Harlan, UK) were used as a model of insulin-resistant type-2 DM.", "Elevations of blood glucose begin at four to six weeks in these mutant mice.", "Age matched lean mice (BKS.Cg-m+/+Leprdb/OlaHsd) were used as control.", "These mice best represent the human model of type-2 diabetes [2,3].", "Total RNA was extracted from the left ventricle of 8-weeks old diabetic and non-diabetic lean mice using Trizol, according to the manufacturer׳s instructions (Invitrogen, UK).", "After confirming the purity and integrity of the total RNA, cDNA was prepared from 1 µg of total RNA by Transcription Kit (Qiagen, UK).", "The activation of apoptotic genes was then evaluated using murine RT-profiler PCR autophagy array (Qiagen, UK) using a light cycler (Roche 480, UK).", "Data were analyzed using the software package from Qiagen and expressed as fold-changes to control.", "Fold change of ≥2 was considered significant [1,4,5].", "The male Wistar rats were killed by cervical dislocation, the heart dissected and rinsed in cold solution A containing (in mM): 137 NaCl, 5 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 20 N-hydroxyethylpiperazine-N′-2-ethanesulphonic acid (HEPES), 16 glucose, 5 Na pyruvate and 1.8 MgCl2 (pH 7.25 with NaOH)+0.75 mM CaCl2.", "The heart was cannulated via the aorta and perfused for 4 min with solution A+0.75 mM CaCl2 (all perfusing solutions were oxygenated and maintained at 37 °C).", "This was followed by a 4-min perfusion with solution A+0.09 mM ethylene glycol-bis (β-aminoethyl ether) N,N,N,N′-tetraacetic acid (EGTA).", "Next the heart was digested with 50 ml of enzyme solution containing: solution A+0.09 mM EGTA, 50 mg collagenase (Worthington Biochemical Corporation, Lakewood, New Jersey, USA.", "Type I), 5 mg protease (Sigma, Poole, Dorset, UK.", "Type IV), with (glutamate loaded) or without (control) 6.4 mM potassium L-glutamate until the tissue felt soft.", "There was a final 4-min perfusion with solution A+0.15 mM CaCl2 before the ventricles were cut down and sliced.", "The sliced ventricles were suspended in approximately 20–25 ml solution A+0.15 mM CaCl2 and shaken for 6 min at 37 °C.", "After filtration, cells were allowed to sediment, the supernatant was discarded, and the remaining cell layer suspended in solution A+0.5 mM CaCl2.", "This sedimentation, removal of supernatant and resuspension step was repeated, but this time the cells were suspended in solution A+1 mM CaCl2.", "This technique typically produced a yield of over 90% rod-shaped cells with the ability to exclude Trypan Blue [6].", "The resulting cells were then washed separately with medium 199 (Invitrogen) supplemented with 0.2% BSA, 10% FBS, 5 mM creatine, 5 mM taurine, 2 mM carnitine, 10 µM cytosine-D-arabinofuranoside (all from Sigma chemicals), ITS and antibiotics (both from Invitrogen).", "After the final wash cells were resuspended in the same medium and plated on laminin coated culture dish according to the experiments [2].", "After isolation 1×106 cardiomyocytes were seeded on a laminin coated T25 flask and allowed to settle for 4 h.", "After 4 h cardiomyocytes were exposed to high glucose (HG, 30 mM) or Mannitol (NG, 30 mM for osmotic control) to simulate diabetic condition in vitro for 48 h.", "After 48 h, the effect of HG treatment on beclin-1 was measured by western blotting [7] and cell survival by caspase-3/7 activity as described earlier [2,8]." ]
[]
Is play a behavior system, and, if so, what kind?
Given that many behavior patterns cluster together in sequences that are organized to solve specific problems (e.g., foraging), a fruitful perspective within which to study behaviors is as distinct ‘behavior systems’. Unlike many behavior systems that are widespread (e.g., anti-predator behavior, foraging, reproduction), behavior that can be relegated as playful is diverse, involving behavior patterns that are typically present in other behavior systems, sporadic in its phylogenetic distribution and relatively rare, suggesting that play is not a distinct behavior system. Yet the most striking and complex forms of play have the organizational integrity that suggests that it is a behavior system. One model that we develop in this paper, involves three stages of evolutionary transition to account for how the former can evolve into the latter. First, play-like behavior emerges from the incomplete development of other, functional behavior systems in some lineages. Second, in some of those lineages, the behavior patterns typical of particular behavior systems (e.g., foraging) are reorganized, leading to the evolution of specific ‘play behavior systems’. Third, some lineages that have independently evolved more than one such play behavior system, coalesce these into a ‘super system’ allowing some animals to combine behavior patterns from different behavior systems during play. Alternative models are considered, but irrespective of the model, the overall message from this paper is that the conceptual framework of the behavior system approach can provide some new insights into the organization and diversity of play present in the animal kingdom.
[ "AGRI", "NEUR" ]
[ "Behavior systems", "Evolutionary history", "Functions", "Locomotor-rotational play", "Mechanisms", "Object play", "Social play" ]
[ "1103", "2802" ]
[ "A common framework for investigating and understanding how animals construct coherent functional sequences of movements has been to view behavior patterns as atomized components that can be combined and recombined in multiple ways to serve particular task demands (e.g., Flash and Hochner, 2005; Kolb and Whishaw, 2015; Skinner, 1938).", "An alternative has been to view behavior patterns, not as discrete components, but as constituents of ‘behavior systems’, defined as structurally and/or functionally coherent packages of causally related and hierarchically coordinated behavior patterns, the expression, organization and coordination of which may be controlled by highly specific environmental stimuli, sensorimotor processes and motivational mechanisms (Baerends, 1976; Tinbergen, 1951).", "Several lines of research support the explanatory and predictive value of the behavior systems conceptualization of behavioral organization.", "For example, rats deprived of food or water, were presented with a rolling ball bearing, which predicted either food or water.", "When the ball bearing predicted food, it was treated as a prey object.", "This was not the case when it predicted water.", "That is, a moving stimulus activated predatory behavior in hungry rats, but not in thirsty rats (Timberlake, 1983).", "Using the same test paradigm with several species of rodents showed that the ability of the ball bearing to activate functional sequences of predatory behavior differed with the degree of carnivory among the species (Timberlake and Washburne, 1989).", "Such findings suggest that particular stimuli do not simply elicit particular behavioral actions, but rather, depending on the context, the stimuli activate particular types of functionally coherent sequences of behavior: in this case, those related to predation.", "In some cases, such behavioral findings are confirmed by physiological analyses.", "For example, as the imminence of a threat by a ground predator increases, chickens progress through a sequence of anti-predator behavior.", "The same sequence of behaviors can be activated in the same order of appearance by electrically stimulating particular brain circuits, independently of the presence of a predator (von Holst, 1973).", "That is, the sequence of actions is bound together in a connected system – a behavior system - in this case, geared for anti-predator defense.", "In other cases, what may be activated are coherent sequences of mating, aggression, nest building and so on (e.g., Adams, 1980; Kortland, 1955; Pfaff, 1999).", "There are different research traditions in studying behavior from a behavior systems perspective; some have focused on the functional connections between the behavior patterns involved (Timberlake, 1994, 2001), some on the causal relationships among the constituent components (Hogan, 1988, 2001), and some on the underlying neural mechanisms (Panksepp, 1998; Teitelbaum, 1982).", "But irrespective of these differences in research traditions, there is a common underlying framework: “That behavior is organized in functional motivational systems lies at the core of the set of ideas constituting the behavior systems perspective” (Burghardt and Bowers, 2017, p.3 42).", "In the present paper, we examine whether the available evidence supports conceiving of play behavior as a ‘play behavior system.’", "Considering that play activity contains behavior patterns that appear to be similar to those observed in other functional behavioral contexts, such as predation, fighting, and sex (e.g., Heymer, 1977; Meyer-Holzapfel, 1956; Millar, 1981), play could be considered a simulation of these behavior systems (Pellis and Burghardt, 2017).", "However, as stated by Burghardt (2006, p. 182): “…whether play…is a separate instinct (or behavior system) or is derived from other systems (such as predatory or fighting), is still an open issue in many species.”", "Despite decades of research, too little is known about the connections between the various forms of play and the differences across the diverse lineages of animals that play (Burghardt, 2005) to evaluate the question at hand from the perspective of any one of the different behavior systems frameworks.", "Consequently, we pursue a more limited goal, that of whether a generic concept drawn from the behavior systems perspective – that is, that there are few independent behavior patterns and few stand-alone actions (Bowers, 2017) - can be used to answer some of the many puzzles associated with this enigmatic behavior.", "Burghardt (2005) combined the various features that have been used in previous attempts to characterize play (e.g., Bekoff and Byers, 1981; Fagen, 1981) into a definition of play involving five criteria, so that for a given behavioral sequence to be classified as play, it must satisfy all five criteria.", "The criteria are: (1) that the behavior is incompletely functional in the context expressed, (2) that it is voluntary or rewarding, (3) that it is, in some ways, modified developmentally or structurally compared with when it is used in its normal, functional context, (4) that it is performed repeatedly, but not necessarily in an invariant form, and (5) that it is initiated in healthy, relatively unstressed animals in a relaxed context.", "Central to these criteria is that they characterize play by when and how behavior patterns are performed.", "Three major forms of play are generally recognized (Burghardt, 1998, 2005): (1) Locomotor play: involves a lone animal engaging in runs, jumps, kicks, whole body rotations, body shakes and sharp turns - behavior patterns that are often associated with anti-predator behavior (e.g., Byers, 1984; Martin and Bateson, 1985; Wilson and Kleiman, 1974); (2) object play: involves a lone animal engaging in carrying, flinging, ripping or otherwise manipulating an inanimate object - behavior patterns that are often associated with predation and foraging (e.g., Gamble and Cristol, 2002; Negro et al., 1996; O’Hara and Auersperg, 2017; Pellis, 1981a); and (3) social play: involves two or more conspecifics wrestling, chasing or otherwise contacting one another - behavior patterns often associated with allogrooming, mating, conspecific aggression and maternal care (e.g., Aldis, 1975; Lancaster, 1971; Pellis and Pellis, 2017a).", "There are some compelling cases of animals combining two or more of these forms of play.", "For example, during object play, animals may use a variety of locomotor movements (Burghardt et al., 2016), and during social play, animals may compete for an object (Shimada, 2012) or incorporate locomotor-rotational movements (Donaldson et al., 2002).", "In some primate species, object-assisted eye-covering play contains behavior patterns typically found in all three types of play, and might have cognitive implications for pretense (Palagi, 2018; Russon and Vasey, 2012).", "Combining different types of play, as illustrated by the above examples, is consistent with a commonly held view of play, which is that during play animals mix together behavior patterns from a number of different behavior systems (Heymer, 1977; Meyer-Holzapfel, 1956; Millar, 1981).", "While in some species a few of the behavior patterns used may be unique to play (see Petrů et al., 2009), most are not (Burghardt, 2005; Fagen, 1981).", "Co-opting behavior patterns from diverse behavior systems would require the ability to disconnect specific behavior patterns from the coherent sequences of behavior patterns embedded in conspecific aggression, predation, mating and so on.", "Thus, engaging in play would not have the functional coherence typically associated with behavior systems (Bower, 2017).", "Rather, play may be more consistent with atomistic views of behavior (see above), whereby animals are able to combine and recombine a variety of independent behavior patterns.", "The fragmentary way that some aspects of behavior emerge during the development of play (Graham and Burghardt, 2010) may be consistent with this view.", "Play is most often associated with juvenile animals (Bekoff and Byers, 1981), a period defined as occurring between weaning and sexual maturity (Pagel and Harvey, 1993).", "During the juvenile period, many behavior systems that are essential in infancy gradually disappear and many that are useful in adulthood gradually emerge (Coppinger and Smith, 1989).", "It is in this transitional age that those sequences of behavior most likely to be labeled play occur (Fagen, 1981).", "In species that can play in multiple ways, the different types of play wax and wane at different ages, and phases of their maturation may overlap (e.g., Barrett and Bateson, 1978; Gomendio, 1988; Pellis, 1981a).", "At some stages of the development of behavior systems, the constituent motor patterns may not only emerge in a piecemeal manner, but also compared to their adult form, they may be incomplete, modified and/or misdirected (e.g., Eilam and Golani, 1988; Golani and Fentress, 1985; Groothuis, 1993; Vestergaard et al., 1990).", "In addition, with multiple systems overlapping in their development, behavior patterns from more than one system can occur concurrently.", "For example, righting from supine to prone on the ground involves several, independent behavior systems that depend on different sensory triggers, such as vestibular and several forms of tactile input (Magnus, 1926).", "During development, there is a serial order in the emergence of these righting behavior systems, but there is also overlap.", "For instance, when body tactile righting first emerges in infant rats, two distinct forms overlap in their development.", "When the rats are turned over onto their backs, tactile stimulation on the upper body triggers righting movements by the forelimbs and tactile stimulation of the hindquarters triggers righting movements by the hind limbs.", "As both are triggered simultaneously, the combined movements by the fore and hind limbs may interfere with one another delaying successful righting (Pellis et al., 1991).", "In the fully mature state, the forequarter righting system is dominant over the hindquarter righting system, so that the latter is only triggered if the former is prevented (Magnus, 1926).", "Based on these developmental considerations, immaturity may account for both the modified execution of particular actions and the mixing of behavior patterns from multiple behavior systems in what may be labeled as play.", "Indeed, the modified performance of behavior patterns evident in play may simply reflect immaturity of an animal’s sensorimotor system (Fentress, 1983).", "From this developmental perspective, play may not be a distinct form of behavior at all, but rather, what appears as ‘play’ may be a byproduct of the maturation of behavior systems and their possible interactions.", "Indeed, with regard to play fighting, one of the most commonly studied forms of play (Pellis and Pellis, 1998), several authors have argued that play fighting is simply the immature expression of aggression (e.g., Cervantes et al., 2007; Hurst et al., 1996; Lazar and Beckhorn, 1974; Taylor, 1980).", "However, for some species, the developmental sequence does not fit this hypothesis of ‘play as immaturity’.", "In some mammalian species with litters of multiple young, the newborns may actively engage in aggression to access preferred nipples (Skok and Škorjanc, 2014).", "That is, aggression precedes the onset of play, which in all such species becomes most prevalent around weaning.", "In some cases, the stakes can be very high indeed.", "In spotted hyenas, the mother digs a den in which she deposits the newborns, with the young being able to move to the entrance to access her nipples.", "However, the mother is too large to get into the den.", "The young agonistically compete for priority access to the available nipples, and the fighting can be fatal (Frank et al., 1991; Smale et al., 1995).", "Thus, for this species, not only does serious fighting precede the onset of playful fighting, but also the serious fighting is sufficiently well developed on first appearance as to be wielded as a lethal weapon (Drea et al., 1996).", "In such a case, claiming that playful fighting represents an immature phase of the development of aggression makes little sense.", "Also counter to the ‘play as immaturity’ hypothesis is that, in many species, play fighting can continue in adulthood (Pellis and Iwaniuk, 2000), and, when it does, while there may be some age differences in performance, the play fighting is recognizably play (e.g., Bauer and Smuts, 2007; Ciani et al., 2012; Cordoni, 2009; Palagi, 2006; Pellis and Pellis, 1992).", "However, for some behavior systems in some species, insufficient activation of the behavior system in adults can lead to incomplete or misdirected behavioral sequences (Kortland, 1955).", "For example, as the mating season approaches, geese may exhibit incomplete sequences of courtship, but even though early in the season courtship does not lead to mating, the constituent behavior patterns are executed in their fully competent adult form (Pellis, 1982).", "Comparisons of how the same behavior patterns are executed by juveniles and by adults during play are needed to determine whether there is a playful form of execution.", "In some species of monkeys, such as Japanese macaques, both juveniles and adults engage in extensive play with objects, especially stones.", "To highlight a few of the behavior patterns involved, these stones can be carried, gathered into piles, shaken, pounded onto substrates, struck and rubbed together (Huffman, 1984; Leca et al., 2007; Nahallage and Huffman, 2007a).", "A detailed evaluation of such stone-directed play behavior in long-tailed macaques indicates that all of the 36 behavior patterns involved belong to the foraging behavior system.", "Moreover, a comparison across three species of macaques revealed differing numbers of stone play behavior patterns, with the differences matching species differences in the complexity of their foraging behavior (Pelletier et al., 2017).", "Among some predatory animals, play with objects or prey is associated with the activation of a foraging behavior system, such as when they are hungry and food is not available or when food (prey) is available, but the animals are inhibited from killing and eating the prey (Biben, 1979; Hall, 1998; Pellis, 1991; Pellis et al., 1988).", "Such misdirected behavior patterns associated with predation may be mistakenly characterized as play.", "However, unlike the case for immature animals, given the sensorimotor competence of adults, their misdirected play should not exhibit the inefficient execution of the behavior patterns that comprise some of the key criteria in Burghardt’s definition of play.", "Long-tailed macaques use a pounding action to strike hard-shelled nuts onto the ground, which, with sufficient repetition, can lead to the shell cracking.", "The monkey is then able to extract the nut and eat it.", "Similar pounding is used with comparable sized stones during object play.", "Both juveniles and adults pound stones, but only adult males are able to crack the nuts successfully.", "If juvenile-typical play involves executing actions in an immature manner, then the kinematics of stone pounding should differ from that seen in the adults.", "Similarly, if stone handling is misdirected foraging behavior in adults, then pounding stones should be structurally similar to pounding nuts.", "Sequences of pounding nuts in which the flesh of the nut was extracted and eaten by adults were compared to sequences when adults pounded comparable sized stones.", "Unlike the nuts that are eaten, the stones that are pounded are neither eaten nor chewed.", "Sequences of stone pounding by juvenile monkeys were also analyzed and compared to both the stone pounding and the nut pounding by the adults (Pelletier, 2017).", "The pounding performed by adults and by juveniles of stones and nuts have the same, basic, three-part sequence.", "From a seated position, the monkey raises the stone (upswing), and modifies its body and hand position to orient the object toward the location of the strike (adjustment).", "Finally, there is the pounding action itself, in which the monkey strikes the object on the ground (downswing) (Fig. 1).", "Measuring a variety of parameters of the actions associated with each phase reveals that adults pounding a food item (i.e., nut) differ significantly on most parameters compared to when the pounding involves a non-food item (i.e., stone).", "This suggests that motorically competent animals modify their movements when using the same pounding action during play relative to how the same actions are executed during foraging.", "Moreover, stone pounding by juveniles - who are unable to crack nuts - resembles adult stone pounding (Table 1), suggesting that the play mode of executing actions in juveniles is not simply a byproduct of immaturity (Fentress, 1983), but rather, is a pattern of execution unique to play.", "There are some differences between adults and juveniles, indicating that the juveniles are in the process of improving their sensorimotor skills, but immaturity does not account for the commonalities in the pounding of stones by juveniles and adults.", "The playful execution of these behavior patterns is play-typical, not a byproduct of immaturity.", "That play is not simply an immature version of a developing behavior system such as aggression, is further supported by findings showing that the opportunity to engage in play fighting is highly rewarding.", "Young rats will learn to take the correct turn in a T-maze so as to gain access to a peer (Humphreys and Einon, 1981) and will move to an enclosure, in a conditioned place preference task, in which the animal has had experience in playing with a partner (Trezza et al., 2009).", "In both of these cases, if the target partner is rendered less playful by the administration of a drug, the effect on the choice of arm or enclosure to enter is muted.", "This suggests that playing is more rewarding than simply seeking non-playful social contact.", "This last point is further emphasized by studies with juvenile chimpanzees (Mason et al., 1963).", "The animals were trained to press levers to access either preferred food or a social partner and for the latter, the access could be limited to touching and being in close proximity or to permit full playful engagement.", "Engagement in play was chosen more often than simple social contact, and the opportunity to play could also trump access to food, even when the animals were hungry.", "These studies, along with a host of others involving manipulation of the brain reward mechanisms, strongly support the view that animals not only find play rewarding, but will, in some circumstances, trade off opportunities for other motivationally salient activities, such as feeding, to engage in play (Vandershuren, 2010).", "Given that immaturity cannot by itself explain what animals do during play and that play has a strong rewarding value for many animals, the conclusion that play constitutes its own behavior system (e.g., Panksepp, 1998; Panksepp and Biven, 2012; Siviy and Panksepp, 2011) begins to look compelling.", "However, there are some limitations to this view.", "First, the play, in all the examples discussed thus far, is what most people think of as play – calves frolicking, kittens wrestling and dogs engaging in a tug-of-war over a stick.", "These forms of play not only meet Burghardt’s five criteria, but also do so by a wide margin.", "What about species that do not play or ones that play but only barely meet the five criteria?", "Second, if play can be conceived as a behavior system, what kind of system would it be?", "It could be a ‘super-play system’ - one that can co-opt and mix behavior patterns from many other behavior systems.", "However, given that there are only a few documented cases of such mixing, it is also possible that there are multiple, independent ‘play behavior systems’, with only some species having the capacity to combine some of these systems.", "Of the 30 or so phyla in the animal kingdom, only three have so far been shown to contain species that play (i.e., meet the five criteria), and within those phyla, not all lineages therein have playful species (Burghardt, 2005).", "Even in the most playful phylum, Chordata, play is not universal.", "First, it has only been reported in the subphylum, Vertebrata, and, within that subphylum, it is sporadic across most classes, only being prevalent in birds and especially in mammals.", "And even within mammals, there are orders that do not play.", "Further, within some playful orders, there are species that do not play.", "Since 2005, although the application of the five criteria-based definition of play has added more species from diverse taxa to the playful side of the ledger, the general picture has not changed (Pellis and Burghardt, 2017; Pellis et al., 2015).", "Closer inspection of the type of play exhibited shows that the three different types of play may appear independently of one another across different lineages (Burghardt, 2005).", "That is, object play can evolve in a lineage independently of either social or locomotor play, and so on.", "For example, object play is either absent or rudimentary among rodents, whereas locomotor play and social play are quite prevalent (Fagen, 1981).", "However, a comparative analysis of social play and locomotor play in murid rodents reveals a negative correlation (Pellis and Iwaniuk, 2004), so that an increase in the complexity of one type of play is accompanied by a decrease in the complexity of the other.", "Further, even within a species, these forms of play can vary independently of one another across different, genetically selected strains (Schneider et al., 2016).", "Finally, studies within species have shown that individuals that exhibit more of one type of play are not more likely to exhibit more of the other types of play (Ahloy Dallaire and Mason, 2016; Lampe et al., 2017; Melotti et al., 2014).", "These phylogenetic considerations suggest that play evolved multiple times in the animal kingdom, with different types of play evolving independently of one another, although a few species that have more than one type of play in their repertoire show evidence that they can mix those types in novel ways (e.g., Burghardt et al., 2016; Shimada, 2012).", "It is in the context of this patchy distribution of play and its various forms, that the questions related as to whether play constitutes a behavior system needs to be considered.", "For example, what the phylogeny shows is that mixing behavior patterns from multiple behavior systems is not a universal defining feature of play, and indeed, may be the exception (Pellis and Pellis, 2017b) rather than the rule (Norscia and Palagi, 2016).", "This would suggest that there are least two levels in the evolution of a ‘play behavior system’.", "First, following the evolution of some type of play in a lineage, the behavior patterns associated with that play are gradually transformed into an organization that has the functional coherence typical of behavior systems.", "Second, some lineages with more than one type of play are able to evolve mechanisms that can combine those different types of play into a super-play behavior system.", "Burghardt (2005) developed a model for the independent evolutionary origins and transformation of play across different lineages of animals.", "This model asserts that, under certain conditions, such as readily available food, surplus energy, a relatively protected environment, prolonged juvenile period and with sufficient neural resources to experience boredom, playful behavior may emerge.", "This incipient or primary process play is the form of play that is most widespread in the animal kingdom, with locomotor, object or social forms predominating in different lineages (Burghardt, 2005).", "The behavior patterns performed in such play may differ little, if at all, from how they appear in their normally occurring functional contexts, but the usual functional consequences of their performance do not arise.", "It is possible that, under particularly benevolent conditions, such play may be maintained in the population even if it is neutral with regard to fitness benefits (Auerbach et al., 2015).", "If conditions are favorable and primary process play provides players with fitness-enhancing advantages by facilitating the performance of the behavior patterns derived from particular behavior systems, then the play is transformed into secondary process play.", "This transformation involves the features of play that are most crucial to those benefits being subjected to natural selection, so that the critical behavior patterns either increase in their frequency and/or are modified in their form to serve that function better.", "Such transformation not only involves the play becoming more distinctly playful, but also requires the evolution of novel, control mechanisms that are capable of modifying the expression of the behavior patterns from the behavior system being simulated (Burghardt, 2005; Pellis et al., 2015).", "Again, when ecological, developmental, life history and psychological factors permit, secondary process play may be further transformed into tertiary process play.", "Functionally, the benefits of tertiary process play may extend beyond feedback on the behavior system being simulated, so that the benefits gained from performing behavior patterns derived from a particular behavior system can lead to improved motor, social and cognitive skills that can be deployed in a variety of situations and across different behavior systems.", "To achieve such a functional transformation requires further modifications as to how behavior patterns are expressed during play and this requires the further evolution of novel control mechanisms that make the play even less like the behavior system being simulated (Burghardt, 2005; Pellis et al., 2015).", "A broad outline of such a transformational process has been characterized empirically for the social play present in the juveniles from sixteen species of murid (i.e., mouse-like) rodents (Pellis and Iwaniuk, 2004; Pellis and Pellis, 1998, 2009; Pellis et al., 2014).", "In murid rodents, social play in the form of play fighting primarily involves attack and defense of body targets otherwise contacted during adult precopulatory behavior, and, across species, this competition can differ markedly in its complexity (Pellis and Pellis, 2009).", "In the simplest forms of such play, one animal may attack the play target of its partner and the partner either does not defend itself, or if it does defend itself, limits that defense to evading contact, thus withdrawing from the attacker (equivalent to primary process play).", "At a more complex level, not only does defense occur relatively frequently, but in this case, many instances of defense involve the use of tactics that promote the occurrence of playful wrestling (equivalent to secondary process play).", "Even greater levels of complexity are achieved by the participants increasing the frequency of launching counterattacks and adopting maneuvers that facilitate role reversals between the partners, so leading to prolonged and repetitive play fighting (equivalent to tertiary process play).", "An example of this highly complex form of play fighting is shown in Fig. 2 for a pair of juvenile rats (for diagrams illustrating the simpler versions of such play, see Pellis and Pellis, 2009).", "From a mechanistic perspective, changes in the complexity of the form of play in murid rodents appear to be accompanied by changes in the neural mechanisms that regulate play (Pellis and Iwaniuk, 2004).", "The decreasing resemblance between what is performed during play and during adult sexual behavior with increasing complexity of play is likely underpinned by changes in such neural control mechanisms.", "The subcortical brain changes associated with changes in complexity remain to be characterized (Pellis and Iwaniuk, 2004; Siviy, 2016), but what has become clear is that the most complex forms of play engage cortical brain mechanisms (Pellis and Pellis, 2016).", "From a functional perspective, there is little evidence for the fitness enhancing effects of the simplest forms of play.", "The most complex forms of play affect not only adult sexual performance, but also cognitive skills useful in non-social and social contexts.", "Studies with rats have been particularly useful in showing empirically these diverse effects of play experience.", "Compared to adult rats that have been deprived of experiencing juvenile-typical experience of play with peers, ones having had such play experience exhibit improved sexual performance (Moore, 1985), are better able to navigate mazes successfully (Einon et al., 1981), have greater impulse control (Baarendse et al., 2013), better social memory (Schneider et al., 2016) and have greater ability to coordinate their movements with a social partner (Pellis et al., 1999).", "The transformative nature of play across lineages and the unique phylogenetic histories of different lineages suggest a model for the origin of ‘play behavior systems’.", "As in the murid rodent model showing the transformation of social play from primary to tertiary process play (Pellis and Pellis, 2009) other forms of play may exhibit similar patterns of transformation within lineages.", "Unfortunately, neither object nor locomotor play has had comparable detailed studies within specific lineages.", "Nonetheless, comparisons across wider swathes of species from different lineages suggest similar patterns.", "For example, as noted earlier, locomotor play frequently involves behavior patterns otherwise seen in anti-predator behavior.", "Within-lineage comparisons show that the complexity with which these behavior patterns can be executed and combined can vary (Pellis and Iwaniuk, 2004).", "However, in some species, locomotor play can involve behavior patterns that are not present in anti-predator behavior, such as running or climbing with the eyes closed, as is done by some monkeys and apes (Kavanagh, 1978; Palagi, 2018; Russon and Vasey, 2012), or the playful pirouettes of chimpanzees (Nishida and Inaba, 2009).", "These latter examples seem consistent with the behavior being tertiary process play in which the actions have become divorced from the underlying functional system from which they may have originally been derived.", "Similarly, object play, which is exhibited widely across lineages of animals (Burghardt, 2005), can vary markedly in the frequency and complexity of its expression (Fagen, 1981).", "In most cases, such play appears to involve manipulative behavior patterns typical of foraging and predation, but this can range from using unitary patterns infrequently to using complex sequences of many behavior patterns often (e.g., Gamble and Cristol, 2002; Negro et al., 1996; O’Hara and Auersperg, 2017; Pellis, 1981a).", "Again, where comparisons are available in a more closely related lineage of species, a similar pattern is revealed (e.g., Nahallage and Huffman, 2008, 2012; Pelletier et al., 2017; Torigoe, 1987).", "Strikingly, some forms of object play can involve elements that go beyond foraging behavior patterns, such as when dolphins blow bubbles (Hill et al., 2017), or when bonobos don plants, straw or blankets as ‘hats’ (Palagi, 2018).", "Again, such examples seem consistent with the behavior being tertiary process play in which the actions have become divorced from the underlying functional system from which they may originally have been derived.", "When the maturation of a particular behavior system in a lineage occurs in a manner that meets the minimum requirements of Burghardt’s five criteria, that lineage may be considered to exhibit play.", "But such primary process play is barely distinguishable from the behavior system from which its behavior patterns are derived.", "However, for secondary and tertiary process play to evolve, novel functional and causal processes are involved.", "In order for such play to exhibit the coherence typically associated with behavior systems (Hogan, 2001; Timberlake, 2001), novel mechanisms are needed, mechanisms that can lead to behavioral and contextual dissociation from the behavior system from which the behavior patterns used during play are derived.", "Such dissociation can be illustrated by object and predatory play.", "As already noted, for some species, play with objects or prey corresponds to the daily cycle of foraging, in that it is most frequent when the animals are most likely to eat, with the play representing misdirected foraging behavior (e.g., Hall and Bradshaw, 1998; Pellis, 1981a, 1991).", "Similarly, when a predator is confronted by a prey that is large or otherwise threatening, the inhibition of predatory attack can lead to the vacillation in contact and withdrawal typically seen in play (Biben, 1979; Hall, 1998; Pellis et al., 1988).", "However, in other species, object play does not cycle with the daily foraging or eating pattern (e.g., Ahloy Dallaire and Mason, 2016) and may include behavior patterns that are not limited to foraging/predatory behavior (e.g., Giljov et al., 2017).", "That is, in the latter cases, object play does not simply reflect activation of the foraging and feeding behavior systems.", "The examples discussed above suggest the following model.", "For all three types of play, it seems likely that different lineages have transformed play from primary through secondary to tertiary processes.", "Therefore, it would seem that multiple behavior systems (e.g., foraging, aggression, sex, anti-predator behavior) have evolved independently into different, specific play behavior systems.", "Again, that for some lineages this occurred for one behavior system, but not others, attests to their independent evolution (Burghardt, 2005).", "Thus, as a first step, there may be multiple play behavior systems that have evolved from their parent behavior systems (e.g., the anti-predator behavior system spawning the locomotor play behavior system).", "Then, for some species that have evolved more than one form of tertiary process play, if they have the necessary neural, cognitive and ecological resources available, novel mechanisms may have evolved to combine different types of play into truly mixed patterns (Pellis and Pellis, 2017b).", "That is, multiple play behavior systems are combined into a super-play behavior system (Fig. 3).", "An alternative way to view the sporadic emergence of play in the animal kingdom is to treat it like migration, which also appears to be idiosyncratic and sporadic in its distribution, but actually relies on common functional and causal processes (Dingle, 2006).", "In this way, it is more like universal behavior systems such as feeding or fighting: that is, the structural components are the same and the functional associations of those components are similar.", "This is unlike our ‘many to one’ hypothesis in which we posit different causal processes leading to convergence of similar outcomes.", "Rather, this alternative would posit parallel evolution in which different lineages use the same core, causal processes to produce comparable outcomes repeatedly.", "There are precedents for such parallel evolution.", "For example, sea slugs appear to use the same conserved neural circuits to evolve, repeatedly, similar behavior patterns across diverse species within the lineage (Katz, 2011).", "Similar parallel evolution may occur across diverse lineages.", "For example, domestication involves similar causal mechanisms to produce a comparable suite of behavioral, neural and physiological traits across diverse lineages (Wilkins et al., 2014).", "Just like there is a ‘domestication syndrome’ or a ‘migration syndrome’, there could be a ‘play syndrome’.", "Such a syndrome would make all play behavior systems one system, but one that may have different behavioral manifestations, depending on the idiosyncrasies of particular lineages.", "Finding commonalities in the neural circuits that are unique to play and then determining how these co-vary across members of a closely related clade that differ in their playfulness would be an important step in identifying such common mechanisms.", "For example, in rats, there are strain differences in the frequency of initiating play fighting and in the likelihood of using different types of behavior patterns for playful defense (Himmler et al., 2014).", "For some strains, reduced playfulness is associated with altered activity of the mesolimbic dopamine system (Siviy et al., 2011).", "This is consistent with the finding that the motivation to engage in play depends on this dopaminergic system.", "Conversely, the rewards gained from playing depend on the brain reward mechanisms that involve endogenous opioids (Vanderschuren et al., 2016).", "Information on how these mechanisms naturally vary across species could be of value in determining how much particular species play and how they play.", "There are limitations in our current knowledge that need to be remedied to be able to use a neurobiological approach.", "The first limitation is that most of our knowledge about the neural controls over play is based on the laboratory rat (Siviy and Panksepp, 2011; Vandershuren et al., 2016).", "What has been shown is that the neural circuit involved spans areas in the forebrain, midbrain and hindbrain (Siviy, 2016).", "Some comparative studies with primates suggest that a similar network may be involved in this clade (Graham, 2011), but this remains to be empirically tested.", "The consistency between rats and primates leads to the second limitation; that, in both cases, this network has been established for animals engaging in play fighting.", "Unfortunately, comparable studies have not been done with any species from any lineage engaging in locomotor or object play, so we do not know if the same circuit is involved across different forms of play and if so, if there are unique causal connections among the components of this circuit for different types of play.", "The third limitation is that components of this circuit are used across a range of behaviors and not just play.", "For example, parts of the prefrontal cortex (PFC) are activated during social play (Gordon et al., 2002; Hehar et al., 2016) and damage to areas of the PFC modifies how they play socially and affects the attractiveness of such damaged rats as play partners (Bell et al., 2009; Pellis et al., 2006).", "But the PFC regulates socio-cognitive skills that influence many aspects of social behavior, not just play (Pellis and Pellis, 2016).", "Similarly, even if the circuits involved in motivating and rewarding play fighting in rats (Vandershuren et al., 2016) were found to have a similar role in object and locomotor play, this would not necessarily mean that there is a common play circuit as these neural systems are utilized in all motivated behaviors (Berridge and Kringelbach, 2013).", "There is much comparative work to be done both across species and across types of play before neurobiology can help resolve the question of what kind of behavior system(s) may constitute play.", "On a more positive note, the details emerging from studies on rats (Siviy, 2016; Vandershuren et al., 2016) are providing a framework for comparative studies on social play in other species (e.g., Burleson et al., 2016; Graham, 2011) and on other forms of play (Riters et al., 2018).", "The behavioral detail available in how animals play across a wide range of species is also deficient.", "For example, what is meant by play involving the mixing of behavior patterns from different behavior systems (e.g., Meyer-Holzapfel, 1956) is ambiguous.", "When animals use behavior patterns simultaneously, as when two Japanese monkeys compete for access to a stick (Shimada, 2012), the mixing is evident.", "Often, however, mixing is assumed when there is a looser temporal association, such as when a piglet jumps and rotates in the air, and, as it does so, crashes into another piglet, which then leads to a brief play fight (Donaldson et al., 2002).", "Alternative explanations, such as incidental contact leading to an activation of a different sequence of behavior, are usually not considered (Pellis and Pellis, 2017b).", "Some detailed studies of play fighting illustrate the problem.", "Play fighting involves animals competing for some advantage over one another (Aldis, 1975), with the advantage sought being the same as that from a particular behavior system being simulated.", "For example, rats compete for nuzzling another’s nape (as in sex) (see Fig. 2), and Australian magpies peck the side of their partner’s head (as in aggression) (Pellis, 1981b).", "The play fighting of some species can involve competing for more than one advantage (Pellis and Pellis, 2017a), and these species give us the opportunity to test whether behaviors derived from different behavior systems are mixed during playful sequences.", "For example, ground squirrels engage in competitive playful interactions in which they attempt either to bite each other on the shoulders (as in aggression) or mount one another (as in sex) (Nunes et al., 1999; Pasztor et al., 2001); grasshopper mice either compete to bite each other on the nape of the neck (as in predation) or lick, groom and nuzzle the sides of their partner’s shoulders (as in precopulatory behavior) (Pellis et al., 2000); and gray mouse lemurs compete to bite each other on the face (as in aggression), groom each others’ faces and upper bodies (as in social affiliation) or mount one another (as in sex) (Pellis and Pellis, 2018).", "Detailed temporal and kinematic analyses of play fighting sequences in all these species show that there is no mixing within a sequence – a play fight starts and ends with attack and defense related to only one type of advantage.", "Once such a play fight is terminated, another involving competition for another advantage may commence.", "Thus, on a broad time scale over the entire duration of a play session or over successive sessions, play fighting involves behavior patterns from multiple behavior systems, but in the moment-to-moment moves and countermoves when engaged in a particular play fight, the animals do not mix behavior patterns from different behavior systems (Pellis and Pellis, 2017a).", "Of course, the sequential pattern of an aggressive play fight being followed by a sexual play fight may still reflect a common play behavior system.", "Indeed, our hypothesized model (Fig. 3) provides a means for this level of mixing.", "The constituent play behavior systems (aggression and sex) may retain sufficient coherence within each system to maintain functional cohesion of the behavior patterns involved, so that aggressive and sex behavior patterns are not interspersed.", "Nevertheless, at the super-play behavior system level, the interspersed sequences of aggressive and sexual play may form part of a seamless session of play.", "Perhaps as dynamic imaging techniques become available that can track brain circuit activity in freely behaving animals (Bermudez-Contreras et al., 2018), objective evidence may be obtained to determine if the participants perceive the overall interaction, one involving sequentially occurring aggressive and sexual play, as one continuous bout of play or as discrete encounters.", "In addition, more detailed studies are needed of species that engage in multiple forms of play to provide a comparative data set on the various ways in which behavior patterns or sequences of behavior patterns derived from different behavior systems can be juxtaposed.", "Many instances of what qualifies as being labeled play has the functional coherence in the organization of how its constituent behavior patterns are ordered to make it look like a behavior system as defined by Burghardt and Bowers, (2017).", "However, given that most of the behavior patterns used in play are co-opted from other behavior systems (e.g., anti-predator behavior, conspecific aggression, sex, foraging), how play originated and how it has achieved the coherence of a behavior system is unresolved.", "Also, since there are multiple forms of play (e.g., social, object, locomotor) that in many lineages have evolved independently (Burghardt, 2005), it is unclear how, in some species, these may coalesce so that they are integrated together in coherent sequences of behavior.", "Finally, no existing theory provides an explanation for how novel behavior patterns - those that are not part of the repertoire of any of the behavior systems simulated during play (e.g., Palagi, 2018; Petrů et al., 2009) - arise and become incorporated in the play of some species.", "The evolutionary-based hypothesis suggested in this paper provides an attempt to answer these questions and does so in a manner that can integrate the vast species differences that exist in the presence and content of play across the animal kingdom.", "Even though the ‘many to one’ hypothesis is coherent and can account for that variation, so may an alternative ‘play syndrome’ hypothesis.", "Empirical limitations in our knowledge about play do not yet permit the construction of formal competing models of the components of how a play behavior system may be organized.", "Nonetheless, by thinking about play from a behavior systems perspective at least two viable hypotheses emerged, hypotheses that may be useful in directing further empirical research." ]
[ "Play behavior is rare occurring sporadically in the phylogenetic tree of animals, with different lineages having evolved distinct forms of play.", "Animals seamlessly weave together behavioral elements from different types of play in the most complex forms of play.", "A phylogenetic model of behavioral transformation is developed to account for how simple forms of play can coalesce into more complex forms.", "The model indicates that lineages with multiple complex forms of play can combine these into a 'super play behavior system'." ]
Superb adsorption capacity of hydrothermally synthesized copper oxide and nickel oxide nanoflakes towards anionic and cationic dyes
The CuO and NiO nanoflakes were synthesized by a hydrothermal reaction. The secondary nucleation and growth of hydroxides resulted in the formation of flake like architectures as the prepared nanoparticles were used as an adsorbent for malachite green oxalate (MGO) and methyl orange (MO). The structure, morphology and surface properties of the nanoparticles were characterized by XRD, SEM and TEM. Effects of the experimental conditions on the adsorption behavior were studied by varying the contact time, initial concentration, initial pH and temperature. The adsorption of MGO increased with increase in the pH, while the MO adsorption showed an opposite trend. The adsorption kinetics was studied in terms of pseudo-first and second-order kinetics, the Langmuir and Freundlich models were also applied to the equilibrium adsorption data. The adsorption process was spontaneous and exothermic in nature and followed the pseudo-second-order kinetic model. Our study indicates that the CuO and NiO nanoflakes can be used as alternative adsorbents for the efficient removal of dyes from an aqueous solution.
[ "MATE" ]
[ "Adsorption", "CuO", "MGO", "MO", "NiO" ]
[ "2501", "2502", "2503", "2504" ]
[ "Water pollution is an important environmental issue that has received major worldwide attention.", "Many industries such as textile, leather, food processing, and dyeing cosmetics generate massive amounts of wastewater.", "Dye chemicals represent one of the most prominent contaminants contained in the wastewater effluents [1].", "The first contaminants that are recognized in water are dyes, which must be removed before discharging them into water bodies.", "Introduction of dyes into aquatic systems is hazardous as they can cause distinct limitations such as the hindrance of light penetration and the mutagenic change increases in chemical oxygen demand (COD) and biological oxygen demand (BOD) of a water system reflecting its low quality.", "Malachite green oxalate (MGO) and methyl orange (MO) are two typical examples of industrially relevant toxic cationic and anionic dyes with known harmful effects on humans.", "Therefore, treatment of effluents containing dyes becomes increasingly important from both toxicological and ecological perspectives.", "Many conventional methods such as precipitation, ion exchange, solvent extraction, biosorption, filtration, and electrochemical treatment have been employed for removal of contaminants from industrial wastewaters [2–4].", "These methods are either expensive or inefficient for treating effluents with high concentration of contaminants.", "They also have significant disadvantages such as incomplete removal, requirements and production of toxic sludge or other waste products that require further disposal.", "The adsorption technique emerges as a competitive method for treatment of dyestuff wastewater due to its easy handling, high efficiency and economic feasibility [5–7].", "The introduction of nanoparticles comprising a variety of shapes, sizes and compositions have led to changes in wastewater treatment approaches.", "Preparation and characterization of novel nanomaterials are important in materials science.", "Nanoparticle based adsorbents possess high surface area in addition to various non-saturated reactive surface atoms, which enable selective and reversible binding of dye molecules [8].", "There are various candidate nano-materials, including carbon nanotubes, nano-wires and nano-particles.", "Adsorbents containing metallic nanoparticles or multi-walled carbon nanotubes have been widely applied to bind and coordinate biological materials, organic pollutants, and various metal ions [9].", "Copper oxide (CuO) and nickel oxide (NiO) nanoparticles have been extensively studied for many years due to their important roles in catalysis, metallurgy, and high-temperature superconductors.", "Several preparation methods of CuO and NiO nanoparticles have been reported [10–14].", "Chemical and physical properties of these materials depend on its composition, structure, phase, shape and size distribution.", "Current efforts are being undertaken to explore the possible utilization of these metal oxide nanoparticles for socio-economic development.", "In this context, we report a cost-effective removal of cationic (MGO) and anionic (MO) dyes from an aqueous solution using low cost, recyclable, and easily available metal oxides (CuO, NiO).", "To the best of our knowledge, such metal oxides have not been much employed, especially in the dye removal process through adsorption.", "A systematic investigation of the effects of initial dye concentration, initial pH, adsorbent dose, temperature of the solution on dye uptake and plausible dye-adsorbent interaction during adsorption is carried out in this study.", "This particular approach has significant advantages such as cost effective, simplicity in equipment, ease in preparation, short reaction time and better repeatability.", "More importantly, the as-prepared CuO and NiO nanoflakes exhibit an excellent dye removal ability in wastewater treatment.", "All reagents including copper(II) nitrate trihydrate (CuNO3·3H2O), nickel nitrate hexahydrate (Ni(NO)3·6H2O), sodium hydroxide (NaOH), Triton X-100 (C14H22O(C2H4O)n), cationic dye (MGO) and anionic dye (MO) were used as absorbates for the adsorption studies.", "All the chemicals used in the experiments were of AR grade and used without any purification.", "Standard solutions (100 mg L−1) of MGO (C52H54N4O12) and MO (C14H14N3NaO3S) were prepared by adding appropriate amounts in the deionized water and further diluted to the required concentrations for adsorption experiments.", "The structures of the above mentioned dyes are given in Fig. 1a and b.", "In a typical procedure, 20 ml of 0.2 mol L−1 NaOH solution was slowly added into a 20 ml of 0.1 mol L−1 copper(II) nitrate trihydrate and nickel(II) nitrate solution containing 0.004 mol L−1 of Triton X-100, with constant stirring.", "After vigorous stirring for 3 h, the mixture was autoclaved at 200 °C for 5 h.", "After the reaction system was naturally cooled to room temperature, the precipitate was separated from solution and thoroughly washed several times, first with deionized water then with absolute ethanol and subsequently dried in an oven at 50 °C for 8 h.", "X-ray diffraction (XRD) patterns were obtained on a Bruker D2 Phaser XRD system.", "Surface morphology (SEM) was studied using scanning electron microscope (JEOL JSM 840A) and Transmission electron microscope (TEM) Philips CM-200 instrument.", "Finally, the BET surface area, total pore volume and average pore size were measured using ASAP 2010 Micrometrics instrument by Brunauer–Emmett–Teller (BET) method.", "Effect of pH on the adsorption capacity of dyes was evaluated by agitating 20 mg L−1 dye solution with 250 mg L−1 of metal oxides for predetermined equilibrium time at pH ranging from 2.0 to 8.0.", "The pH of the solution was adjusted by using 0.5 M HCl or 0.5 M NaOH.", "Similarly the rate of MGO and MO adsorption onto metal oxides was investigated by kinetics and isotherm study.", "Duplicate experiments were carried out for all operating variables studied and only average values were taken into consideration.", "Fig. 2a shows the XRD pattern of CuO nanoparticles.", "The peaks at 2θ = 32.62°, 35.61°, 38.80°, 48.75°, 53.68° and 58.42° can be assigned to (110), (002), (111), (202), (020) and (113) of CuO (JCPDS 80-1916) [12].", "Fig. 2b shows the main peaks at 37.26°, 43.32°, 62.84°, 75.18°, and 79.23°, which correspond to the (111), (200), (220), (311) and (222) planes of NiO nanoparticles (JCPDS 78-0643) [15], the crystallite size has been estimated from the XRD pattern using the Scherrer's equation.", "There are no peaks observed for any impurity, indicating the high purity of the sample.", "The surface morphology of the CuO and NiO nanoflakes was studied by SEM.", "Fig. 3a and b show a uniform growth of the metal oxide nanostructures.", "SEM images with higher magnification show that the grown nanostructures are in the form of nanoflakes which were interconnected with each other and created pores and crevices, yielding large surface areas for easy diffusion of the dyes onto the surface of metal oxides.", "TEM was used to further investigate the microstructure of the as-synthesized CuO and NiO nanoflakes.", "It revealed the formation of nanoflakes with a cubic crystal structure as shown in Fig. 4a and b.", "The color contrast of both oxides is homogeneous, indicating a uniform dense structure and a smooth surface [16].", "This observation is in accordance with the corresponding SEM image.", "Using the BET method, the surface areas of the CuO and NiO nanoflakes were determined to be 69.27 m2 g−1 and 9.72 m2/g respectively, and their total pore volume was 0.023 cm3 g−1.", "Fig. 5 shows the time profile of MGO and MO adsorptions at different initial concentrations in a range from 10 to 40 mg L−1 at pH 7.0 for 90 min.", "The dye adsorption capacities onto CuO and NiO increased with increase in the concentration of dye solutions.", "The adsorption rates were extraordinarily fast in the first 5 min for all the concentrations.", "The adsorption nearly finished within 60 min, indicating the fast adsorption rate.", "The removal efficiencies for dyes initially increased and then decreased, which might be due to the fact that large numbers of vacant active sites were available for the adsorption at a lower initial concentration, and once the sites became saturated, it was difficult to capture the dye molecules because of repulsive forces exerted by the adsorbate molecules.", "In the case of MGO, the removal efficiency was higher than that of MO at low initial concentrations, probably due to smaller spatial prohibition in the molecular structure of MGO and its positive feature, making it more accessible to the adsorption sites of CuO and NiO.", "It is seen that the adsorption capacity (Qt) of both dyes increased with the initial dye concentration.", "This suggests that dye concentration is a major factor in overcoming the mass transfer resistance of the dye between the aqueous and solid phases.", "Fig. 6 demonstrates that a maximum percentage of dye removal was observed at pH 8 for MGO (83.4 and 86%) and at pH 2 for MO (93.2, 84.6%) for CuO and NiO respectively, beyond which it was constant.", "To determine the adsorption mechanism, measurements of zero point charge (pzc) of the adsorbent are crucial, since it is well known that adsorption of a cationic dye is favorable at pH > pzc.", "By contrast, for an anionic dye the favorable adsorption condition is pH < pzc.", "Here we observed that the pzc of CuO and NiO was in the range of 9–10.", "Therefore, in an acidic environment (pH < pzc), a lower adsorption efficiency is expected because of the repulsive forces between the adsorbent and adsorbate.", "Furthermore, in acidic pH, the increased surface excess of H+ ions on the adsorbent implied competition of the H+ ions with cationic dye molecules, which reduced the adsorption of dye molecules.", "However, under alkaline conditions, the electrostatic attraction force increased resulting in a higher % of adsorption of the cationic dye.", "This is attributed to an increase in the number of negatively charged sites, because of deprotonation.", "The Langmuir and Freundlich isotherms for the two dyes are shown in Figs. 7 and 8 respectively.", "The related parameters and correlation coefficients were calculated via a process of linear regression for both isotherm models.", "The corresponding results are presented in Table 1.", "It is seen that the correlation coefficient values for both Langmuir and Freundlich isotherm models are closer to 1.", "The maximum adsorption capacity determined from the Langmuir isotherm model for MGO and MO dyes on CuO is 178 and 158 mg g−1 respectively; similarly for NiO it is found to be 189 and 165 mg g−1.", "The values of KL obtained are in the range of 0–1, indicating that the adsorption of both dyes on the CuO and NiO is favorable in an adsorption process.", "Moreover, the values of n are greater than 1, confirming that the metal oxides are good adsorbents with high adsorption intensity for both dyes.", "Table 2 compares the maximum adsorption capacities of both CuO and NiO nanoflakes for dyes with those of other metal oxide nanoparticles.", "It is seen that the maximum adsorption capacities of our oxides are notably higher as compared to other candiate adsorbents.", "The first-order-rate constant k1 can be obtained from the slope of the plot ln(qe − qt) vs t (Fig. S1).", "The results obtained from applying the first order kinetic model indicated that the correlation coefficient (R2) values of the first-order rate model were not high for the different concentrations of MGO and MO; furthermore, the value of ‘Qe’ was smaller than the value of Qt, suggesting the inapplicability of the pseudo-first-order model to describe the adsorption process.", "Fig. S2 revealed that all the experimental data could fit well to the pseudo-second-order model with high correlation coefficients (R2 > 0.999).", "The values of ‘Qe'cal was also very close to the values of ‘Qe'exp, which indicates that the adsorption process of MGO and MO on CuO and NiO obeyed the pseudo-second-order model.", "Also, the adsorption data do not fit the pseudo-first-order model.", "It can be perceived from Fig. 9 that the temperature adversely affects the removal efficacy of both the dyes on CuO and NiO.", "The adsorption capacity of dyes diminishes as the temperature rises from 303 to 323 K.", "This may be due to the fact that, at higher temperature the solute molecules show an inclination to escape from the solid phase and re-enter the liquid phase as a result of increased mobility [19].", "It is worthy elucidating why CuO and NiO have such a high adsorption performance toward MGO and MO.", "It has been previously suggested that the adsorption of ionic dyes or organic pollutants may be associated with the electrostatic attraction and surface complexation [21,22].", "In the present study, the adsorption of MGO and MO on CuO and NiO can be attributed to electrostatic attraction.", "It is well known that, if the pH of the solution is below the isoelectric point of an adsorbent, the adsorbent will possess a better adsorption capacity.", "The natural pH value (≈7.0) of the dye solution is much lower than the isoelectric point of both CuO and NiO (9–11).", "Therefore, CuO and NiO are expected to become promising materials for rapid and deep treatment of high concentration dye-containing wastewater because of their fast adsorption rate and high capacity desorption of dye molecules at a natural pH [23].", "In this study, the low-cost, environmentally friendly, greater adsorption capacity CuO and NiO nanoflakes were prepared by the hydrothermal method and utilized for the removal of dyes from an aqueous solution by the adsorption process with a removal efficiency >90%.", "The removal efficiency is dependent on the initial concentration, pH and temperature of the solution.", "Based on this study, the optimum conditions for an effective removal of dyes by adsorption onto metal oxide nanoflakes can be fine-tuned.", "Additionally, the adsorption thermodynamics analysis revealed that the adsorbent processes were spontaneous.", "The adsorption of both the dyes fit the pseudo second-order kinetic model.", "Furthermore, an in-depth study on selective interaction phenomena of these dyes with nanoparticles is currently under process, which could be an important tool in remediation technology and nanotechnology as well.", "Excavation of the full potential of CuO and NiO for removing MGO and MO from real industry wastewater samples will also demand further studies." ]
[]
Structural, impedance, dielectric and modulus analysis of LiNi1-x-y-0.02Mg0.02CoxZnyO2 cathode materials for lithium-ion batteries
Mg, Co and Zn co-substituted layer-structured cathode materials LiNiCoxZnyMg0.02O2 (x = y = 0.0, 0.02 and 0.04) were prepared by a solid-state reaction method. The materials were systematically characterized by X-ray diffraction (XRD), field effect scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FT-IR), and electrical impedance spectroscopy (EIS) techniques. XRD analyses revealed the formation of a rhombohedral structure in the prepared materials with a typical α-NaFeO2 layered structure within R3¯m space group. The grain size was determined by FESEM in the range from 3.19 to 3.85 μm for all materials synthesized. The site of the local cation (Li–O) and of the transition metal cations (M–O) in the materials were identified by FT-IR. The complex impedance and modulus studies suggested the presence of a non-Debye type of multiple relaxations in these materials. The dielectric constant was found to increase with increasing Co and Zn concentrations. The ac conductivity studies revealed a typical negative temperature coefficient of resistance (NTCR) behavior, and the conductivity values varied from 1.58 × 10−5 to 8.46 × 10−6 S cm−1. The activation energy determined from the Arrhenius plots at 50 Hz was in the range of 0.23–0.78 eV.
[ "MATE" ]
[ "Dielectric", "Electric modulus", "FESEM", "Impedance", "X-ray diffraction" ]
[ "2501", "2502", "2503", "2504" ]
[ "The lithium-ion battery cathode materials are known to be structured of layered compounds such as lithium transition metal oxides LiMO2 (M = Co, Ni and Mn), spinel compounds like LiMn2O4 and olivine compounds like LiMPO4 (M = Fe, Ni, Co and Mn) [1–4].", "The layered compound of LiCoO2 was commonly used as the cathode material in lithium-ion batteries because of its high volumetric energy density, excellent cyclability and the ease with which it can be synthesized from raw materials.", "LiCoO2 has, however, three major disadvantages, including the high material cost, toxicity and low (only 50%) use of the theoretical capacity.", "Layered LiCoO2 and LiNiO2 exhibit complementary behaviors; LiCoO2 was easy to synthesize, but it is very expensive when compared to LiNiO2.", "Moreover, LiNiO2 shows the better electrochemical performance and is a low cost material.", "The nickel containing compound LiNiO2 and their doped derivatives have been extensively studied.", "LiNiO2 had the advantage of presenting a higher specific capacity for lithium cycling, but it was difficult to prepare in the layered structure due to the tendency of lithium and nickel, leading to the deterioration of their electrochemical performance.", "Many researchers have undertaken the search for new cathode materials to overcome these shortcomings.", "To reduce the cost and to improve the cell voltage and specific energy, other transition and non-transition metals, such as Cr, Mn, Fe, Al, Ca, Mg, Zn, Mn, Co, Ga, Sn, Sr, Ti, Zr, Cu, Rh and rare earth elements Ce and Y were used in LiNiO2 [5,6].", "These materials have been synthesized by various methods, such as sol–gel, combustion, co-precipitation and solid-state reaction etc.", "Among them, the latter method was desirable due to its simple and low cost route of fabrication [7].", "In this case, in order to obtain high performance well-ordered Li[Ni1/3Co1/3Mn1/3]O2 and LiNixMnyCo1−x−yO2 cathode materials, the optimizing and control of material parameters and synthesis procedure are rather important [8,9].", "LiNi1−xCoxO2, Al doped LiNiO2 and Mg doped LiNiO2, have also been extensively investigated for their safety characteristics.", "Thus, electro-inactive non-transition metal ions, partially Mg2+ and Al2+, have been of great interest as depots for cathode materials.", "However, to our best knowledge, another potential non-transition metal ion Zn2+, has not received as much attention as a dopant for layered cathode materials [10].", "In the present work, we report a systematic study of the morphology, structure, dielectric and impedance performance of LiNi1−x−y−0.02CoxZnyMg0.02O2 (x = y = 0.0, 0.02 and 0.04) cathode materials.", "Here Zn and Mg acted as the electrochemically active elements.", "The cathode compositions were synthesized by a solid-state reaction method using appropriate stoichiometric amounts of Li2CO3 (Merck 99.9%), MgO (Merck 99.9%), NiO (Merck 99.9%), CoO (Merck 99.9%) and ZnO (Merck 99.9%).", "A slight excess amount of lithium (5%) was used to compensate for any loss of the metal which might have occurred during the calcination at high temperatures.", "The mixture of the starting materials was sufficiently mixed and after grinding the powder, it was then heat treated in air at 500 °C for 5 h and it was again ground and mixed, and calcined at 750 °C for 20 h. Then, this powder was cooled at the rate of 5 °C/min.", "Finally, the powder was ground and mixed, and calcined again at 850 °C for 20 h in air using a muffle box furnace [11].", "The calcined powder was further annealed at 750 °C for 20 h.", "After being added with polyvinyl alcohol (PVA) as a binder, the powder was reground and then finally pressed at 5 tons/cm2 pressure into a circular disk shaped pellet.", "The pellets were heated up at a heating rate of 5 °C/min and then sintered at 850 °C for 20 h in air.", "Finally they were cooled down at the rate of 5 °C/min to room temperature.", "The sintered pellets were carefully polished on one side to obtain a smooth surface and then washed with acetone.", "After some proper drying, the pellets were coated with silver paste on the opposite surface which then acted as an electrode.", "The powder XRD data of the sample were collected on a Rigaku Cu-Kα diffractometer with diffraction angles ranging from 20° and 80° in an increment of 0.02°.", "Unit cell lattice parameters were obtained by the least square fitting method from the d-spacing and (hkl) values.", "Further, the crystal size of the sample was obtained by applying the Scherrer's equation from XRD pattern.", "The particle morphology of the powders was observed using a field effect scanning electron microscopy image taken from CarlZeiss, EVOMA 15, Oxford Instruments, Inca Penta FETx3.JPG.", "Fourier transform infrared (FT-IR) spectra was obtained on a Shimadzu FT-IR-8900 spectrometer using a KBr pellet technique in the wave number range between 400 and 1200 cm−1.", "The impedance study was performed by a Hioki 3532-50 LCR Hitester in the frequency range 50 Hz to 1 MHz at temperatures between room temperature and 120 °C.", "FESEM images of LiNi1−x−y−0.02Mg0.02CoxZnyO2 (x = y = 0.0, 0.02 and 0.04) samples are shown in Fig. 2(a–c).", "The particles have regular shapes with well defined faces and the microstructure obtained indicates the good crystallinity.", "The electrochemical performance of the Lithium-ion battery depends directly on the particle size, particle size distribution and the morphology of the cathode materials as well.", "The grain growth at the higher calcination temperature was considered as rapid, leading to the larger grain size.", "The calculated particle sizes for x = y = 0.0, 0.02 and 0.04 were 3.19, 3.53 and 3.85 μm respectively.", "The size or the texture of the particles was highly even, which indicates high crystallinity and the absence of defects in the pristine crystallites [16].", "The FESEM images revealed the well crystallized particles with a similar accumulative morphology [17].", "The primary particles of the synthesized samples became well shaped and their size increased with the increase of Co and Zn content.", "The synthesized materials with a smaller particle size with high capacity and uniform particle size distribution enhance the overall battery performance by the uniform depth of charge of each particle [18].", "Fig. 3(a–c) show the FT-IR spectra of synthesized LiNi1−x−y−0.02Mg0.02CoxZnyO2 (x = y = 0.0, 0.02 and 0.04) calcined at 850 °C for 20 h.", "The band found around 536 cm−1, is assigned to Li–O stretching vibration, which indicates the formation of LiO6 octahedra [19].", "The characteristic vibrations of Co–O, Ni–O, Zn–O and Mg–O were 560–590, 579, 611–735 and 630 cm−1, respectively and are listed in Table 2.", "In this work, the broadband located at around 638.69 cm−1 was attributed to the asymmetric stretching modes of MO6 (M = Ni, Mg, Co and Zn) group [20].", "Complex impedance spectroscopy (CIS) technique was used to analyze the electrical properties of a polycrystalline sample and its interface with electronically conducting electrodes in a wide frequency range (50 Hz–1 MHz) at different temperatures (30°–120 °C).", "Finally, Z′ and Z″ are displayed in a Nyquist plot in order to visualize the influence of the parameters.", "The real impedance spectrum Z′ as a function of the frequency and temperature is plotted in Fig. 4(a–c) for LiNi1−x−y−0.02Mg0.02CoxZnyO2 (x = y = 0.0, 0.02 and 0.04) materials.", "The decreased values Z′ have been observed for the rise in temperature at low frequency.", "It also shows a decrease of Z′ with an increase of frequency, forming a plateau like behavior which indicates the increase in conductivity of the material.", "The dispersion at low frequency is attributed to the release of space charge polarization with the increased temperature and frequency.", "This shows that the conduction mechanism has improved with increasing temperature and frequency [21].", "This behavior suggests that the material possesses a negative temperature coefficient of resistance (NTCR) [22].", "It was found that the Z′ values decreased with the increase of temperature, indicating the reduction of the grain size, the grain boundaries and the electrode interface resistance.", "The Z′ values decreased slowly for the frequency depending on the temperature, and continuously with an increase in frequency.", "Fig. 5(a–c) show the imaginary part Z″ of impedance as a function of the frequency and temperature for LiNi1−x−y−0.02Mg0.02CoxZnyO2 (x = y = 0.0, 0.02 and 0.04) materials.", "The relaxation species are possibly attributed to the presence of electrons at low temperatures and of defects/vacancies at higher temperatures.", "It is clearly seen that the curves display broad and low intensity peaks.", "The peak frequency shifts to the high frequency side with the increasing temperature, and the relaxation occurs over several decades of frequency.", "The plots show that Z″ values initially increased, attained a peak (Z″ max) and then decreased with frequency at all measured temperatures.", "Such a Debye-like relaxation peak in the frequency dependence of Z″ usually indicates the presence of space charges since the electrical behavior of space charges is dependent on the frequency.", "It is also observed that the peak value of Z″ decreased with the increased temperature, and the peak position was shifted to the high frequency side.", "Peak broadening with the increase in temperature suggests the presence of a temperature dependent dielectric relaxation phenomenon [23].", "The impedance diagram (Nyquist plot) is shown in Fig. 6(a–c) for LiNi1−x−y−0.02Mg0.02CoxZnyO2 (x = y = 0.0, 0.02 and 0.04) materials for different temperatures.", "All the semicircles exhibited some depression degree instead of the one, centered on the x-axis.", "This can be referred to as the Non-Debye type of relaxation in which there is a distribution of relaxation time [24].", "This non-ideal behavior can be correlated to several factors, such as grain orientation, grain boundary, stress–strain phenomena and atomic defect distribution.", "The presence of two semicircles at higher temperature exhibits the presence of both grain interior (bulk property) and grain boundary effect.", "The contribution peak positioned at low frequency corresponds to the grain boundary response and that in the high frequency range, corresponds to the bulk property of the material [25].", "The depression of the semicircle is considered as an additional evidence of the polarization phenomena with a distribution of relaxation times.", "The assignment of the two semicircular arcs to the electrical response is due to the grain interior and grain boundary, and considered to be consistent with the “brick-layer model” for polycrystalline samples [26].", "The real part of the modulus spectrum variation at different temperatures is shown in Fig. 7(a–c) for LiNi1−x−y−0.02Mg0.02CoxZnyO2 (x = y = 0.0, 0.02 and 0.04) samples.", "It is observed that M′ showed a constant value at higher frequencies while at lower ones, it approached to zero for all temperatures, albeit showing a dispersion in the range of intermediate frequencies which increased as the temperature was increased.", "It is seen that at lower frequencies, M′ approached zero, indicating that the electrode polarization had a negligible contribution to M′ only, and the dispersion is mainly due to the conductivity relaxation.", "The gradual variation of M′ indicates that the relaxation processes are spread over a range of angular frequencies [27].", "The frequency dependence of the imaginary part of the electric modulus M″ at different temperatures is shown in Fig. 8(a–c) for LiNi1−x−y−0.02Mg0.02CoxZnyO2 (x = y = 0.0, 0.02 and 0.04) materials.", "Well defined peaks are seen in the modulus spectra.", "These peaks represent the re-orientation relaxation process of mobile Li+ ions.", "The low-frequency side of the peaks as seen in the region where Li+ ions are capable of performing successful hopping from one site to the next one, whereas the high frequency side of the peak is where Li+ ions perform local motion (reorientation) only [28].", "The position of the peaks in the modulus spectra shifted towards high frequency as the temperature increased, which indicates a thermally activated relaxation process.", "The most probable conductivity relaxation time is determined by the frequency of the peak according to the relaxation.", "It is clearly seen that the values of M″ increased with frequency at each temperature and were constant for both high frequencies and low temperatures.", "Between these plateaus, the polarization effect is evidenced.", "From the M″(ω), we can observe a relaxation process with increased temperature that exhibits a maximum value, with M″max at the center in the dispersion region of M′(ω).", "Fig. 9(a–c) show the frequency dependence of the dielectric constant (ε) at room temperature for LiNi1−x−y−0.02Mg0.02CoxZnyO2 (x = y = 0.0, 0.02 and 0.04) samples.", "The high value of the dielectric constant at low frequency and the low value of the dielectric constant at high frequency indicate a large dielectric dispersion due to the Maxwell–Wagner type interfacial polarization.", "The dielectric constant decreased with increasing frequency and temperature.", "There is a sharp rise in the dielectric constant at low frequency and the shape of the rise changes with the temperature, due to the conducting ion motion.", "The high value of the dielectric constant reflects the effect of the space charge polarization and the conducting ionic motion.", "When an external electric field is applied, the electrons reach the grain boundary through hopping.", "If the resistance due to the grain boundary is high, the electrons pile up at the grain boundaries and induce the polarization.", "This is called space charge polarization [29].", "It can be observed that σac increased with increasing frequency.", "This can be explained in terms of conducting grains separated by highly resistive grain boundaries.", "According to this model, the a.c conductivity at low frequencies exhibited the grain boundary behavior, while the dispersion at high frequency is attributed to the conductivity of grains [30].", "The calculated a.c. conductivity values at different temperatures are shown in Table 3.", "These ionic conductivity values are varied between 1.58 × 10−5 and 8.67 × 10−07 S/cm for x = y = 0.0, 0.02 and 0.04.", "It was observed that the sample with x = y = 0.02 possessed good ionic conductivity compared to those with x = y = 0.0 and 0.04.", "Finally, these results also reveal a high ionic conductivity in samples with low additive concentrations (x = y = 0.02).", "We have successfully synthesized LiNi1−x−y−0.02Mg0.02CoxZnyO2 (x = y = 0.0, 0.02 and 0.04) materials using the solid-state reaction method.", "The XRD studies have revealed single phase formation of the orthorhombic structure in these materials.", "The FESEM images show the grain size in the range of 2 to 1 μm and the presence of agglomeration.", "FESEM micrographs have revealed that the grain size in the synthesized samples increases with increasing Co and Zn content.", "The complex impedance spectroscopy study of the dielectric, impedance and modulus properties of the samples has suggested the multiple relaxation behavior.", "In addition, at high frequencies, the dielectric constant becomes nearly frequency independent.", "Nyquist plots have shown both grain and grain boundary contributions to the impedance.", "The shape of the imaginary part of the modulus suggests that the relaxation processes in these materials are of non-Debye in nature.", "The good ionic conductivity of 1.58 × 10−5 S/cm−1 has been obtained for LiNi0.9Mg0.02Co0.02Zn0.02O2 at 383 K.", "The lowest value of the activation energy of 0.23 eV has been found at 50 kHz." ]
[ "We are preparing cathode materials for lithium ion batteries LiNi1−x−y−0.02CoxZnyMg0.02O2 (x = y = 0.0, 0.02 and 0.04) by using solid state reaction method.", "All the compounds characterized by XRD, FESEM, FT-IR, Impedance and dielectric studies.", "I got good results from conductivity study." ]
Human health risk assessment of antibiotic resistance associated with antibiotic residues in the environment: A review
The extensive use of antibiotics leading to the rapid spread of antibiotic resistance poses high health risks to humans, but to date there is still lack of a quantitative model to properly assess the risks. Concerns over the health risk of antibiotic residues in the environment are mainly (1) the potential hazard of ingested antibiotic residues in the environment altering the human microbiome and promoting emergence and selection for bacteria resistance inhabiting the human body, and (2) the potential hazard of creating a selection pressure on environmental microbiome and leading to reservoirs of antibiotic resistance in the environment. We provide a holistic view of health risk assessment of antibiotic resistance associated with antibiotic residues in the environment in contrast with that of the antibiotic resistant bacteria and discuss the main knowledge gaps and the future research that should be prioritized to achieve the quantitative risk assessment. We examined and summarized the available data and information on the four core elements of antibiotic resistance associated with antibiotic residues in the environment: hazard identification, exposure assessment, dose-response assessment, and risk characterization. The data required to characterize the risks of antibiotic residues in the environment is severely limited. The main future research needs have been identified to enable better assessments of antibiotic resistance associated with antibiotic residues in the environment: (1) establishment of a standardized monitoring guide of antibiotic residues and antibiotic resistance in the environment, (2) derivation of the relationship between antibiotic levels and pathogenic antibiotic-resistance development in different settings, and (3) establishment of the dose-response relationship between pathogenic antibiotic resistant bacteria and various infection diseases. After identification of key risk determinant parameters, we propose a conceptual framework of human health risk assessments of antibiotic residues in the environment. Capsule: A holistic view of human health risk assessment of antibiotic residues in the environment was provided.
[ "BIOC", "ENVI" ]
[ "Antibiotic residues", "Antibiotic resistance", "Exposure assessment", "Health risk assessment" ]
[ "1303", "2300" ]
[ "Antibiotics are a class of secondary metabolites produced by microorganisms, as well as chemically synthesized or semi-synthesized analogous compounds, which could inhibit growth and survival of other microorganisms (Demain and Sanchez, 2009).", "Antibiotics are very useful as therapeutic agents in the treatment of human infectious disease, and nowadays antibiotics are also extensively used in livestock industry and aquaculture (Nisha, 2008).", "Veterinary antibiotics are originally used in prevention and treatment of animal diseases, but they are gradually added as feed additives as prophylactics and growth promoters, which far outweighs their use as animal therapeutics at present (Gelband et al., 2015).", "Antibiotics can enter the water and land environment through various pathways (Ashbolt et al., 2013), such as the discharge of municipal sewage, manufacturing industry, animal husbandry, and landfill leachates of antibiotic disposal.", "Other sources may include runoff from agricultural field containing livestock manure, aquaculture ponds, and urban centers (Fig. 1).", "Particularly, irrigation with treated wastewater and fertilization with livestock manure are high potential pathways for the introduction of antibiotics in the agroecosystem.", "Although the half-life of most antibiotics is not long (hours to hundred days) (Ji et al., 2010), antibiotic residues that remain in the environment can be considered as a ‘persistent’ organic contaminant due to frequent and extensive use of antibiotics and uninterrupted emissions (Hamscher et al., 2002).", "The use of antibiotics in large amounts may pose a strong selection pressure on human and natural microbial system (National Academies of Sciences and Medicine, 2018; Qiao et al., 2017).", "The occurrence of antibiotics in microbial system may lead to genetic or mutational changes in normally sensitive bacteria, allowing the bacteria to survive and further proliferate as antibiotic resistant bacteria (ARB) that carry antibiotic resistant genes (ARGs) (Martinez, 2009).", "Mobile genetic elements (MGE), including plasmids, integrons, and prophages (Martínez et al., 2015), would further enhance the dissemination and promotion of genetic recombination of ARGs by transformation, conjugation, or transduction collectively referred as horizontal gene transfer (HGT) (Vikesland et al., 2017).", "ARGs are becoming recognized as an emerging environmental pollutant (Pruden et al., 2006).", "Concerns over the human health threats of antibiotic resistance associated with antibiotic residues in the environment are mainly (1) the potential hazard of ingested antibiotic residues altering the human microbiome and promoting emergence and selection for resistant bacteria resistance inhabiting the human body namely as human antibiotic resistance (Cho and Blaser, 2012); and (2) the potential hazard of creating a selection pressure on the environmental microbiome and leading to reservoirs of ARGs and ARB referred as environmental antibiotic resistance (Qiao et al., 2017).", "The clinically relevant antibiotic resistances are associated with the increased hospitalization of patient infection induced by such antibiotic-resistant pathogens, which may result in treatment failure and mortality due to the reduced efficiency in therapeutic antibiotic use (Hidron et al., 2008).", "Since antibiotics are extensively used nowadays, risk assessments of human exposure to antibiotic resistance in the environment have attracted increasing attention (Ashbolt et al., 2013).", "Human health risk assessment is to estimate the probability of illness and deaths caused by the infection associated with ARB (Ashbolt et al., 2013; Manaia, 2017), which included four core elements: hazard identification, exposure assessment, dose-response assessment, and risk characterization.", "The major purpose of the present study is to provide a holistic view of health risk assessment of antibiotic residues in the environment, based on the effects on ARB, and to address the main knowledge gaps and future research needs in the assessment framework.", "Once entering into human body, antibiotic residues might interact with the human microbiome, which contains a great number of diverse microorganisms inhabiting the human body (National Academies of Sciences and Medicine, 2018).", "Daily intake of antibiotic residues from the environment may largely enter the human gastrointestinal tract, where approximately 800–1000 different bacterial species and more than 7000 different strains are inhabited (Jernberg et al., 2010).", "Of these microbes, 95% are beneficial bacteria and others are harmful bacteria and opportunistic pathogens (National Academies of Sciences and Medicine, 2018).", "There is a stable micro-ecological balance between these bacteria and between bacteria and the human body over time, with predominant taxa of Bacteroidetes and Firmicutes, and minorities of Actinobacteria, Proteobacteria, Verrucomicrobia and other bacteria phyla that have not yet been isolated or identified (Arumugam et al., 2011).", "Observational, clinical and epidemiological studies have provided increasing evidence that antibiotic exposures are strongly associated with changes in intestinal microbiome composition due to the broad-spectrum influence on the host-associated microbial community rather than one target bacteria (Blaser, 2016).", "Antibiotic therapy could lead to composition changes of intestinal microbiota with an increase in Firmicutes and a concurrent reduction in Bacteroidetes (Pérez-Cobas et al., 2012), and also induce the emergence of antibiotic-resistant bacteria, which could persist in human intestines for years (Andersson and Hughes, 2011; Jernberg et al., 2010).", "Once imbalance of intestinal microbiota occurs, it may lead to the proliferation of harmful bacteria and opportunistic pathogens, and further lead to various diseases such as pseudomembranous colitis, intestinal disorders and colorectal cancer (Damman et al., 2012).", "Even worse, if intestinal bacteria have developed antibiotic resistance, multiplied in large numbers, and evolved into super-bugs, the diseases caused by these bacteria will lead to death due to incurability.", "This is not only a risk to human individuals, but to the global human population.", "A hypothesis has been put forward, that during the course of antibiotic treatment, the taxa which were low in number and vulnerable to antibiotics may be lost, in contrast with that the antibiotic resistant bacteria would be survived and accumulated (Blaser, 2016).", "If the taxa serve special metabolic functions, the collateral damage to the host may further lead to human metabolic perturbations and alteration of immunologic development, which may affect adiposity and bone growth (Cho et al., 2012; Cox et al., 2014).", "This is in particular important in early-years of human life, when the adult microbiome has not been formed and mainly inherits from mother (Blaser, 2016).", "Compared to the proactive avoidance measure in medicine, the intake of antibiotic residues from the environment is inevitable for children especially after weaning and beginning to consume staple food, which may exert a great influence in the early human life with a critical window in the development of microbiome.", "Both epidemiological and experimental studies have suggested that the effects of antibiotics may be cumulative in the human individuals, and thus it can expect that the effects of environmental antibiotic exposure on human microbiome may cumulate across generations (Sonnenburg et al., 2016).", "However, the select and shape influences on human microbial community structure with chronic exposure to antibiotic mixtures and possible interactions among themselves are not fully understood.", "This critical and important relationship between antibiotic residue exposure and human microbiome and function in terms of human health will require further research.", "Antibiotic residues could accelerate the emergency and evolution of ARB and ARGs in the environment (Qiao et al., 2017), and the risks associated with the environmental antibiotic resistome refer to the transmission of environmental ARB and ARGs to humans (Manaia, 2017).", "Antibiotic residues in the environment create selection pressure on the environmental microbiome and thus generate environmental reservoirs of ARB and ARGs, the phylogenetical bacteria diversity of which belong to phyla such as Actinobacteria, Proteobacteria, or Bacteroidetes, which often possess the capacity to produce or metabolize antibiotics as well as to transfer environmental ARB and ARGs to humans (Forsberg et al., 2012; Manaia, 2017).", "However, the transmission of environmental ARGs to human could be relied on carrying by pathogenic ARB or human commensal ARB both of which are most likely to be members of the classes Gammaproteobacteria and Betaproteobacteria and the phyla Actinobacteria and Firmicutes (Li et al., 2015; Manaia, 2017; Vaz-Moreira et al., 2014).", "Pathogenic and the human commensal ARB have the ability to colonize and proliferate in human body, and pathogenic ARB have the ability to penetrate into tissues and cause acute immune reaction and infectious disease.", "Most commensal ARB are not pathogens, but they could cumulatively reside in the human body and may harbor crucial virulence genes and therefore cause a disease, or they could transfer the genes conferring crucial virulence to other commensal human microbiota.", "There are about 200–220 types of antibiotic in the natural environment, out of more than 250 types of semisynthetic or modified natural product-based drugs produced (Bérdy, 2012).", "Antibiotics can be divided into 10 main categories according to different functional chemical structures: aminoglycoside, β-lactams, lincosamides, macrolides, polypeptides, quinolones, sulfonamides, tetracyclines, chloramphenicols, and others.", "In these categories, there are about 120 antibiotics available in human health care and livestock husbandry (Wang et al., 2015a).", "Since antibiotics are extensively used nowadays, biomonitoring of human exposure to antibiotic residues in the environment has become increasingly important.", "Antibiotics have frequently been detected in surface water and wastewater at concentrations generally ranging between 0.01 and 1.0 μg/L (Le Page et al., 2017).", "That means antibiotic residues have already entered the major sources of drinking water for most people, the occurrence of which may be persistent for several months and may not be removed completely by traditional disinfection technologies in drinking water treatment.", "However, there is relatively scarce information regarding antibiotic occurrence in drinking water.", "Quinolones, chloramphenicols, sulfonamides, and macrolides appeared to be the main antibiotics detected in drinking water with the highest concentrations and detection frequencies, with ciprofloxacin exhibiting the highest detected concentration up to 679.7 ng/L (Wang et al., 2010).", "Based on the analysis from the existing data, the concentration of antibiotic residues of tap water in Guangzhou City (maximum concentrations: 7.9–679.7 ng/L) was higher than that in 42 other cities in China, and Chinese tap water may contain a higher residual antibiotic concentration than other countries (Benotti et al., 2008; Boleda et al., 2014; Simazaki et al., 2015; Zuccato et al., 2000) (Fig. 2A).", "To our best knowledge, there is currently no report on the concentration of antibiotic residues in the air.", "However, residual antibiotics have been detected in airborne particles originating from pig-fattening farms, cattle feed yards, and pharmaceutical companies (Farshad et al., 2016; Hamscher et al., 2003; McEachran et al., 2015).", "Six different antibiotics including tylosin, three tetracyclines, sulfamethazine, and chloramphenicol were detected ranging from not detected (ND) - 12.5 mg/kg in dust samples collected from a pig-fattening farm in Germany during 1981–2000 (Hamscher et al., 2003), while the concentrations of three tetracyclines, tylosin and monensin detected in dust downwind of feed yards in the Southern High Plains, USA ranged from 0.5 to 4.6 mg/kg (McEachran et al., 2015).", "Penicillin was detected in airborne dust from a pharmaceutical company with a mean concentration of 6.6 mg/m3 (Farshad et al., 2016).", "However, there is a lack of information related to antibiotics associated with dust from public areas where general population comes into contact.", "Antibiotics could be introduced into soil through wastewater or reclaimed water irrigation, manure-amended application, or sludge landfill.", "The concentrations of antibiotics varied greatly across soils from different sources from μg/kg to mg/kg (dry weight) (Fig. 2E).", "Generally, antibiotic residues exhibit higher concentrations in manure amended soils than those of wastewater or reclaimed water irrigated soils.", "In addition, tetracycline antibiotics in soils showed higher residual levels than quinolones and sulfonamides, with the highest concentration (11.0 mg/kg) of chlorotetracycline detected in livestock manure and amended soils in Northern China (Huang et al., 2013), while sulfonamides showed a higher detection frequency (33.33–100%) in wastewater irrigation soils in Beijing and Tianjin, China (Chen et al., 2014).", "Many antibiotics have been used in food-producing animals for therapeutic, prophylactic and growth promotion purposes, and therefore trace quantities of antibiotics may be present in meat products as residues (Cheong et al., 2010; Tavakoli et al., 2015; Yamaguchi et al., 2015).", "Antibiotic residues have been found in pork, chicken, beef, and swine liver in different countries (Fig. 2B).", "It seemed that sulfonamides and tetracyclines were presented at higher concentrations and detection frequencies in meat products than quinolones, while aminoglycoside and β-lactams were also commonly detected in meat products.", "The antibiotic residue concentrations in meat products in Vietnam (Yamaguchi et al., 2015), Iran (Tavakoli et al., 2015), and Malaysia (Cheong et al., 2010) were higher than those in China when comparing residual levels of the same antibiotics.", "Antibiotics are widely used as an antibacterial agent into dairy products and diary livestock feed which may induce deposition of antibiotic residues in milk (Fig. 2C).", "Forty-three antibiotics were analyzed in raw milk samples collected from 10 provinces in China through ELISA (enzyme-linked immunosorbent assay) method, and the results showed that the average concentrations of 3 fluoroquinolones and 18 sulfonamides in raw milk in China were 8.52 and 7.64 μg/kg, respectively, with the highest concentrations detected in samples collected from Guangdong, followed by Tianjin (Zheng et al., 2013).", "The highest concentration of chloramphenicol (3629.2 μg/L) was noted in milk samples from Shanghai, China (Wang et al., 2017a), while the highest concentration of penicillin G (353 μg/L) was obtained in milk from Nigeria (Khanal et al., 2018).", "Laying hens fed or treated improperly with antibiotics may produce eggs contaminated with antibiotic residues.", "In an investigation conducted in Jordan, the concentration of chlortetracycline was up to 8589 μg/kg in egg white and 808 μg/kg in egg yolk (Alaboudi et al., 2013), respectively, while the highest concentration of enrofloxacin (1485 μg/kg) was detected in chicken eggs in Vietnam (Yamaguchi et al., 2017).", "Since chemical fertilizers are restricted for organic vegetables and grains, manure rich in plant nutrients is considered to be a good alternative in organic farming.", "However, the use of antibiotics may end up in livestock or poultry manure with a high residual concentration due to the incomplete absorption by animals.", "Therefore, antibiotic residues may accumulate in crops through uptake from manure-amended cropland.", "Concentrations of antibiotic residues in vegetables and grains in China are shown in Fig. 2D. High concentrations of three quinolones have been detected in vegetables from one of the largest vegetable bases in Shandong Province, China, of which the detected concentration was in the range of 18.2–658.3 μg/kg for norfloxacin, 2.0–32.3 μg/kg for enrofloxacin, and 2.5–27.5 μg/kg for ciprofloxacin, respectively (Li et al., 2014).", "This was significantly higher than that in crops from the Pearl River Delta in Southern China (Pan et al., 2014).", "Quinolones and chloramphenicol showed higher concentrations in vegetables than those of tetracycline and sulfonamides.", "In general, the antibiotic distribution in plant was in the order of sequence of leaf > stem > root, which was consistent with the investigation of antibiotic residues in vegetables irrigated by pharmaceutical wastewater from Lahore, Pakistan with a mean detected concentration range of 0.02–0.85 μg/kg (Hussain et al., 2016).", "The relatively high concentrations of antibiotic residues in vegetables and grains suggested human exposure to antibiotics through the consumption of edible crops should not be neglected.", "The use of certain antibiotics as veterinary pharmaceuticals is approved for aquaculture, such as oxytetracycline, thiamphenicol, erythromycin, sarafloxacin, and trimethoprim (Serrano, 2005).", "Therefore, antibiotics may enter into the aquatic products mainly by oral administration and can also be taken up from the surrounding water or sediments with comparatively lower levels.", "Antibiotic residues have been detected in aquatic products collected in China (He et al., 2016; Song et al., 2016), Vietnam (Uchida et al., 2016), India (Swapna et al., 2012), Italy (Chiesa et al., 2018), and Korea (Won et al., 2011) (Fig. 2F).", "Quinolone and sulfonamide antibiotics were the most detected antibiotics in fish and shrimp, with a higher concentration and detection frequency.", "In general, the most developed economic regions in China, Tai Lake and Pearl River Delta, showed a higher antibiotic residue contamination in fish and shrimp (mean concentration: 0.1–120.58 μg/kg fresh weight) than that of Vietnam (0.02–47.23 μg/kg fw) and Italy (055–3.59 μg/kg fw).", "According to the above data summary, there is a huge biomonitoring data gap on the residual levels of antibiotics in human exposure sources in the environment, and different detected antibiotic compounds and analytical methods which hindered direct comparison of antibiotic residue concentrations among different studies.", "For example, bioanalytical method including ELISA (Zheng et al., 2013) and microbiological detection test kits (Aalipour et al., 2013) have been used, but most studies employed chemical analytical methods based on liquid chromatography-based mass spectrometry coupled with different detectors such as mass spectrometry, diode array detector (Salama et al., 2011), spectrofluorometric (Pena et al., 2004), and UV detector (Cheong et al., 2010).", "Furthermore, most previous studies only focused on < 20 antibiotic compounds, while up to 95 antibiotic compounds have been investigated in the environment or animal-derived products, 72 antibiotic compounds of which exhibited detectable concentrations (Table S1).", "Possible antibiotic residues in the environment may occur beyond the 72 antibiotics compounds.", "Therefore, monitoring a wider range of antibiotic residues using unified analytical methods are urgently needed with regard to the establishment of a standardized monitoring guide of antibiotic residues in the environment, which is the prerequisite for a more comprehensive exposure assessment of antibiotic residues in the environment.", "Previous studies monitored 72 antibiotic residues in drinking water, airborne dust and soil particles, and food (Table S1) with the highest reported concentrations in these matrices being up to 679.7 ng/L (Wang et al., 2010), 11.0 mg/kg (Hamscher et al., 2003) and 15.1 mg/kg (Chen et al., 2015), respectively.", "These data strongly indicate possible human health risks via drinking water, inhalation, ingestion and dermal contact of particulate matters, and food consumption, due to the unintended intake of antibiotics.", "A multisite biomonitoring-based study on the measurement of 18 antibiotics in urine samples of more than 1000 children in east China showed that the antibiotics were detected in 58.3% of the urine samples (Wang et al., 2015a).", "Especially, the detection of antibiotic residues in the children's urine samples which were used exclusively in animal husbandry strongly indicated that food might be the primary exposure source (Wang et al., 2016a), such as chloramphenicol which has been banned in animal-derived food in western developed countries, Japan and China (Nordkvist et al., 2016).", "However, the current comprehensive data on human exposure to antibiotic residues in the environment is still very limited.", "Furthermore, human exposure assessments of antibiotic residues through inhalation and ingestion of drinking water and food have not been fully explored and comprehensively evaluated across the globe.", "Human daily exposure to antibiotics from drinking water has been estimated in several studies which collectively suggested a limited role of drinking water in total human exposure to antibiotics.", "Therefore, the most likely dominant source would be food intake (Leung et al., 2013; Simazaki et al., 2015; Wang et al., 2016a).", "To our best knowledge, only a few studies have so far estimated the daily intake of antibiotic residues through food consumption.", "The median daily intake among general population in Korea through consumption of drinking water, and consumption of beef, pork, chicken, dairy products and fish was estimated to be 679.3, 88.8, 619.2, and 49.7 pg/kg/day for sulfamethazine, trimethoprim, enrofloxacin, and roxithromycin, respectively (Ji et al., 2010).", "The daily intake of a mixture of 21 antibiotics was estimated to be 0–0.0125 μg/kg/day for women and 0–0.0111 μg/kg/day for men in Shanghai, China, respectively, based on exposure sources of only meat products derived from livestock, poultry and aquaculture rather than full consideration of all the exposure sources (Wang et al., 2017a).", "The estimated daily intake was about 100-fold lower than the result obtained from back-calculation based on the concentration of 21 antibiotics detected in urine, that the daily antibiotic intake of 56.4% in children and 37.4% in pregnant women was in the range of 0–1.0 μg/kg/day, while that of 22% in children and 4.1% in pregnant women was > 1.0 μg/kg/day (Wang et al., 2017b; Wang et al., 2017b, 2016a).", "Although the reverse dosimetry calculation method may have great uncertainty, the lower external exposure estimation in the study based on the exposure pathway of meat product consumption may greatly underestimate human daily exposure to environmental antibiotic residues due to insufficient consideration of all the exposure sources (Wang et al., 2017a).", "However, a different result was obtained from a similar survey conducted in Hong Kong, China that the estimated daily intake of single antibiotic using the reverse dosimetry calculation method with the antibiotic concentrations in children urine ranged from 7.7 × 10−4 (ofloxacin) to 0.045 (oxytetracycline) μg/kg/day, which was 10 times lower than that based on food consumption (Li et al., 2017).", "This may be due to the relatively small investigation scale of 31 urine samples in the study.", "It is noteworthy that most antibiotic residues in food are heat stable in cooking process, suggesting that cooking process could not completely remove antibiotic residues in food (Li et al., 2017; Manu, 2014).", "Current studies on potential hazards of antibiotics on intestinal microbiome are mostly based on the short-term effects by therapeutic clinical or self-medication generally with a single type of antibiotic, which is used at a high-dose exposure level of mg/kg/day.", "Compared with the exposure of antibiotic targeting specific pathogens during disease treatment, exposure to antibiotic residues in the environment presented a different exposure mode: long-term exposure to a mixture of antibiotics with a high proportion of broad-spectrum veterinary antibiotics used in animal husbandry，at a low-dose exposure level of μg/kg/day.", "The detected trace amounts of antibiotic residues in the environment may pose a low risk when considered individually.", "However, chronic exposure to the mixture of hundreds of residual antibiotics may become a more powerful stressor on the selection of antibiotic-resistant bacteria than the antibiotics used in therapeutic purposes, similar to the difference between the toxicity processes of long-term exposure to low dose and short-term exposure to high dose.", "This may pose a high potential risk to human by the ARB infection associated with human microbiome.", "Further research is needed to assess the overall human exposure to antibiotic residues in the environment, especially through food consumption, and to clarify the exposure characteristics and the main exposure pathways.", "Gastrointestinal absorption of orally administered antibiotics mainly in the range of 30–90% derived from pharmacokinetics experiments with high oral doses used for therapy (MacGregor and Graziani, 1997).", "Few studies reported absorption of antibiotics intake through inhalation, ingestion of drinking water and food consumption, and the corresponding gut bacteria metabolic response.", "This is due to the technical challenge to analyze metabolites of the re-ingested antibiotics at the environmental level in the highly heterogeneous gastrointestinal system.", "Exposure of different single gut bacteria species to xenobiotic mixtures including antibiotics at human relevant levels, demonstrated that the gut bacteria metabolism could be significantly perturbed in a species-specific manner (Zhang et al., 2018).", "The absorption and metabolism of re-ingested antibiotics should be further investigated due to the importance of their role in selection and maintenance of ARB and ARGs within human body when taking total concentrations of active antibiotics into account.", "ARB and ARGs have been suggested as emerging environmental contaminants due to the undesirable increased prevalence impacted by human activities and the potential health threats induced by transmission from environment to human (Pruden et al., 2006).", "The highest ARB and ARGs burden sources are sewage water discharged from home and hospitals, antibiotic manufacturing facilities, and animal feedlots (Vikesland et al., 2017).", "There is a growing interest in the dissemination of antibiotic resistance in the environment, which has been reviewed (Qiao et al., 2017).", "Based on culture-dependent methods, ARB have been reported in water, soil and atmosphere related to antibiotic contaminated hotspots, with high microbial density, nutrient content and antibiotic concentration.", "This would enhance the selection and propagation of ARB and ARGs, especially from wastewater treatment plants, livestock farms and hospitals (Qiao et al., 2017).", "For example, based on the antimicrobial susceptibility test, it was indicated that about more than 50% Staphylococcus spp. isolated from the atmosphere of a sewage treatment plant in Poland exhibited resistance to penicillin, with 20% to erythromycin, and 11% to rifampicin, 6% to tetracycline, 3% to gentamicin, 3% to cefoxitin, and 1% to levofloxacin, but totally susceptible to chloramphenicol (Małecka-Adamowicz et al., 2016).", "19–75% of the Enterococcus spp. and 13–94% of the Streptococcus spp. recovered from the indoor air of a large-scale swine-feeding operation contained macrolide, lincosamide, streptogramin, or tetracycline resistant genes (Sapkota et al., 2006).", "The percentages of Enterobacteriaceae (Aeromonas, Enterobacter, Escherichia or Shigella, and Klebsiella) isolated from the effluents of a municipal wastewater treatment plant in China, resistant to chloramphenicol, penicillin, cephalothin, ampicillin, rifampicin, and tetracycline were 69%, 63%, 55%, 47%, 11% and 2.6%, respectively (Huang et al., 2012).", "Due to accessible isolation from different settings, Escherichia coli (E. coli) has also been widely used to indicate bacteria resistance.", "E. coli isolates from chicken, swine and cattle in animal farms in Shandong Province, China presented resistance to 12, 10 and 1 antimicrobial agents with a percentage of 51.61%, 25%, and 30%, respectively (Lu et al., 2010).", "Almost 50% of E. coli isolates had resistance to nine antibiotics from surface water of Wenyu River Basin in Beijing, China (Hu et al., 2008), and the resistant percentages to multiple classes of antibiotics ranged from 64% to 98% of isolates from manure waste discharged from livestock and poultry farms in Beijing and Hebei Province, China (Yang et al., 2004).", "The high prevalence of antibiotic resistance among bacteria especially pathogenic bacteria indicates a tremendous potential threat on human health.", "The different phenotyping of antibiotic-resistant bacteria may arise from different genes conferring resistance to different antibiotics.", "Genotyping profile of bacteria, namely ARGs, is a more sensitive indicator that facilitates the understanding of the mechanism of antibiotic selection for ARB, and the action mechanism of antibiotic resistance.", "Based on culture-independent method, thousands of ARGs in the environment could be identified and monitored by quantitative polymerase chain reaction (qPCR) and shotgun sequencing methods (Vikesland et al., 2017).", "Tetracycline (tet) and sulfonamide (sul) resistance genes are the most reported in effluent and sludge from wastewater treatments, surface water and sediments, livestock manure and manure-amended soils, which were considered to be commonly widespread in the environment due to the extensive use of the corresponding antibiotics.", "In addition, more recent attention has been paid to the occurrence of other ARGs, including macrolide resistance genes (erm), quinolone resistance genes (qnr), and beta-lactams (bla) (Qiao et al., 2017).", "In the present study, by consulting the relevant literatures, we summarized the detectable ARGs in the environment which add up to 56 genes including 3 MGEs (intI1, intI2, and Tn916/1545) with an abundance range of ND to 6.7 × 1010 (Zhang et al., 2009) copies/mL in surface water, 7.4–9.7 × 1010 (Luo et al., 2010) copies/g in sediments, ND to 6.6 × 1011 (Cheng et al., 2016) copies/g in manure, 3–7.9 × 102 (Xie et al., 2018) copies/m3 in air, ND to 1.4 × 1011 (Rahman et al., 2018) copies/g in soil, and ND to 7.8 × 1010 (Li et al., 2016) copies/mL in sewage and ND to 7.6 × 1011 (Wang et al., 2015b) copies/g in residues from sewage treatment plants.", "The detail information can be found in Table S2.", "However, these studies may be limited to the qPCR method of tracking antibiotic resistance, which is generally only able to examine a few ARGs at a time.", "It has been demonstrated that high-throughput sequencing-based metagenomic analysis could profile the full complement of DNA without a preselection of ARGs and yield a significantly higher diversity of ARGs than that by qPCR alone (Vikesland et al., 2017).", "However, qPCR arrays have been improved to simultaneously track up to 285 ARGs in the environment to overcome such weakness (Zhu et al., 2013).", "Similarly, as with antibiotic residue monitoring, different detected ARGs and especially the interpretation of data methods which based on total ARGs abundance, relative abundance of total ARGs, or diversity of ARGs hinders the direct comparison of ARGs levels in the environment among different studies.", "Furthermore, the ARGs database are still incomplete which can impact both qPCR and high-throughput sequencing approaches as they are intended to look for previously discovered genes.", "Therefore, further supplements and improvements of the ARGs database are still needed for a more comprehensive exposure assessment of antibiotic residues in the environment.", "Although a significant acceleration of selection and expansion of antibiotic resistance caused by antibiotic residues in the environment has been regarded as a common occurrence (Vikesland et al., 2017), the relationship between ARGs and their corresponding antibiotic concentrations or a different antibiotic class was found to be inconsistent.", "There are different results of strong, weak, or no correlation generated from different studies (Qiao et al., 2017), which may be due to different environmental fates related to ARGs and antibiotics themselves in different environmental settings (Wang et al., 2016b).", "It is noteworthy that horizontal co-transfer of ARGs and MGEs (Sun et al., 2016), and co-selection for resistance to unexposed antibiotic classes have been observed through metagenomic analysis (Lundström et al., 2016; Murray et al., 2018).", "The cross-resistance phenomenon is likely to be due to the transferable multi-resistance genes, indicating that HGT may partly contribute to the lack of direct correlation.", "MGEs are strongly associated with HGT, which could take along ARGs to new hosts to generate new resistant strains.", "It has been suggested that class 1 integron (intI1) consists a good indicator of antibiotic resistance associated with human pollution, due to the positive correlations with ARGs and human pollution (Gillings et al., 2015).", "With respect to this hypothesis, we integrated 72 sets of ARGs data all of which included 13 groups of genes: 7 tet genes (tetM, tetO, tetQ, tetW, tetA, tetB, tetX), 3 sul genes (sul1, sul2, sul3), 2 erm genes (ermB, ermC), and intI1 gene.", "Correlation analysis among the 13 group of ARGs was conducted based on SPSS (version 25.0), and the results show that intI1 is significantly correlated with the other 12 resistant genes (Pearson's correlation coefficients: 0.382–0.926, p < 0.05).", "There is a lack of information on human exposure to ARB or ARGs in the environment.", "Attempts have been made to estimate the daily intake of ermB, tetW, qnrS and intI1 via the inhalation of fine Particulate Matter (PM2.5), drinking water and ingestion of soil particle (Xie et al., 2018).", "Results showed similar daily intakes of ermB and intI1 by the three exposure pathways, but intake of tetW through drinking water or ingestion of soil particle was at least 2 fold higher than that by inhalation of PM2.5 (Xie et al., 2018).", "However, this calculation did not take into account the bioavailability of ARGs, nor other potential exposure routes such as contacts with water, farm animals, etc.", "Given the human consumption patterns, ARB or ARGs may be killed or thermal degraded via ingestion of drinking water and food, inhalation and dermal contact with particulate matters (soil, dust and airborne particles) may be therefore the main pathway in human intake of ARB or ARGs.", "Further studies are urgently needed to consider human exposure to ARB and ARGs.", "Bacterial responses to antibiotics are concentration-dependent with diverse biological responses in bacteria at different concentrations as a longstanding assumption (Gullberg et al., 2011; Bernier and Surette, 2013; Sandegren, 2014) (Fig. S1).", "The high concentrations of antibiotics would completely inhibit growth of the susceptible bacteria, exhibiting antimicrobial activities.", "When the concentrations are lower than the inhibitory concentrations, bacteria may trigger different cellular responses or alter gene expression as well as induce gene mutation and HGT, leading to antibiotic resistance.", "Dose-response assessment of the relationship between the evolution and emergence of antibiotic resistance and the antibiotic concentration requires a metric to indicate the potential of antibiotic concentrations to promote the development of ARB in complex bacterial communities.", "The Minimum Inhibitory concentration (MIC), based on bacteria lethality is used as the common metric of bacteria population resistance.", "Acinetobacter, Burkholderia, Stenotrophomonas, Neisseria, Enterococcus, Staphylococcus, Streptococcus, and Haemophilus have been suggested as bacterial indicators to assess MICs of antibiotics in clinical settings (Patel, 2017; Reller et al., 2009).", "MICs are generally based on the inhibition of bacterial growth rate at different gradient antibiotic concentrations and are measured by broth dilution test or disk diffusion test (Reller et al., 2009) (Fig. S1).", "The broth dilution test is to inoculate standard bacteria suspensions in different liquid media containing a gradient of antibiotic concentrations at 35 °C overnight, and MIC is the minimum concentration that can inhibit bacteria growth rate which is determined by visual observation of the turbidity of each medium.", "Disk diffusion test is to place disks containing different antibiotic concentrations on the surface of solid agar medium inoculated with a standard bacteria suspension at 35 °C overnight.", "The zone diameters of growth inhibition around each of the antibiotic disks are measured and interpreted according to the criteria published by the Clinical and Laboratory Standards Institute (CLSI) (Patel, 2017) or the US Food and Drug Administration (FDA) (Reller et al., 2009).", "It is a qualitative method to derive susceptibility classification as susceptible, intermediate, or resistant relative to an MIC value (Reller et al., 2009).", "The EUCAST database (European Committee on Antimicrobial Susceptibility Testing) has published the data of MICs for more than 100 antibiotics/antibiotics combinations and for clinically relevant bacteria species (Bengtsson-Palme and Larsson, 2016; EUCAST, 2018).", "In addition, based on the MICs for 111 antibiotics from the public EUCAST database, predict no effect concentrations (PNECs) for resistant selection were assessed with an assessment factor of 10 (Bengtsson-Palme and Larsson, 2016), corresponding to the median MIC/MSC (Minimal Selective Concentration) ratio estimated by Gullberg et al. (2011).", "The use of MICs is more relevant for clinical breakpoints of therapeutic process, but it is inadequate to assess bacterial antibiotic susceptibility and resistance evolution with the low levels of antibiotics.", "MSC of bacteria is defined as the concentration below MIC that favor the sensitive variant and would not compensate for growth impairment of the resistance mutation (Kraupner et al., 2018).", "MSC is a more sensitive metric to reflect the selective pressures at lower antibiotic concentrations than MICs (Sandegren, 2014).", "It has been proposed to obtain MSC in a two-strain competition system including susceptible and resistant genotypes as the lowest antibiotic concentration that could give a more competitive advantage of growth rate to the resistant strains than the susceptible strains (Fig. S1).", "Although MSC could better reflect enrichment possibility of resistant bacteria where low concentrations of antibiotics are present, the MSCs obtained from competition experiments are highly depended on the selected susceptible and resistant strains, the latter of which is generally the susceptible mutant carrying specific resistant-genes based on genetic modification technology (Sandegren, 2014).", "What needs to be firstly emphasized is that the composition and structure of bacterial community differ greatly in environmental and clinical settings.", "In addition, antibiotic residue concentrations are highly variable in different environmental media and human body in which bacteria could evolve different mechanisms to respond accordingly.", "This strongly implies that the relationship between the antibiotic concentration and the probability of the emergence of antibiotic resistance may be different in different settings.", "Therefore, the reliability of the MSCs measured according to two closely related strains is not likely to be directly applicable when extended to more complex microbial communities.", "Aware of the limitation of MSCs derived from single-species competition experiments, selective concentrations of antibiotics in complex communities have been assessed in recent studies, including those of tetracycline (≤ 1 μg/L) in complex aquatic bacteria biofilm (Lundström et al., 2016), ciprofloxacin (1 μg/L) in E.coli in complex aquatic bacteria biofilms (Kraupner et al., 2018), and cefotaxime (0.4 μg/L) in sewage-derived bacteria communities (Murray et al., 2018).", "It is worth noting that Murray et al. (2018) observed a selection plateau as antibiotic concentration increased over a 2-order-magnitude range.", "This indicated that bacterial selection responses to antibiotics may not occur in a concentration-dependent manner in complex bacteria communities, presumably due to a cross protective effect that the resistant bacteria in the community may break down the extracellular antibiotics to the benefit of the nearby susceptible bacteria.", "However, MICs are generally based on the function of growth rate of a single clinical isolate, which is actually not sensitive and representative enough to reflect the evolution of bacteria antibiotic resistance.", "In contrast with MICs, MSCs have been assessed using both phenotypic endpoints (culture-based method), taxonomic endpoints (culture-based method), and genotypic endpoints (metagenomic analysis and qPCR method) (Lundström et al., 2016; Kraupner et al., 2018; Murray et al., 2018).", "Genetic resistance markers have been demonstrated to be more sensitive than phenotypic endpoints (Lundström et al., 2016).", "Several approaches have been proposed to define the genetic resistance of antibiotic, including total ARG abundance, relative abundance of total ARGs, diversity of ARGs, and specific ARGs of interest.", "It is essential to emphasize that in a health risk assessment framework, link-bridges should be established to enable direct comparison and conversion of antibiotic resistance in different environmental compartments, which is also further relevant to human exposure and subsequent infections.", "With respect to health risk assessment, relative abundance normalized to bacterial 16S rRNA genes (type of pathogens) or pathogenic bacteria number (infection dose of pathogens) is recommended due to the correlation with human infection by antibiotic resistant pathogens.", "Further specific ARG encoding to last resort antibiotics (e.g, carbapenems, vancomycin, colistin, etc) normalized to bacterial 16S rRNA genes or pathogenic bacteria number is also of great concern due to the correlation with treatment failure of antibiotic resistant pathogen infection.", "Furthermore, HGT is the core challenge of assessing the spread of antibiotic resistance in the environment.", "MGEs are of the greatest concern due to the association with HGT, which are recommended to be included in the target genes in the response assessment.", "High levels of co-selection were found in selection for antibiotic resistance in complex microbial communities exposed to a single antibiotic (Lundström et al., 2016; Kraupner et al., 2018; Murray et al., 2018), indicating the presence of multi-resistance plasmids which may colonize multiple genes and could confer resistance to a wide range of different antibiotics (Gullberg et al., 2014).", "Selection of antibiotic resistance at very low concentrations indicates a high potential selection of antibiotic resistant bacteria in or on human bodies and the external environment.", "To obtain a representative set of species or a prevailing species in different settings relevant for risk assessment is challenging due to the difficulty in cultivating the vast majority of environmental and human bacteria.", "Therefore, holistic parameters of antibiotic resistance evolution are needed, to show the emergence and dynamics of ARB and ARGs in different environmental and human compartments as condition changes such as pH value, temperature, oxygen level, and oxidation reduction potential which may be related to the spread of pathogenic resistance.", "In our opinion, holistic parameters should include three factors: antibiotic resistance definition, representative bacteria genotypes, and living conditions.", "The use of either different representative bacteria structure in different settings or the same bacteria indicator is a feasible choice.", "It has been suggested that E. coli may be a good indicator of resistance since it is ubiquitous in the environment and serves as a representative of many pathogens.", "The reliability of E. coli representing the emergence and dynamic of antibiotic resistance evolution should be critically validated in different settings as different living conditions may impact the antimicrobial activities of antibiotics and the evolution of bacterial antibiotic resistance in different environmental or human compartments.", "This could be achieved by setting up different simulation systems as close as possible to imitate the living conditions, including pH value, temperature, oxygen level, oxidation reduction potential, etc.", "If the relationships between antibiotic concentrations and ARG abundance in different settings have been observed, further relationships between ARGs and ARB should be established in order to identify pathogens since that the risk of antibiotic resistance to humans must be assessed based on ARB rather than ARGs.", "Thus, in the conceptual model proposed to assess the health risk of antibiotic resistance associated with environmental antibiotic residues, pathogenic ARB would be the end-of-the pathway for environmental antibiotic resistance transmission to humans (Manaia, 2017).", "These recommendations attempt to sharpen the focus of antibiotic resistance in the environment to derive the relationship between antibiotic levels and antibiotic-resistance development and suggest a standardized method of resistance testing, taking into account of antibiotic resistance definition, representative bacteria genotypes, and living conditions, which could be directly applicable in different environmental and human settings.", "This is a pre-requisite for obtaining a global view of antibiotic resistance dynamic evolution impacted by antibiotic residues in environmental and human compartments, and further evaluating the health risk of antibiotic resistance associated with antibiotic residues in the environment.", "The overall goal of health risk assessment of exposure to antibiotic resistance is to estimate the number of infection diseases caused by antibiotic-resistant bacteria driven by environmental antibiotic residues and the number of deaths resulting from those infections.", "Pathogenic ARB are the most crucial component for assessing risks, the emergence probability of which could be predicted on account of antibiotic concentrations in human and environmental compartments, based on the presumptive quantitative relationship between antibiotic concentration and ARB development in different settings.", "Further estimation of pathogen risks also requires dose-response assessments that quantify the relationship between pathogen dose and the likelihood of infection.", "This would require the capacity of pathogenic ARB to infect human body at species or even strains level to characterize the exposure risk, which could be designated as the number of bacterial cells that are required to infect a host (Leggett et al., 2012).", "The pathogen-specific estimates of morbidity and mortality from antibiotic resistant infections have been suggested for each pathogenic ARB, according to the surveys of healthcare-associated infections and deaths in hospitals (Roberts et al., 2009).", "Therefore, on the combination with the probability of emergence of pathogenic ARB and the antibiotic resistant pathogen-specific likelihood of infection and death, the probability of infection and death driven by antibiotic residues in the environment would be derived.", "In the present study, a coherent conceptualized model is suggested to undertake human health risk assessment of antibiotic resistance in the environment (Fig. 3).", "The model established is to assess the risks of infection and deaths arising from human exposure to antibiotic resistance driven by environmental antibiotic residues.", "It takes into account of compartment-dependent aspects of antibiotic concentration-dynamic resistance response on the basis of experimental and modeling approaches.", "However, given much of the data required to work on the model is severely limited, it would be very important to conduct extensive research to fill these data gaps.", "As progress is made, the assessment model would be very helpful to form targeted policies in monitoring of antibiotic residues in the environment and mitigating the dissemination of environmental sources of antibiotic resistance.", "This would also provide a theoretical basis for reducing health risks of human exposure to antibiotic resistance in the environment." ]
[ "A quantitative framework is proposed to assess the impact of antibiotic residues on human health.", "A standardized monitoring guide for antibiotic residues is presented for exposure assessment.", "Co-resistance and cross-resistance complicate the dose-response relationship for ARGs.", "Pathogenic ARB, not ARGs, is the end component to assess the health risk of antibiotic resistance." ]
A note on a design protocol for deoxygenation of water
Selection of a water deoxygenation method presents some challenges due to the broad range of options including physical, chemical, electrochemical and biological techniques. This communication presents the design of an apparatus developed for efficient and simple deoxygenation of water for the purpose of electrochemical corrosion studies in a lab environment. The design is modular and can be scaled to meet processing needs. The operation of the apparatus is based on the combination of nitrogen bubbling and surface absorption to achieve complete and uncontaminated deoxygenation of water. Tests conducted on this apparatus revealed that pressurisation of the apparatus enhances economy and performance while increasing nitrogen injection rate accelerates deoxygenation. However, a point was identified, at which a balance between economy and performance is achieved and further injection rate increase suffers from diminishing returns.
[ "CHEM" ]
[ "Deaeration", "Deoxygenation", "Water" ]
[ "1603" ]
[ "Removal of dissolved oxygen from water is an important process used in a number of industries in order to mitigate issues such as corrosion, poor heat transfer, oxidation of food and pharmaceuticals or even gas bubble disease in fish [1].", "There are a number of physical, chemical, electrochemical [2] and biological methods for deoxygenation of water available.", "Physical methods include thermal degassing, vacuum degassing, nitrogen purging, desorption and degassing through a membrane [3–8].", "Chemical methods include the use of reagents such as sodium sulphite [9], Wood's metal alloy [10], or hydrazine [11] and biological methods include the use of glucose oxidase [12] and yeast [13].", "All the aforementioned methods come with shortcomings such as low efficiency, water contamination, high capital and operating cost, low production rate, production volume limitations, and complicated protocols.", "Quite often a combination of methods is selected to work around the shortcomings of individual methods.", "The selection of the appropriate method or combination of methods that fit the intended purpose is essential to achieve economical, rapid and simple deoxygenation of water.", "According to ASTM G119-09 (2016) Standard Guide for Determining Synergism between Wear and Corrosion [14] and Stack et al. [15–17] an essential part of determining wear and corrosion synergism is achieved by conducting tests under cathodic protection to quantify the corrosion-free mechanical degradation contribution.", "However cathodic protection alone does not fully eradicate the effect of corrosion, but in conjunction with deoxygenation of water it allows for a truly corrosion-free test to be conducted without the need to reduce the potential to values at which hydrogen redox reaction occurs and affects the mechanical properties of the specimen.", "This communication aims to present the design of an apparatus that is capable of complete deoxygenation of water for the purpose of corrosion studies in a lab environment.", "The deoxygenation setup was fabricated using a 105 l water tank that sits inside a steel cage designed to prevent deformation of the tank (Fig. 1).", "The tank is fitted at the top with a pressure gauge to monitor pressure, a ball valve to regulate pressure and a pressure relief valve to maintain a gauge pressure of 3.5 PSI.", "At the lower part of the tank there is a ball valve that acts as the water inlet and outlet and at the bottom of the tank sits a ceramic fine bubble diffuser connected to a regulated nitrogen supply.", "All components are widely available and allow for quick and easy assembly of the apparatus.", "The apparatus is sealed and purged with oxygen-free nitrogen of 99.98% purity (BOC) to ensure maximum efficiency [4].", "Nitrogen purging is conducted at a rate of 1 l/min for 20 min.", "By displacing the atmospheric air with the injected nitrogen the interfacial area between nitrogen and the water is increased, leading to an increased rate of gas absorption [3].", "Deionised water, at room temperature (20 °C) with an average DO concentration of 6.5 ppm, is then introduced in the rig at a rate of ~5 l/min while nitrogen injection continues at a rate of 1 l/min, amounting to a total use of 29, 34 or 39 l of nitrogen for the preparation of 45, 70 or 95 l of water respectively.", "At the completion of the process water DO concentration has been reduced to an average of 5.6 ppm.", "The injected nitrogen is broken down into micro-bubbles by the submerged sintered ceramic bubble diffuser with a mean pore diameter of 37 μm.", "Although for a fixed number of bubbles, big bubbles offer greater area for bubble-liquid gas exchange and are more effective than their smaller counterparts, for a fixed gas supply the bubble number and residence time increases with decreasing size leading to smaller bubbles being more effective deoxygenators [1,3].", "The pressure relief valve keeps the system at a constant gauge pressure of 3.5 PSI.", "The pressure build-up accelerates the absorption of nitrogen through the water surface.", "The absorption through the surface is further enhanced by the surface agitation caused by the breaching bubbles.", "After a predetermined amount of time dependant on injection rate the solution is fully saturated with nitrogen and the DO levels have been reduced to 0.00 ppm as measured using a HI-2400 (Hanna Instruments) dissolved oxygen meter and Rhodazine-D reagent ampules (CHEMetrics).", "In order to determine the most efficient, and economical way of deoxygenation, and further expand the understanding of the process, nitrogen purging was conducted for 45, 70 and 95 l of water, using injection rates of 1, 2, 3, 4 and 5 l/min, under pressure and in an open environment.", "Fig. 2 displays a trend of rapid deoxygenation at the initial stages of processing.", "However, the rate of deoxygenation steadily declines with decreasing dissolved oxygen (DO) concentration.", "The reduced DO concentration leads to less N2 bubbles – DO interaction which slows down the rate of deoxygenation.", "This trend is present for all injection rates and environments.", "In Fig. 3 the processing time required to achieve complete deoxygenation of 70 l of water in an open and a pressurised environment was plotted for a range of injection rates.", "It is evident that the pressurisation of the environment (3.5 PSI gauge pressure) accelerates the deoxygenation of water.", "At 1 l/min injection rate, pressurisation reduced the processing time by 30%, however at the higher injection rates of 2, 3, 4 and 5 l/min the effect was reduced down to 7%, 4.5%, 5.5% and 12% respectively.", "In both the open and pressurised environment an increase in injection rate led to a decrease in the processing time.", "In both environments the degree of reduction in processing time is diminished at injection rates beyond 3 l/min.", "Fig. 4 displays the deoxygenating performance of 45, 70 and 90 l of water in a pressurised environment (3.5 PSI gauge pressure).", "It can be seen that although the volume increase from 45 to 70 l is substantial (~60%) it is not accompanied by an analogous increase in the deoxygenation duration.", "In fact, the largest increase in the deoxygenation time was that of 1 min for the 1, 4 and 5 l/min injection rates while the duration for the 2 and 3 l/min injection rates remained constant.", "Minor duration increases are also noticed when comparing the processing time of 45 and 90 l.", "The 100% increase in volume leads to a 3, 2, 2, 4, and 8 min increase for the 1, 2, 3, 4 and 5 l/min injection rates respectively.", "Looking at the processing duration increase in terms of percentage, a trend is revealed.", "There is a 2.7%, 3%, 4.6%, 11.7% and 28.5% increase for the 1, 2, 3, 4 and 5 l/min injection rates respectively.", "It is clear that the effect of volume increase becomes more noticeable with greater injection rate values.", "The overall insignificant gains in the processing time with volume increase, can be interpreted by examining the geometry of the apparatus.", "Water volume increase only led to an increase in the height of the water column while the cross sectional area was left unaffected.", "The N2 bubbles will travel the full height of the column regardless and it appears that their DO-removal properties are barely affected by traveling distance.", "Looking at the consumption data in Fig. 5, it is clear that even the minor pressurisation of 3.5 PSI (gauge pressure) greatly affects consumption.", "Pressurisation reduces the nitrogen consumption by an average of 12.5%.", "Consumption in the pressurised environment follows an almost linear relationship with injection rate whereas in the open environment nitrogen consumption decreases with increasing injection rate until it reaches a low point at 3 l/min and further increase causes an increase in consumption.", "The N2 consumption data for the deoxygenation of 45, 70 and 95 l (Fig. 6) reveal an overall higher N2 consumption for greater water volumes.", "However, the N2 consumption per litre of water data portray staggering differences (Fig. 7).", "The deoxygenation of 45 l displays the highest consumption of N2 per litre of water ranging from 3.07 to 3.76 l/l, while deoxygenation of 95 l is the most economic option with consumption values ranging from 1.59 to 2.31 l/l.", "Furthermore, consumption increases with increasing injection rate, although for both the deoxygenation of 45 and 70 l, the consumption value for 3 l/min injection rate is slightly lower than that for the 2 l/min injection rate.", "It is clear that increasing the volume leads to a greater overall economy while lower injection rates also offer greater economy at the expense of processing time.", "Deoxygenation in this apparatus is achieved by two physical mechanisms; nitrogen bubbling and surface absorption.", "By pressurising the environment, the mechanism of surface absorption is enhanced.", "The enhancement of surface absorption accelerates processing time and enhances economy by making the process less dependent on continuous nitrogen injection.", "In the open environment, nitrogen bubbling is the main mechanism of deoxygenation.", "Based on the consumption (Figs. 5, 6 and 7) and performance (Figs. 3 and 4) data, the system appears to reach a critical point at the 3 l/min injection rate.", "At 3 l/min, a critical number of bubbles is generated that fully engulfs the volume of water achieving optimum deoxygenation; further increase in injection rate marginally shortens the processing time and mostly increases consumption.", "However, it must be noted that this critical point is dependent on a number of parameters unique to this apparatus.", "There are several alternative deoxygenation techniques that offer comparable results such as that by Lausier et al. [5] who by using a nitrogen powered permeator achieved a reduction in DO from 8.37 to 0.7 ppm with one pass and down to 0.12 ppm with an additional pass, or that presented by Polyanskii et al. [7] who by using a sorption membrane achieved DO concentration of less than 0.01 ppm.", "However, nitrogen purging was selected as it is a very efficient and simple technique which can also be seen in the comparison of water deaeration techniques by Butler et al. [3].", "By plotting the DO measurements (Fig. 8), it can be seen that the system achieves rapid deoxygenation even after only 5 min.", "Its high efficiency can be attributed to the extremely high injection rate for a small volume of water (1.5 l/min for 1 l of water).", "The use of higher purity nitrogen, based on a very small sample size, appears to have no effect and follows the same trend of deoxygenation.", "The process appears to slow down at around the 0.2 ppm mark, behaviour also observed in the author's apparatus, which in this case could be due to oxygen reabsorption from the surface or due to the oxygen present in the nitrogen gas.", "A larger sample size is required to attain a conclusive image of the system's performance.", "This communication presents the design of an apparatus that is capable of rapid, complete, simple and economical deoxygenation of small batches of water for the purpose of corrosion studies in a lab environment.", "It employs the combined effect of two physical methods without the use of energy or chemical reagents that contaminate the water.", "The design is modular and can be scaled to adjust production batch size.", "It also allows for the user to optimise its performance and economy by adjusting nitrogen injection rate and pressure.", "However, as every other deoxygenation process examined, this apparatus has two minor limitations.", "The production of deoxygenated water is non-continuous and the produced water is saturated with dissolved nitrogen.", "Although nitrogen is inert and in temperatures below 150 °C its effect is limited to nitrogen-fixing bacteria but in temperatures above 150 °C it may be involved in abiotic redox reductions.", "Tests conducted on the apparatus revealed that:", "The rate of deoxygenation by N2 purging is proportional to the DO concentration.", "Efficiency and economy can be enhanced by designing a tall processing column with a small cross sectional area.", "Maximising batch size and maintaining low injection rates can also enhance economy at the expense of processing time.", "Applying pressure enhances surface absorption which in turn enhances economy and accelerates deoxygenation.", "Increasing the N2 injection rate accelerates the deoxygenation process, however after a certain point, 3 l/min for this specific apparatus, there are diminishing returns." ]
[ "A design for complete, rapid, and economical deoxygenation of water is presented.", "Deoxygenation is achieved by a combination of N2 purging and surface absorption.", "The rate of deoxygenation by N2 purging is proportional to the DO concentration.", "An increase in pressure allows for greater economy and reduced processing time.", "Maximising batch size and maintaining low injection rates can enhance economy." ]
Examination of a soot model in premixed laminar flames at fuel-rich conditions
The primary objective of the present work is to collect and review the vast amount of experimental data on rich laminar premixed flames of hydrocarbon fuels reported in recent years, and to analyze them by using a detailed kinetic mechanism, identifying aspects of the mechanism of PAH and soot formation requiring further revisions. The kinetic assessment was hierarchically conducted, with the progressive extension of the core C 0 -C 2 NUIG mechanism up to the CRECK kinetic mechanism of PAH and soot formation. This mechanism is here adopted to evaluate and analyze the extensive amount of experimental data collected. Therefore, it provides a kinetic guideline, useful to critically compare and unify flames involving similar fuels and/or conditions from different sources. The relevant effect of soot particles formation on heavy PAHs concentration is also discussed, together with the kinetic analysis highlighting systematic deviations and critical issues still existing in the present model. The model performances were evaluated using the Curve Matching approach (Bernardi et al., 2016). Considering the challenges of quantitative PAH measurements and associated uncertainties, this extensive database is a further value of this paper and is beneficial for improving reliability of kinetic models in a wide range of conditions.
[ "CENG", "CHEM", "ENGI" ]
[ "Experimental database", "Laminar premixed flames", "PAH", "Soot precursors" ]
[ "1500", "1606", "2210" ]
[ "Polycyclic aromatic hydrocarbons (PAH) are carcinogenic compounds harmful to human health.", "In addition to this, PAHs are also precursors of soot by means of radical addition reactions on double bonds, cyclization, and formation of resonantly stabilized radicals, finally resulting in incipient soot [1].", "The formation of soot has been intensively studied through different modeling approaches [2–4].", "However, all of them require predictive and comprehensive detailed kinetic mechanisms able to correctly describe PAHs formation for a wide range of fuels and operating conditions.", "PAH growth and soot formation are typically investigated in nearly sooting or sooting premixed laminar flames [5].", "A large number of experimental studies of rich premixed laminar flames have been presented in the past decades, providing a massive amount of detailed information on temperature and concentration profiles for individual flames.", "The last effort to collect and review several flames for different hydrocarbon fuels dated back to over eight years ago and consisted of 22 flames [6].", "Since then, advances in analytical techniques and additional measurements drove improved predictive capabilities of kinetic models.", "These advancements motivate the comprehensive collection here reported which is used to gain significant insights on current knowledge of PAHs and soot chemistry.", "This work provides the basis for further improvements of the CRECK soot model by means of a hierarchical approach to the modeling of PAHs kinetics in flames.", "The motivations and objectives of the present work are:", "To organize an extensive data collection of literature measurements in rich laminar premixed flames of different hydrocarbon fuels;", "To critically assess the model performance through extensive comparisons with experimental data by means of a statistical analysis using the curve matching (CM) approach [7].", "Simulations of all the flames are done with the updated CRECK kinetic mechanism of PAH and soot formation [8,9], which includes the reference C0–C2 chemistry developed at NUIG [10].", "These simulations are useful both for the evaluation of the consistency of experimental data and for identifying limitations in kinetic models.", "To our knowledge, this work constitutes the first effort toward such an extensive and systematic analysis on PAHs chemistry, also highlighting the effect of soot formation on the yields of intermediate PAHs.", "Table 1 summarizes the experimental conditions of the laminar flames here investigated.", "The database provided in the Supplementary Material (SM) accounts for 60 flames, for more than 20 different C1–C10 fuels, in a wide range of equivalence ratios (ϕ = 1.00–3.06) and pressures (P = 0.03–1.00 atm).", "The gas-phase high temperature CRECK mechanism (244 species and ∼6000 reactions) implements a C0–C3 core mechanism obtained from the H2/O2 and C1/C2 subsets from Metcalfe et al. [10], C3 from Burke et al. [11], and heavier fuels from Ranzi et al. [12,13].", "This important step toward the unification of core mechanisms allows to more effectively focus the attention on specific features of heavier and more complex molecules such as PAHs.", "The thermochemical properties were adopted, when available, from the ATcT database of Rusic [14] or from Burcat's database [15].", "Unavailable properties of some species were obtained from group additivity method [16].", "The kinetic mechanism with thermodynamic and transport properties is attached as Supplementary Material to this study.", "With the aim to update the detailed kinetic mechanism towards PAH and soot, the dominant molecular weight growth pathways described according to the HACA mechanism [17], were revised based on the recent theoretical study by Mebel et al. [18].", "To fit this detailed and accurate description to the constrained size of the CRECK model [12,13], many isomer species were lumped, where appropriate.", "Regardless, the relative branching ratios are taken into account in the lumped mechanism, treating reactions with explicit forward and backward rates.", "In fact, as already discussed elsewhere [8,19], the CRECK mechanism involves only 20 lumped species and 16 radicals for the progressive growth from naphthalene to C20 species, which are the first soot precursors (BIN1).", "Additionally to HACA mechanism, the mass growth through light and heavy resonantly stabilized radicals (from propargyl C3H3, up to indenyl (C9H7)) is also considered [12].", "The soot mechanism, based on a discrete sectional model, is then coupled to the PAH sub-mechanism to describe the evolution from gas-phase to solid particles.", "The whole range of carbon atoms of heavy PAH and soot particles (from 20 up to more than 108 C atoms) is divided into 25 sections with a spacing factor of two.", "25 classes of lumped-pseudo species called “BIN” are considered with three different hydrogenation levels.", "The first four BINs (up to 160 carbon atoms) are heavy PAH, while heavier BINs are soot particles (up to 4 × 104 carbon atoms) and then soot aggregates.", "Thus, the soot kinetic model involves 150 molecular and radical species for describing the formation of soot particles up to ∼0.2 µm, i.e. 3.2 × 108 carbon atoms.", "The soot kinetic model consists of surface growth, nucleation, coagulation and oxidation processes.", "The surface growth reactions include HACA mechanism, as well as gaseous species and PAH condensation reactions.", "The condensing species are molecules and radicals of aromatic starting from benzene and phenyl radical.", "The complete soot sub-mechanism consists of ∼400 species and 25,000 reactions.", "Further details on the soot model are available in [8,9].", "Numerical simulations were performed using the 1-D laminar flame solver implemented in the OpenSMOKE++ suite of programs [20].", "The mixture-average diffusion coefficient was used in the simulation.", "Thermal diffusion (Soret effect) is also included in species transport equations.", "Solution gradient and curvature coefficients of 0.05 and 0.5 were assigned to ensure the smoothness of the calculated profiles.", "The measured temperature profiles were imposed to the simulations to account for the unknown conductive and radiative heat losses.", "Assessing the overall performances of the kinetic model over the wide set of experimental flames is a challenging task, because of the large number of species profiles.", "The Curve Matching approach [7] was here adopted to evaluate the agreement between experimental data and model predictions.", "It performs quantitative and qualitative error comparisons by the transformation of discrete experimental data and model predictions into two different continuous functions.", "It provides individual error indices, which account not only for square errors, but also for the shape of the species profiles.", "An integrated error index (Eflame) of each flame (average over all species) and an integrated error index (Especies) of each species (average over all flames) are also provided.", "Error indices range between 0 (accurate model) and 1 (inaccurate model).", "Before discussing PAHs formation in sooting flames, it is first important to assess the model reliability in predicting the main combustion features of key PAH precursors such as cyclopentadiene and single ring aromatics.", "Comparisons of model predictions with experimental data for the laminar flame speed of acetylenic species, cyclopentadiene, benzene and toluene are therefore reported in the SM.", "These comparisons confirm and emphasize the role of resonantly stabilized radicals in reducing laminar flame speed [12].", "The interest in cyclopentadienyl radical is also due to its role in naphthalene formation through self-recombination reactions.", "Moreover, the interest in phenyl radical chemistry is due to its relevance in the HACA mechanism.", "All the 60 flames were simulated, with and without the soot sub-mechanism, and detailed results and comparisons with the experimental measurements are reported in the SM.", "As reported in Table 1, the integrated error index (Ei) varies from ∼0.15 in the case of n-heptane (Flame 58), up to >0.5 for ethylene Flame 26, indicating a very poor predictive capability.", "A preliminary analysis of the error index trend does not seem to indicate systematic deviations depending on fuels, equivalent ratios, or soot yields.", "Due to space limitations, in the attempt to obtain the most significant, effective and general overview, scatter plots between the maximum predicted and measured value of measured species are here reported as a global measure of model performances, together with the error index of each species.", "The SM contains the complete set of comparisons, also grouping the same species in different flames.", "Figure 1 shows the parity diagrams of maximum experimental and predicted concentrations of major species in rich flames, for different fuels.", "As expected, CO is the primary component in rich conditions.", "Predicted CO profiles agree within 20% with the measurement and do not show systematic deviations.", "The model captures CO2 predictions quite well, also for C6H6/O2/Ar at ϕ = 1.25 (Flame 46, Yang et al., 2015), where CO is largely converted to CO2, after reaching its maximum concentration.", "A similar degree of accuracy is found for H2 and H2O, even if larger deviations are observed in most of CH4 flames where both species are under-predicted.", "The scatter diagrams of CH4 and the sum of C2H4 and C2H2 are also satisfactory.", "Larger uncertainties are indeed observed for the parity diagrams of individual species C2H4 and C2H2, as reported in the SM.", "Similar deviations are also observed using other literature models [21,22].", "Although C2H2 predictions are more accurate for atmospheric flames, more scattered results are observed at lower pressures, where over- or under-predictions up to a factor of ∼3 are highlighted.", "C2H4 seems systematically over-predicted in CH4 and C2H6 flames, highlighting shortcomings in both C2H6 formation and dehydrogenation pathways within the C0–C2 mechanism.", "C3H4s (propyne and allene) and benzene are correctly predicted by the model, whereas diacetylene (C4H2), despite its lower concentration, deserves greater attention.", "Large under-predictions of C4H2 are in fact observed in atmospheric flames, whereas minor discrepancies are obtained at low pressures.", "Similar disagreements are observed with KAUST [21] and Blanquart et al. [22] mechanisms (see SM).", "The lack of consistent findings, together with the possible relevance of C4H2 in soot formation mechanism, suggests the need of further investigating acetylenic species kinetics.", "Figure 2 shows the scatter plots of PAHs clearly highlighting the higher propensity of C3+fuels to form heavier species.", "The overall prediction of PAHs in atmospheric flames is satisfactory, but most of the low-pressure benzene flames show an overestimation of phenylacetylene (C6H5C2H) and underestimation of naphthalene (C10H8).", "Similar deviations are also obtained with [21] and [22] (see SM).", "This suggests that pressure dependent rates of acetylene addition might require further revision.", "It has to be noted that Fig. 2 shows the scatter plots of heavier species as predicted with the complete kinetic mechanism, also including the soot model.", "Together with the previous comments on the observed deviations at low pressures, it is worth noting the general over-predictions, at high pressures.", "The lumping of heavy species can partially explain these deviations.", "In facts, lumped phenanthrene groups not only anthracene, but also C13 and C15 homologous species.", "Figure 3 shows the error index of different species, as averaged over the different flames, versus the number of available data.", "The convergent behavior suggests the importance of the number of measurements to improve model reliability.", "Good predictive capabilities are shown for benzene and gaseous species for which not only the availability of many reliable data, but also the information gained from quantum chemistry ensure a deeper knowledge.", "Although it is intuitive that accounting for soot formation leads to a reduction of PAH concentration [23], the scope of this section is to quantify this effect, by comparing model predictions with and without soot mechanism.", "Figure 4 clearly shows this relevant effect on benzene profiles from CH4 [24], C2H4 [25], and C2H6 [26] flames.", "The inclusion of the soot significantly reduces not only the maximum but the entire benzene yields along the space domain (dashed lines in Fig. 4), with an improved agreement in all the three cases.", "Soot presence affects the benzene yield in Flame 4 and 5 (CH4/O2/Ar – ϕ = 2.4–2.6 – Melton et al. [24]).", "On the contrary, this effect is negligible when considering the benzene formation in Flame 1 (CH4/O2/Ar – ϕ = 2.5 – Marinov et al. [27]), as reported in the SM.", "By comparing the temperature profiles of these flames, it is possible to note that the maximum temperature of Flame 1 (Tmax = 1600 K) is about 100 K lower than the maximum temperature of Flames 4 and 5, even though the flame conditions are very similar.", "By correcting and assuming a higher flame temperature profile for Flame 1, it is possible to observe the soot influence and to achieve a better agreement with experimental data (see SM).", "This critical analysis of the consistency of similar flames shows one of the useful aspects of the database.", "Relevant variations of C10H8 and mainly of heavier PAHs are also observed, as reported in the SM.", "Figure 5 shows the predicted reduction of the error index of major PAH in sooting flames at different equivalence ratios.", "The reduction percentage is calculated by comparing error indices with and without soot model.", "As expected, the soot chemistry allows to reduce the average error indices, with average reductions of about 10%, increasing at richer conditions.", "The 33% reduction of the error index of pyrene at ϕ = 2 – Flame 31 [26] is not very significant, because of its very low concentration (<1 ppm).", "Close to non-sooting conditions the differences decrease to less than 1%.", "The blue line shows the average reduction of error index, increasing for higher soot propensity conditions (i.e. higher equivalence ratios).", "This improvement in error index confirms the important role of PAH condensation and soot growth processes.", "Additionally, the model performance evaluation suggests that validation of PAH without the soot sub-mechanism is misleading, particularly in rich flames.", "As demonstrated in [3], the coupling of different PAH mechanisms to the same soot model provides significantly different predictions.", "The database reported herein is also useful for the selection of the most appropriate PAH mechanism and for the verification of PAH consumption by soot model.", "To verify the overall performances of the model in predicting soot volume fraction, Fig. 6 shows a comparison with data collected in CH4 and C2H6 flames by Melton et al. [24,26], as well as in the C2H4 flame of Carbone et al. [25].", "Good agreement is observed for CH4 and C2H6 flames.", "While soot volume fraction from CH4 is consistently under-predicted mainly for short distances from the burner, for the ethane cases a better agreement is observed, with maximum deviations of a factor of ∼2 for the intermediate ϕ = 2.4 case.", "A reasonable agreement with the experimental data is observed in the C2H4 flame of Carbone et al. [25].", "The overall reasonable agreement between experimental data and model predictions allows to rely on the analysis of the PAH-soot interaction through the surface growth reactions.", "Figure 7 schematically shows the relative contributions of HACA mechanism and PAH condensation reactions to the formation and growth of soot particles, obtained from the soot model at different pressures.", "Acetylene is mostly involved in the HACA mechanism with the growing of PAH species.", "At atmospheric pressure, incipient soot particles form near flame region and react with aromatic radicals via chemical condensation.", "PAHs also interacts with soot particles, in the post-flame region.", "Thus, the role of C2H2 and HACA mechanism in the successive soot growth decreases, because of the preferential addition to most abundant PAH than to the heavy particles present in lower concentrations.", "Mainly in very rich flames at high pressure, benzene and heavy PAHs condensation reactions on soot particles play a relevant role.", "Because of their relative concentration, benzene contribution is the dominant one, followed by phenylacetylene and naphthalene.", "At lower pressure instead, acetylene addition dominates over PAH condensation, as PAH concentration strongly depends on pressure [18].", "This pressure effect is in agreement with the observations with Guo et al. [28].", "However, the different relative role at atmospheric condition from this work is due to the concerned PAH in the model and PAH chemistry.", "In addition to pressure, the two competitive pathways depend on temperature and equivalence ratio.", "In particular, lower temperatures and higher equivalent ratios increase the relative role of PAH condensation compared to C2H2 addition.", "Mainly in methane and ethane flames, it is important to highlight that acetylene is also involved in benzene formation, via the methyl addition to form propargyl radical.", "Accurate experimental temperatures are crucial for kinetic studies especially in rich flames where soot fouling and disturbances can affect measurements [27].", "Flame 27 (C2H4) [29,30] is chosen to demonstrate the temperature effect.", "The simulated temperature profile is obtained from Menon et al. [30] that used a spectral line reversal technique up to ∼10 mm and two‐color pyrometry technique, where the presence of soot perturbs the substantial absorption of radiation from the lamp.", "This confirms the challenges of accurately measuring temperatures in sooting conditions.", "Figure 8 shows the comparison between measurements and model predictions.", "The model satisfactorily agrees with the experimental measurements when using the original temperature profile.", "Model predictions obtained perturbing the temperature profile of ±50 K are also reported.", "These temperature variations strongly impact soot formation.", "At higher temperatures, soot volume fraction increases of a factor of ∼2.", "This is justified by higher yields of C2H2 from C2H4 dehydrogenation, enhancing PAHs production.", "Conversely, the sooting tendencies decrease with temperature.", "These results highlight the importance of accurate temperature measurements.", "Besides the obvious impact of temperature on kinetics, thermodynamic effect is also expected on PAH and soot formation at high temperatures.", "The upgraded CRECK kinetic model, hierarchically obtained by assembling the C0-C2 NUIG mechanism [10], the kinetic mechanism of heavy fuel combustion including PAH formation [12], and the soot model [8,9]was used to simulate the whole set of rich laminar premixed flames.", "Besides the important steps toward a unified core mechanism, this extensive validation provides a significant overview of the current understanding of the molecular weight growth kinetics leading to nanoparticle formation under high-temperature sooting conditions.", "The flame database extends the previous one of laminar premixed flame speeds [12], and constitutes an added value to this work and a valuable source of information for the whole combustion community.", "Moreover, the overall kinetic mechanism including soot formation is furtherly validated over this database, and is provided as Supplementary Material.", "This comprehensive validation shows regions of fuels/conditions where model predictions are quite accurate (CO, CO2, H2, C2H2, C6H6, etc.) and more importantly, highlights areas requiring further attention (C4H2 and other polyyne species).", "This mechanistic study clearly highlights the important role of PAHs as surface growth species, largely contributing to the successive soot formation.", "The effect of the soot formation model was also quantitatively investigated, highlighting its importance on heavy PAHs concentrations.", "The reduction in the average error index of intermediate PAHs shows that neglecting the successive soot formation leads to deviations up to 10%.", "The better qualitative and quantitative agreement obtained by using the complete kinetic model proves that the soot sub-mechanism is necessary to properly analyze rich sooting flames.", "Finally, this paper demonstrates that not only kinetic model uncertainties and simplifications, but also experimental uncertainties in temperature and PAHs measurements significantly affect predicted soot yields.", "Therefore, more significant insights can only be obtained from a comprehensive mechanism validation relying on all the possible target flames, coming from different facilities, for different fuels and experimental conditions." ]
[]
Isoprenoid emission variation of Norway spruce across a European latitudinal transect
Norway spruce (Picea abies) is one of the dominant tree species in the European boreal zone with the capacity to grow over large areas within Europe. It is an important emitter of biogenic volatile organic compounds (BVOCs), which can act as precursors of photochemical smog and ozone and contribute to the formation and growth of secondary organic aerosols (SOA) in the atmosphere. Isoprenoid emissions were measured from Norway spruce trees at seven different sites, distributed from Ljubljana in Slovenia to Piikkiö in Finland. Four of the sites were part of a network of genetically identical spruce trees and contained two separate provenances. The remaining three sites were part of other networks which have been used to conduct studies in the European boreal zone. There were minimal differences in the standardized emission rates between sites and across latitudes. The emission profile differed between provenances and sites, but there were not any distinct patterns which could be connected to a change in latitude. By using genetically identical trees and comparing the emission rates between sites and with genetically different trees, it was observed that the emission patterns were mostly influenced by genetics. But in order to confirm this possible stability of the relative emission profile based on genetics, more studies need to be performed. The effects of branch height, season and variation between years on observed emission pattern variations were also investigated. There were indications of potential influences of all three factors. However, due to different experimental setups between measurement campaigns, it is difficult to draw any robust conclusions.
[ "EART", "ENVI" ]
[ "Biogenic volatile organic compounds", "Emission profile", "Genetic diversity", "Latitudinal gradient", "Picea abies" ]
[ "1902", "2300" ]
[ "Biogenic volatile organic compounds (BVOCs) are emitted in large variations from different types of plants, where some of the most prominent compound groups are isoprene (ISO, consisting of one C5 unit), monoterpenes (MTs, consisting of two C5 units) and sesquiterpenes (SQTs, consisting of three C5 units) (Kesselmeier and Staudt, 1999; Holopainen, 2011).", "The primary functions for plants to release BVOCs are to attract pollinators, defend against herbivores and pathogens, serve as signals in plant-plant communication and to give protection against abiotic stresses such as high temperature, high irradiation or oxidative stress (Dudareva and Pichersky, 2008; Laothawornkitkul et al., 2009; Vickers et al., 2009; Holopainen and Gershenzon, 2010; Loreto and Schnitzler, 2010; Peñuelas and Staudt, 2010; Maffei, 2010).", "But once released into the atmosphere, BVOCs can influence the atmospheric chemistry and Earth's radiative balance (Di Carlo et al., 2004; VanReken et al., 2006; Beerling et al., 2007; Paasonen et al., 2013).", "BVOCs can act as precursors of photochemical smog and ozone, but also contribute to its destruction depending on the levels of NOx in the atmosphere (Chameides et al., 1988; Atkinson, 2000; Peñuelas and Staudt, 2010).", "Furthermore, they are important in the formation and growth of secondary organic aerosols (SOA) and for enhancing the production of cloud condensation nuclei (CCN) (Claeys et al., 2004; VanReken et al., 2006; Spracklen et al., 2008; Paasonen et al., 2013; Ehn et al., 2014).", "Both SOA and modifications of cloud properties have the potential to affect incoming solar radiation and thus influence global warming (VanReken et al., 2006; Laothawornkitkul et al., 2009; Paasonen et al., 2013).", "Forests are significant sources of BVOCs, in particular of isoprene and MTs (Kesselmeier and Staudt, 1999; Holopainen, 2011; Laffineur et al., 2011).", "One of the largest vegetation zones in the world is the boreal zone, consisting of approximately 33% of the total worldwide forest cover across Eurasia and Northern America (FAO, 2001).", "Boreal forests emit less BVOCs in comparison to temperate or tropical regions on a global scale, but are important contributors to regional emission budgets (Guenther et al., 1995, 2012; Acosta Navarro et al., 2014; Sindelarova et al., 2014).", "The emission budgets are influenced by the high reactivity rates of BVOCs, which depend on the structure of the BVOCs released and the chemical composition of the air at the local scale where BVOCs are emitted (Atkinson and Arey, 2003).", "The emission from the boreal zone is often characterized by MTs, but there can be considerable isoprene and SQT emissions as well (Tarvainen et al., 2007; Rinne et al., 2009).", "The boreal zone yields substantial amounts of SOA in the atmosphere, which plays an important role in the global radiation balance (Claeys et al., 2004; Tunved et al., 2006; Bonn et al., 2009).", "Norway spruce (Picea abies) is one of the dominant tree species of the European boreal zone, but it is also widely distributed throughout other parts of Europe (Janson et al., 1999; Grabmer et al., 2006; Filella et al., 2007; Skjøth et al., 2008; Kivimäenpää et al., 2013).", "Norway spruce is a low or moderate monoterpene emitter and a low isoprene emitter (Janson et al., 1999; Kesselmeier and Staudt, 1999; Simpson et al., 1999; Grabmer et al., 2006).", "It has the capacity to emit BVOCs from storage structures in resin ducts, needles and the bark, but there can also be a significant contribution from de novo emissions (Ghirardo et al., 2010).", "Leaf- and canopy scale emission models can be used to investigate the effect of BVOC emissions on atmospheric chemistry and aerosol formation.", "As BVOCs have different reactivity with OH radicals and ozone due to their different molecular structures (Atkinson and Arey, 2003), it is of importance to consider the composition of the emissions from different plant species (Niinemets et al., 2010).", "At a spatial-, biome- or global-scale, the emissions are typically determined by dividing the plants into plant functional types (PFT) for which an average emission potential has been determined.", "However, the emission capacities for different plant species within a PFT can vary greatly and the parameterization chosen for a PFT might vary for different models (Guenther et al., 2006; Arneth et al., 2010; Niinemets et al., 2010; Monson et al., 2012).", "The vegetation has a capacity to adapt their emission patterns over environmental gradients, which leads to large uncertainties between emissions and geographical location (Niinemets et al., 2010; Bourtsoukidis et al., 2012; Harrison et al., 2013) that are difficult to include in models.", "The observed geographic variability may be related to adaptation to local growing conditions, hybridization or to existing genetic variation (Staudt et al., 2004; Bäck et al., 2012; Steinbrecher et al., 2013), but may also be affected by microclimatic conditions, adaptations at different canopy heights and by seasonal development (Keenan et al., 2009; Niinemets et al., 2010; Grote et al., 2013).", "The importance and impact of genetic diversity and growing condition adaptations on the BVOC emission patterns has been highlighted by several studies.", "Nerg et al. (1994) studied Scots pine (Pinus sylvestris) seedlings grown at different latitudes in Estonia and Finland, and reported latitudinal effects on terpene concentration in pine shoots.", "Bäck et al. (2012) measured BVOC emission from Scots pine trees and found that there existed different chemotypes attributed to genetic variation within the same stand.", "Persson et al. (2016) measured genetically identical trees of English oak (Quercus robur), European beech (Fagus sylvatica) and Norway spruce and found that the relative compound contribution remained fairly stable between individuals of the same species.", "These studies highlight an existing uncertainty regarding the importance of genetic variation for observed BVOC emissions relative to the effect of local environmental conditions across latitudes (Kesselmeier and Staudt, 1999; Bäck et al., 2012; Persson et al., 2016).", "The aims for this study are (I) to investigate the potential variability for the emission rates of Norway spruce across a latitudinal transect in Europe and (II) to examine how the observed emission patterns are influenced by genetic diversity and height within the canopy, but also time of season and variation between years will be considered.", "Measurements were performed at seven sites along a latitudinal transect across Europe, stretching from Ljubljana in Slovenia to Piikkiö in Finland (Fig. 1).", "The sites were chosen as they represent both the boreal ecosystem and cover a wide range where spruce has the capacity to grow.", "It is a collaboration of four separate projects with different measurement campaigns, where one to five campaigns were performed per site.", "Each campaign lasted between one and three weeks.", "The details of location, tree height, tree age, sampling date, number of performed campaigns, measurement year, annual average temperature (°C), annual total precipitation (mm), average Photosynthetically Active Radiation (PAR) and average temperature (°C) during campaigns can be found in Table 1.", "Four of the sites, namely Ljubljana (Slovenia), Grafrath (Germany), Taastrup (Denmark) and Piikkiö (Finland), are part of the International Phenological Garden (IPG) network in Europe which have been used to perform long-term phenological observations on naturally occurring plant species (Chmielewski et al., 2013).", "The advantage of the network is that all plants are clones, which means the genetic variation between plants is negligible.", "Ljubljana, Grafrath and Piikkiö were established between 1962 and 1965, whilst Taastrup was begun in 1971 (www.agrar.hu-berlin.de).", "Within the IPG network, there are two provenances of Norway spruce which are divided into early spruce (SE), with an early budburst, and late spruce (SL) which experiences budburst approximately one week later.", "The remaining sites did not contain genetically identical trees, but are part of other networks used to perform studies in the European boreal zone.", "Hyltemossa research station is located in southern Sweden, whilst Norunda research station is located about 30 km north of Uppsala in Sweden.", "Both sites are run by the Integrated Carbon Observation System in Sweden (ICOS).", "Hyltemossa is dominated by Norway spruce with a small fraction of Downy birch and Scots pine (www.icos-sweden.se), whilst Norunda is part of a boreal forest dominated by 80–123-years old Norway spruce and Scots pine (Lagergren et al., 2005).", "The site in Skogaryd is located in south-western Sweden, approximately 50 km from the west coast and covers an area of roughly 30 km2.", "The site is part of Swedish Infrastructure for Ecosystem Science (SITES) (Shendryk et al., 2014) and mainly contains coniferous trees, dominated by Norway spruce and Scots Pine (Wallin et al., 2015) (Fig. 1).", "For the IPG network sites and Hyltemossa, samples were collected from needle chambers with a volume of 270 cm3 and which were connected to a portable infra-red gas analyzer (IRGA; 6400-22L lighted conifer chamber; LI-6400, LICOR, Lincoln, NE, USA).", "The needle twigs were acclimated to 1000 μmol m−2 s−1 PAR and 400 μmol CO2 mol−1 air for approximately one hour before BVOC sampling.", "Relative humidity was maintained close to ambient conditions between 50 and 65% by removing excess water vapor from the ingoing air stream when necessary.", "Leaf temperatures were kept constant between 10 and 15 °C in October and 20–30 °C in April–August, based on the anticipated average daily temperature.", "Sample air was collected from the chamber outlet.", "The measurements in Skogaryd and Norunda were performed on a single tree and used 13-L cylindrical and transparent chambers consisting of Teflon and stainless steel (Haapanala et al., 2009).", "To avoid stress induced emissions from the trees, the chambers were installed one day before the measurements were initiated.", "Purge air was continuously flowing into the chambers with a flow rate of 4–6 l/min.", "Temperature and humidity were measured inside and outside the chambers (Campbell Scientific CS215, USA) together with PAR (Li-Cor Li-190, USA).", "In Skogaryd, two chambers were mounted on a scaffold at two heights (2.5 m and 3.5 m).", "In Norunda, one chamber was installed 20.0 m above the ground, whilst another chamber measured the emission rates at a lower level (3.0 m in June and 11.0 m in July).", "In all campaigns, a hydrocarbon trap (Alltech, Associates Inc., USA) containing MnO2-coated copper nets was fixed between the ambient air inlet and the chamber inlet to absorb all the VOCs and ozone from the incoming air stream.", "Sample air passed through stainless steel cartridges (Markes International Limited, Llantrisant, UK) packed with adsorbents Tenax TA (a porous organic polymer) and Carbograph 1TD (graphitized carbon black).", "The air samples were extracted by using flow-controlled pocket pumps (Pocket Pump, SKC Ltd., Dorset, UK).", "The sampling flow rate was 200 ml min−1 and the collected volume for each sample was between 5 and 6 L. Blank samples were also collected in order to acknowledge possible background contamination in the ambient air.", "After BVOC measurements were performed, the needles inside the chambers were harvested, dried until the biomass weight was constant and weighed to get the dry weight (g(dw)).", "All measurements were performed during daytime (8:00–17:00).", "A total of 556 cartridges were collected and analyzed.", "All cartridges were sealed with Teflon-coated brass caps and stored at 3 °C until analysis.", "For all sites except Norunda, the BVOCs samples were analyzed by gas chromatograph-mass spectrometer following thermal desorption at 250 °C at the University of Copenhagen (for details, see van Meeningen et al., 2016).", "For standardization, pure standard solutions of isoprene, α-pinene, camphene, δ-phellandrene, limonene, eucalyptol, γ-terpinene, linalool, aromadendrene and α-humulene in methanol (Fluka, Buchs, Switzerland) were injected into adsorbent cartridges in a stream of helium.", "When quantifying BVOCs without a standard solution, α-pinene was used for MTs and α-humulene was used for SQTs.", "The chromatograms from the analysis were identified with the mass spectra in the NIST library and analyzed with the MSD Chemstation Data Analysis software (G1701CA C.00.00 21 Dec 1999; Agilent Technologies, Santa Clara, CA, USA).", "For the samples collected in Norunda, the analysis of sample tubes was done by the Finnish Meteorological Institute (Atmospheric Composition Unit, Helsinki, Finland).", "The samples were desorbed at 300 °C, and the standard solutions were camphene, 3-carene, 1,8-cineol, limonene, linalool, myrcene, α-pinene, β-pinene, terpinolene, longicyclene, isolongifolene, β-caryophyllene, aromadendrene, α-humulene, 2-methyl-3-buten-2-ol (MBO), p-cymene and bornylacetate.", "Isoprene was calibrated using a gaseous standard.", "The significant difference between sites, provenances and years was analyzed by a Kruskal-Wallis test on all of the different groups.", "If the test resulted in significant differences, a Mann-Whitney U test followed by Bonferroni correction was performed to determine significant groups.", "Differences in the emission rates between different heights were tested using Mann-Whitney U-tests.", "Partial least squares regression (PLS) was performed to investigate possible connections between factors such as ambient and past average temperatures, height, latitude, PAR and season (X-variables) and the emitted BVOCs (Y-variables).", "Season was defined as the number of active growing days where the average daily temperature had exceeded 5 °C for five consecutive days.", "Analysis was performed using SIMCA (Umetrics, version 13.0.3.0, Umeå, Sweden).", "The total average normalized emission rate of BVOC was 2.74 ± 2.96 μg g(dw)−1 h−1 (mean ± standard deviation), of which MT (1.50 ± 1.91 μg g(dw)−1 h−1) was the most commonly emitted BVOC group followed by isoprene (0.94 ± 1.84 μg g(dw)−1 h−1) and SQTs (0.30 ± 0.88 μg g(dw)−1 h−1).", "The average release of isoprene, MTs and SQTs between sites are in line with previous studies on Norway spruce (Table 2).", "The sites Taastrup, Skogaryd and Norunda had different emission rates in comparison to remaining study sites.", "Taastrup had a higher total MT emission, due to high emission rate contribution from needle branch measurements done in 2013.", "Skogaryd had a total emission which was a fifth of the average total emission rate from all measured sites.", "But it should be noted that the site was only visited in October, whilst the other sites had a majority of their measurements taken between May and August.", "Norunda had the highest emission of isoprene, mostly originating from the lower parts within the canopy (Table 2).", "A total of 23 terpene compounds were detected from the measurement sites, which were isoprene, 17 MTs and five SQTs.", "The most emitted compounds from a majority of the sites were α- and β-pinene and limonene.", "Other compounds with high emission rates were camphene and 3-carene.", "Early spruce had in general a higher emission of α- and β-pinene whilst late spruce emitted more isoprene and limonene.", "For the sites Hyltemossa and Skogaryd, about a third of the emissions came from pinene, whilst Norunda had high emission rates of isoprene.", "Average isoprene emissions for Grafrath was 0.64 μg g(dw)−1 h−1 and for Norunda 2.58 μg g(dw)−1 h−1, which was significantly higher in comparison to the majority of the study sites (P < 0.05).", "MT emissions from Norunda were 1.02 μg g(dw)−1 h−1, which was significantly less in comparison to Grafrath (1.60 μg g(dw)−1 h−1).", "Taastrup had an average MT emission of 2.63 μg g(dw)−1 h−1, which was higher compared to Ljubljana (1.17 μg g(dw)−1 h−1) and Norunda.", "Average SQT emissions were significantly lower for Grafrath (0.10 μg g(dw)−1 h−1) and Norunda (0.38 μg g(dw)−1 h−1) in comparison to the other sites.", "In regards to separate compounds, Taastrup had high emission rates of pinene (0.91 μg g(dw)−1 h−1) and low emission rates of camphene (0.13 μg g(dw)−1 h−1), whilst Norunda differed in 3-carene emission due to low emission rates (<0.01 μg g(dw)−1 h−1).", "Limonene emission varied most between sites (Fig. 2).", "There were little changes in the emission patterns of specific compounds with changes in latitude.", "Within the IPG network, the contribution of limonene in relation to the total BVOC emission increased slightly with latitude; from 12% in Ljubljana to 23% in Piikkiö.", "The relative emission of pinene on the other hand ranged between 16 and 23% between samples and was fairly similar between the IPG sites.", "For the sites Hyltemossa, Skogaryd and Norunda, the relative percentage of limonene was lower, with 16% of the total emission at Hyltemossa and 7–8% for Skogaryd and Norunda.", "For total BVOC emission, α-pinene and β-pinene had a relative contribution of 30–32% for Hyltemossa and Skogaryd and 10% for Norunda.", "In the case of Norunda, isoprene contributed with 64% to the total emission.", "But if only MTs were considered, the relative compound contribution of pinene would be in range of both Hyltemossa and Skogaryd.", "Fig. 3 shows the average emission profile of individual MTs for each of the sites.", "As the emission rates and emission profiles varied between the study sites, a PLS was used to investigate if there was any specific climatic factor which had a higher tendency to affect the observed emission patterns.", "It should be noted that the aim of this PLS-analysis was to investigate the connection between the compound emissions (isoprene, MTs and SQTs) and the climatic conditions experienced on site, and not to build a good prediction model for each of the studied compounds.", "The PLS-analysis revealed that the samples were separated according to latitude and measurement height on the y-axis and temperature on the x-axis (Fig. 4a).", "Height and latitude were strongly correlated.", "In Fig. 4a, data from higher latitudes and canopy heights are positioned towards the bottom whilst lower latitudes and canopy heights are positioned at the upper part of the figure.", "On the x-axis of this figure, data points with higher temperatures are positioned to the right whilst lower temperatures are positioned to the left.", "The number of active growing days (season) was not as strong driver as temperature on the x-axis, but is part in separating sites with measurements taken in October (Ljubljana and Skogaryd in 2015) from the sites where measurements were done in the middle of the summer (Fig. 4a).", "The PLS-analysis also revealed that the individual compounds are poorly explained by the model (Fig. 4b); the included climatic factors explained 7.8% of the total emission variation (Fig. 4).", "The sites within the IPG network were used to study how the BVOC emissions from Norway spruce might have adapted to local growing conditions without considering genetic variation.", "Grafrath was significantly different (P < 0.05) from the other sites in regards to its isoprene emissions.", "The average emission from Grafrath (0.64 μg g(dw)−1 h−1) was two to four times higher in comparison to all remaining sites and provenances, except for late spruce in Taastrup (0.50 μg g(dw)−1 h−1).", "The isoprene emission rate from early spruce at Taastrup (0.22 μg g(dw)−1 h−1) was low and significantly different from Ljubljana (0.32 μg g(dw)−1 h−1) and Grafrath.", "Total MT emission from early spruce in Grafrath (1.88 μg g(dw)−1 h−1) and late spruce in Taastrup (2.95 μg g(dw)−1 h−1) were significantly different from Ljubljana (1.17 μg g(dw)−1 h−1) and late spruce in Piikkiö (0.70 μg g(dw)−1 h−1) due to higher average MT rates (Fig. 5).", "There were no significant differences in SQT emissions.", "In regards to separate compounds, late spruce in Taastrup had higher emission rates of α-pinene (1.44 μg g(dw)−1 h−1) and lower emission rates of limonene (0.82 μg g(dw)−1 h−1) in comparison to many of the other sites (Fig. 5).", "There were no significant emission differences between sites for camphene and 3-carene.", "The importance of genetics and latitudinal adaptation was further investigated by comparing the emission pattern variation between genetically identical trees and genetically different trees over similar latitudinal ranges.", "The sites Taastrup and Piikkiö were chosen for comparing genetically identical sites and the sites Hyltemossa and Norunda for the genetically different sites.", "These four sites were chosen as the latitudinal differences between sites are relatively similar, with approximately one latitude degree difference between the two sites compared.", "For Norunda, the emission rate from 20 m up in the canopy was used for the comparison.", "For early and late spruce in Taastrup (n = 25) and Piikkiö (n = 19), there were significant differences (P < 0.05) in the emission of isoprene, 3-carene, limonene, other compounds, total MT emission and the total BVOC emission.", "However, if only late spruce was considered (n = 19 for Taastrup and n = 10 for Piikkiö), then there were no statistical differences (P > 0.05).", "This comparison could not be done for early spruce as there was only one tree available in Taastrup and too few measurements from Piikkiö.", "For the sites Hyltemossa (n = 27) and Norunda (n = 72), there were significant differences (P < 0.05) in the emissions of isoprene, 3-carene, other compounds and total emission of MTs.", "When the genetically identical trees from Taastrup and Piikkiö were compared with the emissions from Hyltemossa and Norunda, all compounds except for the total BVOC emission were significantly different, with an average emission rate of 2.76 ± 1.88 and 1.73 ± 1.55 μg g(dw)−1 h−1 for Taastrup and Piikkiö and1.76 ± 1.36 and 1.75 ± 1.59 μg g(dw)−1 h−1 for the Hyltemossa and Norunda.", "At Taastrup (2013) and Norunda (2014), samples were taken at different heights within the canopy in order to investigate possible emission pattern variations.", "Focus has been on the emissions of isoprene, MT, SQT and dominant MT compounds (α-pinene, β-pinene, camphene, 3-carene and limonene) from different canopy heights (Table 3).", "For early spruce in Taastrup, there was a significant difference (P < 0.05) for limonene emission, between canopy level of 2.0 m (2.47 μg g(dw)−1 h−1) in comparison to 5.5 m (1.38 μg g(dw)−1 h−1).", "Camphene emissions were higher at 2.0 m (0.07 μg g(dw)−1 h−1) in comparison to 12.5 m (0.02 μg g(dw)−1 h−1).", "The remaining compounds showed no statistical difference (P > 0.05) between canopy heights.", "For late spruce in Taastrup, camphene emission was significantly lower at 2.0 m (0.01 μg g(dw)−1 h−1) than at 12.5 m (0.1 μg g(dw)−1 h−1), whilst limonene emission was lower at 2.0 m (0.57 μg g(dw)−1 h−1) than at 12.5 m (2.32 μg g(dw)−1 h−1) and at 5.0 m (0.69 μg g(dw)−1 h−1) than at 12.5 m. For Norunda, all BVOC emissions between 3.0 m and 20.0 m height within the canopy were significantly different.", "The emissions of isoprene (3.79 μg g(dw)−1 h−1) and MT (1.51 μg g(dw)−1 h−1) at 3.0 m height were higher in comparison to the emissions at 20.0 m above the ground (isoprene 0.98 μg g(dw)−1 h−1 and MT 0.60 μg g(dw)−1 h−1).", "The total MT emission was not significantly different for canopy levels of 11.0 m and 20.0 m, but there was a significant difference for the compounds β-pinene (0.1 μg g(dw)−1 h−1 for 11.0 m and <0.01 μg g(dw)−1 h−1 for 20.0 m) and 3-carene.", "It should be noted though that no 3-carene emission was on 11.0 m due to a high 3-carene concentration in the blank samples (Table 3).", "Within the IPG network, Ljubljana and Grafrath were visited in 2014 and 2016 at approximately the same time within the growing season.", "Comparisons between years were done on the main BVOC groups and the emission rates were not significantly different (P > 0.05) between measurement years.", "Taastrup was visited between June and August in 2013–2016.", "Isoprene emission was significantly different for both 2013 (0.19 μg g(dw)−1 h−1) and 2015 (1.12 μg g(dw)−1 h−1) in comparison to remaining measurement years (P < 0.05).", "In 2013, the emission of isoprene was low in comparison to the remaining measurement years, whilst 2015 had a higher emission rate.", "MT emission was significantly different in 2013 (4.17 μg g(dw)−1 h−1) and between 2014 (1.57 μg g(dw)−1 h−1) and 2015 (0.69 μg g(dw)−1 h−1) (P < 0.05).", "Both provenances of spruce had MT emission rate three to four times as high in 2013 as the average emission rate in the other years.", "There were also differences in MT emission rates between 2014 and 2015, where the average emissions in 2014 were higher in comparison to the emission rates in 2015 (Fig. 6).", "In regards to seasonal development, most of the BVOC measurements were taken between May and August, but for Ljubljana (2015) and Skogaryd, measurements were performed in October.", "The average standardized emission rates in October were approximately a third to a sixth of the total average standardized emission rates measured between May and July (Table 1; Fig. 6).", "For Ljubljana, where the emission profile in October and May were compared, early spruce had a higher contribution to the total emission of camphene (43.1%) and limonene (23.72%) in October, whilst in May there was a higher contribution of isoprene (17.64%) and SQTs (23.6%).", "Late spruce had a higher emission of isoprene (43.06%) and SQT (36.02%) in October and a higher contribution to the total emission of pinenes (20.48%) and other compounds (24.83%, which for Ljubljana were tricyclene, terpinenes, eucalyptol and linalool) in May.", "Both provenances showed a decrease in the total emission contribution of other compounds, but without a decrease in the number of emitted compounds (data not shown).", "Norway spruce and Scots pine are important BVOC emission contributors in European boreal ecosystems (Rinne et al., 2009).", "In comparison to Scots pine, less emission studies have been undertaken on Norway spruce despite it being one of the most common coniferous tree species growing in Europe (Rinne et al., 2009; Yassaa et al., 2012).", "This study presents the average standardized emission rates of Norway spruce within the European boreal zone, but also over a larger latitudinal range across Europe.", "The emission rates across the sites were in range of other earlier studies (Janson, 1993; Christensen et al., 2000; Janson and de Serves, 2001; Hakola et al., 2003; Grabmer et al., 2006; Yassaa et al., 2012; Bourtsoukidis et al., 2014; Hakola et al., 2017) and there was minimal emission rate difference between the southernmost site and the northernmost site.", "Taastrup, Skogaryd and Norunda were sites which differed most from the other sites in regards to their emission patterns.", "Even though these sites are located in the northern range of this study, the observed emission rate differences are not necessarily due to their geographical location.", "The total MT emission rate at Taastrup in 2013 was higher and significantly different from the emission rates at the other sites.", "The measurement years between 2014 and 2016 were in range of the other visited sites.", "BVOC emission at Skogaryd was only measured in October and had lower emission rates in comparison to sites measured during the growing season.", "Norunda had the highest average isoprene emission, with high emission rates at 3.0 m and 11.0 m in the canopy.", "This is most likely due to a strong emission response during high irradiation events in different parts of the canopy.", "Apart from limonene, the emission rates of other compounds in the southernmost sites Ljubljana and Grafrath were not significantly different from each other.", "The spruce tree in Norunda differed most from the trees in the remaining sites, both in regards to BVOC emission groups and for separate compounds.", "One reason could be that the tree in Norunda was standing in a much denser forest in comparison to the other sites, leading to a possible difference in shade acclimation.", "Both local climate and genetic diversity have been shown to impact observed emission pattern fluctuations (Komenda and Koppmann, 2002; Semiz et al., 2007; Niinemets et al., 2010; Bäck et al., 2012; Steinbrecher et al., 2013), but it can be difficult to separate latitudinal adaptations and genetics apart.", "By using the IPG network, it was possible to further investigate the impact of possible adaptations to local growing conditions from two provenances of spruce without considering genetic diversity.", "The two provenances of Norway spruce were slightly different in regards to their emission profiles.", "Whilst early spruce had a higher relative emission of α- and β-pinene, late spruce emitted more isoprene and limonene.", "Apart from limonene emission, which increased slightly with increasing latitude, there were no distinct emission pattern changes within the same provenance and between IPG sites.", "This relative compound similarity across latitudes would suggest that the genetic material of the tree determines the emission profile of the compounds and that adaptation to local growing conditions do not happen instantly, but may have developed over generations.", "The importance of genetic diversity was further investigated by comparing the BVOC emissions of genetically identical trees from Taastrup and Piikkiö with genetically different trees in Hyltemossa and Norunda.", "When all sites were compared, there was a statistical difference for all compounds and sites except for the total amount of BVOC emissions.", "But when the same comparison was performed on genetically identical trees, there were significant differences for all compounds and sites.", "These results highlight that the sites show less of a difference in emission patterns with a change in latitude, most likely due to acclimation to local weather and growing conditions.", "The non-significant difference between sites and genetically identical trees further suggest that adaptation to local environment do not happen instantly, but may take place over a longer time.", "In order to confirm this possible similarity of the relative emission profile, similar emission comparisons with more sites and over a longer latitudinal range are suggested.", "The potential similarity in the total emission rates across a latitudinal gradient and relative emission profile might be of use in atmospheric emission models.", "In general for many emission models, the emission potentials are calculated for isoprene, the sum of all MTs and the sum of all SQTs as they represent a significant emission proportion from trees (Arneth et al., 2010; Niinemets et al., 2010).", "The results presented here provide average standardized emission rates of these three compound groups from spruce over a vast latitudinal range.", "However, for models which focus on atmospheric chemistry, it is important to also consider the emission profile, as the reactivity of compounds varies by several orders of magnitude due to their molecular structure (Atkinson and Arey, 2003; Niinemets et al., 2010).", "As the composition might vary due to short-term environmental impacts and genetic diversity between trees, it is difficult to give specific emission ranges of different compounds.", "However, the observed similarity of emission rates between IPG network sites, which also have had the possibility to acclimate to the local growing conditions, show that the composition seen at different sites can be considered to remain fairly stable with the progression of time.", "Apart from the studied effects of latitudinal adaptation and genetic diversity on observed emission pattern fluctuations, the effect of measurement height within the canopy, change with season and yearly differences have also been investigated.", "In Taastrup and Norunda, emissions were measured at three levels within the canopy.", "Whilst Taastrup only showed emission pattern differences between levels for a few compounds, Norunda showed statistically significant differences both for compound groups and separate compounds for all measured canopy heights.", "A possible explanation for the dissimilarity in emission response could be the amount of shade adaptation at different heights and sites.", "Persson et al. (2016) explained that the insignificant emission difference between canopy heights in Taastrup could be caused by the wide spacing between trees in the phenological garden.", "This results in relatively high levels of irradiation at lower canopy levels, which minimizes the difference in light adaptation between needle branches from the upper and lower parts of the canopy.", "In Norunda, the trees were planted with less space in between trees and therefore have quite distinct differences in available light at different canopy levels.", "Furthermore, the shape and the color of the needles in Norunda were different between canopy heights, with thicker, greener and a higher percentage of newly produced shoots at the upper canopy in comparison to lower levels.", "The emission pattern change in regards to time of season has shown to have a substantial effect on observed emission rates (Janson, 1993; Hakola et al., 2003, 2012, 2017; Tarvainen et al., 2005).", "Seasonality has also shown to influence not only the emission amounts, but also changing the compounds’ emission profile (Janson, 1993; Hakola et al., 2003).", "The majority of the measurements in this study were performed between May and August, which is in the middle of the growing season.", "But in Ljubljana and Skogaryd in 2015, measurements were performed in October which is outside the main growing season.", "In comparison to both the emission differences between campaigns within Ljubljana and between the remaining sites, the emission rates in October were less than one third of the emissions reported during the middle of the growing season.", "The trees in Ljubljana also showed a change in emission profile for both provenances of spruce and season.", "Early spruce changed from high emissions of isoprene and SQT in May to emitting a higher relative contribution of camphene and limonene in October.", "Late spruce had high relative compound contribution of pinenes and other compounds in May and emitted more isoprene and SQT in October.", "However, even though the emission profile changed with the progression of the season, the number of compounds emitted did not change considerably.", "Skogaryd has only been measured in October and as it is unknown what the emission capacities are during summer season, which makes comparisons of emission profile with other sites unrealistic.", "However, the differences in emission patterns with remaining sites points to a potential influence of season which would be recommended to study further.", "Apart from a seasonal change in BVOC emission patterns, there might also be differences between measurement years (Hakola et al., 2017).", "Hakola et al. (2012) compared the emissions from a boreal forest in Hyytiälä between seasons and years.", "They found that apart from summer, when the emission differences were rather small, the emission profile could differ between measurement years.", "A similarity between summer season emissions was also indicated in this study for Ljubljana and Grafrath, which showed no significant difference between measurement years.", "But as only two summer seasons were compared, the emission similarities during the summer period in general cannot be quantified.", "For Taastrup, measurements were conducted for four years in a row at approximately the same time in July.", "For the total emission rates, the emissions were on average higher in 2013 compared to the other years.", "For the summer season of 2013, Persson et al. (2016) reported a distinct period without rainfall which might have caused drought stress to the spruce trees.", "Even though the volumetric water content of the soil was not measured, other indications such as dry needles shedding from lower branches whilst being handled suggested that the trees were in some state of water shortage.", "In a study by Lappalainen et al. (2009) in a coniferous forest, MT concentrations started to decline soon after a summer drought.", "This drop in emissions might be connected to senescence or needle shedding.", "The emission rates also differed between measurement years.", "In 2013, the emission of isoprene was significantly lower in comparison to other measurement years, whilst MTs were higher.", "2015 was also a year which differed from remaining measurement years, with high isoprene emissions and lower MT emissions.", "In relation to other measurement years, 2015 was almost 2 °C colder than average for July (data not shown) and this may have had an influence on the observed emission patterns.", "Even though the reasons for variable emission patterns only can be speculated in this case, there are indications that past weather events might have had an influence on the emission pattern variations between years.", "Several sites which are part of different measurement campaigns were studied in order to get a better representation of the possible growing range of Norway spruce.", "The inclusion of different measurement campaigns made it possible to investigate different influential aspects of the emission variation, such as genetic diversity, latitudinal adaptations, potential emission differences between summer and autumn, height within the canopy and between measurement years.", "But as the different measurement campaigns were not originally set to cover all of the above mentioned aspects, the amount of data collected is not sufficient to quantify their separate importance on observed BVOC emissions.", "Furthermore, the inclusion of different chamber measurement techniques between studies adds further uncertainty to emission variations between sites.", "The needle chambers used together with LI-6400 has a disadvantage that the plant needs to be handled for each sample, which might increase the risk of mechanical stress induced emissions.", "The disadvantage of the transparent branch chambers is that the climatic conditions inside the chamber are more difficult to control, which might have indirectly provided with more stress to the branch inside the chamber due to different climatic conditions.", "Apart from the influence of genetic diversity, adaptations to different growing conditions, height, season and measurement year on observed BVOC emissions, the emissions have also been influenced by other factors which have not been thoroughly studied in this setup.", "In the performed PLS analysis, only 7.8% of the emissions could be explained by temperature, PAR, height, latitude and time of season.", "Factors which were not included in the analysis but which have shown visible effects on the emission rates are drought, heat, herbivore attack and mechanical damage of the needle twigs or branches.", "Therefore, more studies investigating how the emission patterns in situ are influenced by these short-term effects would be needed.", "In order to compare the emission rates between sites, all measurements were normalized according to the algorithms presented by Guenther et al. (1993).", "For the normalization of monoterpenes and sesquiterpenes, a β-value of 0.09 was used.", "But it has been shown by Duhl et al. (2008) that the β-value could be different between SQTs and MTs when the standardized emissions are calculated.", "Previous results have indicated that β for SQTs could be in the range 0.05–0.29 (Duhl et al., 2008).", "However, since many factors need to be taken into account when choosing an appropriate β-value, such as plant species, season and specific compounds, it is hard to decide which value should be used.", "For the studies reported in Table 2, the β-value for MTs is between 0.01 and 0.19 and for SQTs 0.02–0.17.", "As the main purpose of this study was to compare the emission pattern variability for spruce influenced by latitude, genetics and height, the same value for both MTs and SQTs was chosen to simplify the analysis.", "Norway spruce is an important coniferous source of BVOCs which can be found over a large range in Europe.", "This study measured the emission rates of genetically identical and genetically different Norway spruce trees at seven European sites.", "The results showed that the standardized emission rates from different sites within Europe did not vary considerably with a change in latitude.", "The emission profile differed between provenances and sites, but the emission profile difference was less distinct for genetically identical trees.", "The study suggests that the spruce isoprenoid emission is potentially more determined by genetic diversity than by adaptation to local growth conditions.", "This possible stability in isoprenoid emissions could be used to improve the parameterization in different emission models.", "It was also indicated that the observed emission rates were influenced by canopy height, time of season and measurement years.", "But due to different experimental setups between measurement campaigns, it is not possible to quantify the effect of the separate factors.", "More comprehensive measurements involving the effect of canopy height, season and measurement years would be needed in order to better understand the emission pattern fluctuations." ]
[ "Isoprenoid emission from Norway spruce were measured at seven sites in Europe.", "There were little differences in standardized emission rates across Europe.", "The emission profile differed between sites, but was less distinct for cloned trees.", "Emission patterns were potentially influenced by tree height, season and year." ]
Initial spreading of a mega feeder nourishment: Observations of the Sand Engine pilot project
Sand nourishments are a widely applied technique to increase beach width for recreation or coastal safety. As the size of these nourishments increases, new questions arise on the adaptation of the coastal system after such large unnatural shapes have been implemented. This paper presents the initial morphological evolution after implementation of a mega-nourishment project at the Dutch coast intended to feed the surrounding beaches. In total 21.5 million m3 dredged material was used for two shoreface nourishments and a large sandy peninsula. The Sand Engine peninsula, a highly concentrated nourishment of 17 million m3 of sand in the shape of a sandy hook and protruding 1 km from shore, was measured intensively on a monthly scale in the first 18 months after completion. We examine the rapid bathymetric evolution with concurrent offshore wave forcing to investigate the feeding behaviour of the nourishment to the adjacent coast. Our observations show a large shoreline retreat of O (150 m) along the outer perimeter of the peninsula, with locally up to 300 m retreat. The majority (72%) of the volumetric losses in sediment on the peninsula (1.8 million m3) were compensated by accretion on adjacent coastal sections and dunes, confirming the feeding property of the mega nourishment. Further analyses show that the morphological changes were most pronounced in the first 6 months while the planform curvature reduced and the surf zone slope flattened to pre-nourishment values. In the following 12 months the changes were more moderate. Overall, the feeding property was strongly correlated to incident wave forcing, such that months with high incoming waves resulted in more alongshore spreading. Months with small wave heights resulted in minimal change in sediment distribution alongshore and mostly cross-shore movement of sediment.
[ "ENGI", "ENVI" ]
[ "Alongshore feeding", "Beach nourishment", "Coastal safety", "Longshore transport", "Mega nourishment" ]
[ "2212", "2305" ]
[ "Sand nourishments are nowadays widely applied to enhance coastal safety and to increase beach width (e.g. Burcharth et al., 2015; Castelle et al., 2009; Dean, 2002; Kuang et al., 2011; Luo et al., 2015; Ojeda et al., 2008; Roberts and Wang, 2012; Yates et al., 2009).", "With the increasing pressure on the coastal zone in terms of population and the (projected) relative sea level rise, the number of nourishment projects and the total volume has increased greatly, e.g. for the US East Coast alone Valverde et al. (1999) report an increase in annual nourishment volume over all projects combined from ~ 1 106 m3/year in the 1920s to ~ 12 106 m3/year in the 1990s.", "The first nourishment projects, executed roughly before the 1970s, targeted specific local weak spots along the coast (Hanson et al., 2002; Valverde et al., 1999).", "The added sand was mostly placed on dunes or beaches and the success rate of the nourishment was predominantly quantified by the proportion of material remaining in the project area over time (e.g. Elko and Wang, 2007; Leonard et al., 1990).", "The cross-shore size of these nourishments, i.e. the nourished volumes per meter alongshore, was typically small (order of 100 m3 per m) if these were used for beach maintenance.", "Following up on these initial beach and dune nourishments, shoreface nourishments have been carried out in the last quarter of the 1900s as an economical alternative for some locations (Grunnet and Ruessink, 2005; Mulder et al., 1994; Niemeyer et al., 1996).", "The cross-shore size of these nourishments is typically larger, i.e. in the order of 400–600 m3/m (e.g. Ojeda et al., 2008; van Duin et al., 2004).", "The positive effect of a shoreface nourishment on the beach and dunes can be subdivided into two hypothesized effects: the wave attenuation function and the (cross-shore) feeder function (Mulder et al., 1994).", "The wave attenuation function is formed as waves break on the nourishment during storm conditions, thus reducing the wave energy and longshore wave driven currents higher up the profile.", "The second, feeder effect is caused as a gradual landward movement of sand is stimulated, thus causing a seaward movement of the coastline.", "Although in practice it can be difficult to delineate between both effects, measurements (Hoekstra et al., 1996) and modelling (van Duin et al., 2004) of experimental nourishments suggest that both effects are present.", "At present, sandy mitigation strategies using frequent (every 3 to 10 years) beach and shoreface nourishments are carried out as the solution for several populated sandy coasts with a structural coastal recession (Cooke et al., 2012; Hamm et al., 2002).", "For locations with a large annual sand deficit however large quantities of sand need to be supplied or frequent re-nourishments are needed.", "It is questionable whether such frequent re-nourishing has not a detrimental effect on the fauna of the nourished site (e.g. Janssen et al., 2008; Peterson and Bishop, 2005; Speybroeck et al., 2006).", "Such potential negative effects and the upscaling to a more regional approach have given the incentive to look for better methods.", "In this light, the concept of nourishing with the intention to feed adjacent coasts by means of alongshore diffusion in the form of concentrated (mega) nourishments have been proposed as an alternative (Stive et al., 2013).", "In this approach a large volume of sand is placed at a single location with the intention to feed a larger alongshore stretch of coast over time.", "Under the combined natural forces of wind, waves and tides the sediment is expected to be redistributed in along and cross-shore directions, hence enhancing the safety of a longer stretch of coast.", "In the Netherlands, a concentrated mega-scale nourishment called the Sand Engine was implemented in 2011 as a large sandy hook with the size of approximately 17 million m3 placed in an area of 2.5 km in the alongshore direction and 1 km in the cross-shore.", "In contrast to previous nourishments with cross-shore sizes of 400–2000 m3 per meter alongshore and small transitions with the surrounding coastlines, the Sand Engine peninsula design project has a highly concentrated nourishment volume (up to 10.000 m3/m) and sharp coastline angles with the intention to redistribute rapidly by means of alongshore diffusion of sand.", "An evaluation of such a feeder nourishment project cannot solely done in terms of the amount of sand that remains within the project area (as done with more traditional nourishments; e.g. Dean and Yoo, 1992; Elko and Wang, 2007; Verhagen, 1996), but requires a broader view on the coastal section, including the rate of spreading and the magnitude of the accumulation of sediment on adjacent coastal sections.", "The objective of the current paper is to present the morphological evolution of the Sand Engine in the first 18 months after construction.", "Special attention is on the feeding character of the nourishment, assessing spatial and temporal redistribution of sediment.", "Although it is known that this response in the first period after completion is vital for the total evolution of the project, it is often poorly recorded due the lack of frequent monitoring data (Elko and Wang, 2007).", "We performed a high resolution monitoring study with frequent surveys to capture this behaviour in the first period after completion.", "In this paper we discuss the feeding of the mega nourishment pilot by focusing on the planform adjustment of the nourished peninsula (Section 4.2), cross-shore profile and volume changes at the Sand Engine and along adjacent coasts (Section 4.2), quantifying the proportion of eroded sand on the peninsula with respect to the accretion on adjacent sections (Section 4.3) and relating the feeding behaviour on a monthly timescale to the incoming wave power and the shape of the peninsula (Section 4.4).", "The Sand Engine nourishment was executed along the ‘Westland’ coastal cell, a 17 km stretch of coast between the harbour entrances of Scheveningen and Rotterdam (Fig. 1).", "This southern part of the Dutch coast is subject to structural erosion, the coastline migrated landward by about 1 km in the period 1600–1990.", "After a retreat of O (300 m) in the 18th century, construction of rubble mound groynes was initiated (van Rijn, 1997).", "Yet, as this coastal stretch remained erosive despite the groynes sand nourishments were introduced as mitigation measure in the 1970s.", "Since then, nourishments have been implemented more frequently, especially from 1990 onwards when the implementation of the ‘Dynamic Preservation Act’ dictated that the 1990 coastline position had to be maintained at all costs (van Koningsveld and Mulder, 2004).", "At the site of the Sand Engine, the first nourishment was implemented in 1986.", "Since then it has been re-nourished eight times prior to the construction of the Sand Engine.", "In all, approximately 55 million m3 of sand was added until present to the 17 km Westland coastal cell for mitigation of erosion and land reclamation.", "In the last years the nourishment volumes in this stretch have increased to ~ 1.7 106 m3 per annum (~ 100 m3/m alongshore/year).", "The net yearly-averaged alongshore sediment transport in the middle of the Westland coastal cell is estimated to be 3.8 105 m3 northward excluding pores, resulting from the gross transports in both directions, i.e. 7.6 105 m3 northward and 3.8 105 m3 southward (van Rijn et al., 1995).", "Prior to the Sand Engine pilot project the beach was O (150) m wide with a mild lower shoreface slope (1:300) (Fig. 1a).", "The median grain size around the shoreline in this area is O (250 μm) (Wijnberg, 2002).", "The slope of the profile in the intertidal and surfzone (+ 1 to − 4 m) prior to all the nourishment works (averaged over 1965 to 1985) was 1:55 in this coastal stretch.", "The profile on the Holland coast generally contains multiple nearshore subtidal bars, migrating offshore in cycles with return intervals of 4 to 16 years (Ruessink et al., 2003).", "At the Westland coastal cell however only a single bar offshore of the groyne heads was observed (Wijnberg and Terwindt, 1995) without a net annual migration.", "Possibly the less prominent bar behaviour and cyclic migration at this site was influenced by the presence of the groynes.", "Near the waterline a seasonal pattern is present in the beach width and the cross-shore volume of the supra tidal beach, related to the seasonality in the wave forcing (Quartel et al., 2008).", "The southern Dutch coast faces the shallow (20–80 m deep) North Sea basin, and as a result the wave climate is wind sea dominated with annual mean wave height Hs of 1.3 m and wave periods typically in the order of 5–6 s (Wijnberg, 2002).", "Energetic storm events in autumn and winter are often from the south-west and north, causing wave incidence to be highly oblique.", "Storms with a return period of once in a year have a significant wave height offshore of about 4 m.", "The mean tidal range at the location of the Sand Engine is 1.7 m and the horizontal tidal velocities have an amplitude of O (0.5 m/s).", "The Sand Engine project consists of a mega nourishment peninsula and two flanking shoreface nourishments with a total dredged sediment volume of 21.5 million m3.", "The main part, the mega nourishment, was shaped as a large hook-shaped peninsula with the outer tip curved towards the north (Fig. 2).", "This design fulfilled best the multi-disciplinary requirements of safety in combination with living quality and innovation (Stive et al., 2013).", "Early predictions of the development of the peninsula suggested lee side erosion on both sides; two flanking shoreface nourishments were proposed to mitigate this effect (Deltares, pers.", "comm.).", "About 19 million m3 of dredged sediment was planned for the curved peninsula, with 2 million m3 and 0.5 million m3 for the northern and southern additional shoreface nourishments respectively.", "The present paper focuses on the evolution of the peninsula and the term Sand Engine is from hereon used to indicate the peninsula only.", "In the design of the Sand Engine the most seaward position of the shoreline was foreseen to protrude ~ 1000 m from the original shoreline.", "The cross-shore slope of the peninsula was 1:50, such that the toe of the nourishment was at − 8 m and 1500 m from the original shoreline.", "The alongshore footprint of the Sand Engine peninsula was planned to be 2000 m.", "In the design, the tip of the peninsula was curved northward creating a sheltered area that was intended to be a nurturing ground for ecology (Fig. 2f).", "Furthermore, the design contained a small (~ 8 ha) lake at the base of the peninsula (see Fig. 2f).", "This lake was intended to prevent the freshwater lens in the dunes to migrate seaward, which could endanger the water extraction from the more landward dune area.", "The construction of the Sand Engine took place from March 2011 to July 2011 (Fig. 2).", "The Sand Engine was constructed using trailing-suction hopper vessels, used to either dump or spray sand in intermediate waters or to pump sand ashore.", "The construction started with the building of a narrow cross-shore seaward dam, such that alongshore losses from this initial build out would remain within the project domain as much as possible.", "The outer rim of the peninsula was completed last (Fig. 2e, f).", "The sediment for the nourishment was dredged 5 to 10 km offshore of the site and was prescribed to be of similar properties to the surrounding coast.", "Regular grain size analysis during construction (after each 0.5 million m3 of nourished sediment) showed an average D50 of 281 μm.", "The bed elevation data were measured prior and after construction of the peninsula by the dredging contractor (Fig. 3a, b).", "The plan view topographies displayed in Fig. 3 were based on multiple surveys on different dates, and thus do not provide a perfect snapshot of the topography and bathymetry at a single instant.", "However, the difference between both topographies (Fig. 3 c) highlights the large quantity of added sediment after the implementation of the nourishment.", "The added volume per profile is an order of magnitude larger than regular nourishments at this part of the coast, exceeding 1.104 m3 per m alongshore near the most seaward point of the Sand Engine (Fig. 3 d).", "By integration over the common areas of both surveys an added volume of 17.02 million m3 was found in situ, of which 4.04 million m3 (24%) was located above 0 m NAP (NAP being the Dutch datum at ~ MSL).", "This added volume based on surveys was O (10%) smaller than the 19 million m3 of dredged sediment that was transported for the curved peninsula, due to the losses during construction.", "After construction the highest point on the Sand Engine peninsula was 7.3 m above NAP, just seaward of the small lake (Fig. 3 b).", "Following the completion of the peninsula in July 2011, an intensive monitoring programme was initiated.", "As part of this monitoring programme, the area around the Sand Engine peninsula has been surveyed nearly monthly to capture and investigate the adaptation to the large intervention.", "Surveys span the beach and shoreface area from the dunefoot (~ + 5 m NAP) to beyond the − 10 m NAP contour.", "The surveyed domain was 4.7 by 1.6 km in along- and cross-shore direction, respectively.", "The transect grid consists of 59 full cross-shore transects with an average alongshore spacing of 80 m (Fig. 4, red lines).", "The transect definition for these transects was identical to the so-called ‘JARKUS’ high-density profiles used for the annual surveys executed by the Dutch Ministry of Infrastructure and Environment (e.g. Wijnberg and Terwindt, 1995).", "In the nearshore region the transect distance has been refined with 68 shorter transects to obtain an alongshore transect spacing of O (40 m) on dynamic parts of the beach and surfzone.", "Furthermore, additional alongshore transects and oblique transects were measured on the northern side of the peninsula to capture the evolution of the lagoon entrance.", "Surveys were executed using three different techniques, all based on the real-time kinematic differential global positioning system (RTK-DGPS) surveying technique.", "Sub-aerial parts were measured using the GPS system mounted on a 4WD quad bike (Fig. 5a).", "For the sub-aqueous parts of the transects the GPS was mounted on a waverunner/jetski and combined with single beam echo sounder (Fig. 5b).", "Runnels and narrow tidal channels with small water depth were surveyed using the GPS mounted on a wheeled pole (Fig. 5c).", "The vertical accuracy of the bed elevation measurements was of order 5, 10 and 3 cm for the three different techniques respectively (Huang et al., 2002; Ruggiero et al., 2009; van Son et al., 2010).", "The surveys of the full domain were executed during 3 days of calm conditions approximately every month.", "To facilitate the analysis of cross- and alongshore displacements, the bed elevation data were rotated to a local shore-orthogonal coordinate system with its origin at the beach entrance ‘Schelpenpad’.", "The collected x, y, z point data of all three survey platforms were combined and interpolated to a 10 by 10 m grid by means of linear interpolation.", "The resulting 17 bathymetries are depicted in Fig. 6.", "The monthly surveyed bed elevation data do not incorporate the dune face and first dune row.", "To estimate the sediment volume loss towards the dunes biannual Lidar flights of the sub-aerial domain were used.", "These Lidar data were available for July 2011, May 2012, October 2012 and May 2013 and sampled on a 5 by 5 m grid.", "Concurrent wave height Hs,0, direction θ0 and period Tm02,0 offshore in the months after construction were obtained from a wave station (‘Europlatform’) located 40 km offshore at a water depth of 32 m (Fig. 7).", "The wave height has a seasonal signal with the highest waves from September to December (Northern Hemisphere autumn).", "The high-energy wave events after construction were mostly from the south and west sectors, similar to the long term averaged wave climate (Wijnberg, 2002).", "The maximum recorded daily-averaged offshore wave height over first 17 months was 4.4 m during the storm on January 5, 2012 (Fig. 7a).", "In contrast, during summer months wave heights were low, with the lowest average wave height Hs,0 over a period between two surveys being 0.7 m.", "An overview of the morphological development is given in Fig. 6 and illustrates a rapid, predominantly alongshore redistribution of sediment.", "The initial sharp angle in the shoreline on the southern side of the peninsula accreted over time, resulting in a more gradual transition between the nourishment and the adjacent coast (near alongshore location y = − 500 m, Fig. 8a).", "The accretion in this transition zone was primarily in the inter- and subtidal zone (between the − 6 m and + 2 m NAP) resulting in a 250 m wide barred inter-tidal zone.", "The limited change above the + 2 m contour suggests that the shape transition was primarily due to marine processes.", "On the opposite (northern) side of the peninsula the lagoon entrance dynamics dominated the morphological behaviour.", "Soon after the implementation of the peninsula a (small) spit-like feature developed, pinching the lagoon entrance over the following months (Fig. 6).", "In the first 4 to 5 months, this spit mostly elongated in the cross-shore direction towards the adjacent coast and in a later stage it elongated to become a large inter-tidal shoal of 300–400 m across (Fig. 8b).", "The widening of the spit can be attributed to a seaward expansion but also to a landward widening.", "The maximum elevation of the spit and shoal were slightly below the high water level, such that they flooded during high tide (and storms).", "The channel landward of the shoal discharged the flow into and from the lagoon and strong flow velocities of over 1 m/s were observed here during rising and falling tide in the spring of 2012.", "This channel contained medium 3-D sub-aqueous dunes of O (1 m) length and O (0.5 m) height, similar to those found in tidal channels (Ashley, 1990).", "As the shoal migrated landward, this channel was forced slightly into the existing beach, similar to the behaviour observed with ebb tidal shoal bypassing (Cheung et al., 2007; FitzGerald, 1982).", "Towards the end of the investigated period the channel started to meander causing a steep embankment in outer bends.", "The largest morphological changes (> 0.5 m) over the total period were restricted to the zone − 7 to + 3 m NAP (Fig. 8c), which gives an indication of the closure depth on these time scales.", "Small accretion zones can be discerned on the seaward edges of the small lake at the interior of the peninsula and the lagoon.", "These are thought to be due to eolian sediment transport under influence of the (predominantly) western winds and the large bare sandy surface area upwind of these regions.", "The large morphological changes after installation of the peninsula resulted in a transformation of the shape of the artificial peninsula and a reduction of the curvatures along the perimeter (Fig. 8).", "These changes in the planform shape were quantified by examining the shoreline, which was taken as the most seaward cross-shore position of the 0 m NAP isobath, x0m(y).", "The remaining shoreline expansion was quantified by x0m(y) with respect to the 0 m NAP isobath measured prior to construction, x0m,pre(y).", "To avoid disturbances from smaller scale topographic features (e.g. ridge runnels and swash bar rip channels) a 5 point (i.e. a 50 m) running average filter was applied to x0m(y).", "The resulting planform contour is shown in Fig. 8 for the first and last survey, and the temporal evolution is displayed in Fig. 9a.", "From August 2011 to the December 2012 the maximum cross-shore extent of the Sand Engine reduced by 132 m, i.e. from 906 m to 773 m (~ 15% decrease).", "Most of the locations along the outer perimeter of the peninsula (alongshore locations − 100 to 1800 m) were subject to retreat of O(150 m) as shown in Fig. 9a. Larger values were found near the northern edge of the peninsula (alongshore y = 1800 m) where the initial coastline curvature was the strongest.", "At this location the 0 m NAP isobath shifted landward over 300 m in the 17 months investigated.", "Simultaneously, the alongshore extent of the peninsula increased: the shoreline prograded by up to 200 m over a stretch of 480 m northward of the peninsula and 710 m southward, resulting in an increase in the alongshore extent of the Sand Engine from 2450 to 3640 m (~ 50% increase).", "The planform analysis shows that shoreline progradation can be observed by ~ 1200 m in the adjacent coastal sections.", "This provides a first estimate of the area of positive influence, but cross profile sediment movements were however not taken into account in this analysis.", "To assess whether the shoreline movement can be taken as a good proxy for the area which shows accretion by the nourishment the alongshore distribution of sedimentation and erosion was computed, integrating the bed level changes per cross-shore profile to obtain sediment volume changes per meter alongshore.", "The profile integrated volume change (Fig. 9b) shows a larger spread of sediment than the shoreline (Fig. 9a), revealing that the shoreline motion underestimates the spreading of sediment slightly in this case.", "Fig. 9b also reveals accretion on both sides of the nourishment and the expansion over time.", "Especially on the northern side this is visible as the northern limit of the accreting profiles was shifting from y ~ 2100 m after 3 months to y ~ 3000 m after 17 months.", "By then, the accretional profiles extended over 4.5 km along the entire stretch of the surveyed domain.", "The most southern profiles (near y = − 1300 m) show substantial accretion at the border of the survey domain, indicating that the survey domain may not be fully encompassing the influence region of the Sand Engine.", "For future evaluation of the feeding beyond the edge of the survey domain an ongoing monitoring campaign with less frequent surveys but increased alongshore scale was initiated (de Vries et al., 2014).", "During the first 8 months the Sand Engine evolved from the man-made strongly asymmetric (γy ~ 1, Fig. 11c) shape to a near symmetrical shape (γy ~ 0.2).", "This also caused a substantial reduction in RMSE (Fig. 11d), implying the evolution into a Gaussian shape.", "After this first period γy remained ~ 0.2 and RMSE was about constant.", "The rapid shape resemblance to a Gaussian distribution function corresponds well to the conceptual picture of planform evolution and diffusion of a rectangular nourishment as originally presented by Pelnard-Considère (1956) (in: Dean, 2002).", "To summarize, the data show that, despite its particular initial shape the Sand Engine peninsula quickly transformed to a diffusive form with a smooth shoreline, a rapid decrease in cross-shore extent and a large alongshore increase in width to both sides.", "The timescale of the adjustment was about a year, after which changes to the shape became less pronounced.", "The overall ratio of the feeding behaviour was quantified using volume budgets of the peninsula and the adjacent sections.", "To that end, the survey domain was separated into 4 different surface areas (Fig. 12, insert): 1) the peninsula only, 2) the adjacent coast on the northern side, 3) the adjacent coast on the southern side and 4) the sum of changes in all three areas combined showing the net loss/gain in the total survey domain.", "The surface areas for volume calculations have a landward edge at the dune foot and were trimmed seaward at about the − 10 m NAP, as here the bed level changes were small (see Fig. 8).", "Over the 17 months we observe a loss of 1.8 106 m3 on the peninsula alone (Fig. 12, red line), amounting to 11% of the 16 106 m3 of added volume during the construction in this area.", "The majority of the erosion took place during the first 6 months, and especially during December 2011 and January 2012.", "For a more traditional evaluation of a nourishment, the remaining volume in this area would be a measure for the success of the project (e.g. Dean, 2002; Dean and Yoo, 1992; Elko and Wang, 2007; Verhagen, 1996).", "However as the Sand Engine nourishment was intended to feed the adjacent coasts it was essential to combine the loss with the accretion volumes in the adjacent coastal sections.", "Both adjacent coastal sections show accretion (Fig. 12, blue and magenta lines) although a slightly larger volume was found in the northern section.", "This is in line with existing knowledge on net northward sediment transport on this part of the coast (e.g. van Rijn, 1997); however, it must also be noted that the accretion on the southern side was likely to extend beyond the southern border of the measurement domain (see Fig. 9 b).", "Therefore, the accretion to the south of the peninsula might be underestimated.", "In total, an increase in sediment volume of 1.3 106 m3 was found at both sides combined.", "This implies that losses on the peninsula were for a large part (72%) compensated for by accretion the adjacent coastal sections.", "Overall, the surveyed area experiences a loss of sand of 0.5 106 m3, a loss which was primarily growing during the energetic winter months.", "These losses could be attributed to the southern boundary being very close to the peninsula and the accretion observed at this boundary.", "Furthermore these losses can be due to consolidation of the subsoil, washing out of fines, transport towards the dunes, transports to offshore (< − 10 m NAP) or more northward.", "To examine the feeding towards the dunes, the Lidar surveys were analysed.", "A small foredune area was selected landward of the areas used for the volume budgets on beach and shore face in Fig. 12.", "This foredune area with the first dune row captures the region around the dune face between approximately + 4 and + 10 m NAP and was on average 50 m wide in the cross-shore direction.", "The biannual Lidar surveys were not executed simultaneously with other surveys but provide data on the bed elevation in the dunes every 6 months.", "The increase in volume in the dune area was about 0.1 106 m3 in the first 18 months, i.e. ~ 15 m3 per m alongshore per year.", "This magnitude corresponds well to values reported by (van der Wal, 2004) (14 m3/m/year) for nourished beaches and de Vries et al. (2012) (0–40 m3/m/year) at the Dutch coast.", "Thus at least 20% of the losses in the survey domain can be attributed to eolian transport towards the dunes.", "The previous two sections examined the overall redistribution of sand to adjacent coastal sections in terms of changes in shape and volume of the peninsula.", "The main objective of this section is to analyse the variations in the redistribution on a monthly timescale, as the time series of the volume change of the peninsula revealed that the erosion volumes varied from one survey period to another (Fig. 12, red line).", "Plan view images of the bed level changes in between consecutive surveys (Fig. 13) show that not only the magnitude but also the alongshore patterns in bed level change varied with time.", "For a time period with energetic (oblique) wave action, as found during autumn and winter, the sedimentation and accretion varied along the perimeter (Fig. 13a).", "The convex tip of the Sand Engine eroded while the adjacent concave coastline sections predominantly accreted.", "These conditions therefore resulted in a smoothing of the outline and feeding from the nourishment to adjacent coasts.", "The pattern of erosion along the convex coastline and sedimentation along the concave adjacent sections confirms the schematic example of sediment redistribution of nourishments under small-angle waves (e.g. Dean, 2002; Elko and Wang, 2007).", "Contrary to the period with energetic wave action, the changes during a month with mild wave action were nearly uniform along the perimeter of the peninsula (Fig. 13b).", "For the specific time period with mild wave action shown here, the sedimentation erosion pattern bears resemblance to the cross-shore generation of a summer berm (e.g. Inman et al., 1993; Quartel et al., 2008), with sedimentation just above mean sea level and erosion below mean sea level.", "The examples in Fig. 13 suggest a relation between wave conditions and the patterns in feeding, but the level of dependency cannot be quantified easily as the observed differences from one survey to another do not solely originate from the environmental conditions.", "More generally the controls for the changes to the feeding on a monthly timescale can be subdivided into three categories: 1.", "the environmental conditions (e.g. wave height), where more energetic wave conditions yield larger gradients in transport (e.g. Splinter et al., 2010); 2.", "the cross-shore profile (e.g. cross-shore slope of the surf zone), where steeper slopes yield larger transports (Kamphuis, 1991; Mil-Homens et al., 2013) and 3.", "the planform shape (e.g. shoreline curvature), where strongly curved coasts experience more lateral dispersion (Dean, 2002; Dean and Dalrymple, 2002).", "During the first period after construction of a nourishment none of these categories were likely to be invariant and therefore we examined parameters of the three categories side-by-side.", "First, the cross-shore profile changes are shown in detail to show the adaptation of the man-made profile to a more natural profile for a transect south (Fig. 14 a), in the centre (Fig. 14 b) and north of the peninsula (Fig. 14 c).", "The adjacent coast south of the Sand Engine peninsula was subject to a large (1000 m3/m) increase in sediment (Fig. 9 b).", "The majority of the sand was deposited in the surf zone and upper shoreface between 0 and − 6 m NAP, indicative of the active zone on this timescale.", "Beyond − 6 m, the bed level changes were small.", "Rapidly after construction a subtidal bar formed, which growed vertically in the first months and then moved slightly downslope (Fig. 14a; yellow and green lines near x = 500 m).", "In the 18 months investigated here no signature was present of cyclic bar behaviour as found on other parts of the Dutch coast.", "However, as typical timescales of cyclic bar behaviour is 4–16 years (Ruessink et al., 2003), this has to be evaluated after further continuation of the monitoring.", "In the supra-tidal profile small variations can be observed (< 0.2 m above the 3 m NAP contour), but overall the upper beach hardly changed.", "Towards the centre of the Sand Engine the profile primarily eroded (Fig. 14b).", "The steep convex profile implemented by the contractor (black line) adjusted to a concave erosional profile, with the slope the profile reducing quickly, i.e. from 1:32 to 1:45 in the first 5 months for the zone + 1 m to − 4 m.", "After 18 months the mean sea level isobath retreated by about 150 m and the profile slope was 1:53, similar to the natural slope of 1:55 prior to the nourishments.", "The reshaped profile mimicked a compound double concave profile with the top concave part of the profile running from the berm top (+ 3 m NAP) to mid-surf zone (− 2.5 m NAP) and a second continuous concave slope downward from − 2.5 m NAP.", "As a result the overall average slope in the zone + 1 m to − 6 m NAP reduced greatly, yet the part around and above mean sea level remained very steep (< 1:20).", "A similar adjustment of the profile was previously reported at other nourishments (e.g. at Hunting Island, USA; Kana and Mohan, 1998).", "North of the Sand Engine peninsula the profiles were influenced by the formation of the spit and the tidal channel connecting the lagoon (Fig. 14c).", "Similar to the profiles south of the peninsula, a subtidal bar formed.", "The seaward slope of the bar matched the slope of the shoreface nourishment executed in this coastal stretch.", "Higher up the profile a large intertidal bar with a cross-shore width of more than 200 m formed because of the spit feature.", "This bar migrated slowly landwards (50 m in the last 12 months) and forced the channel into the supra-tidal beach, similar to the observations of channel movement by Cheung et al. (2007) at a natural spit.", "All profiles show the formation of a smooth compound concave up profile with a tipping point at the subtidal bar crest (at app.", "-3 m NAP).", "Such a compound profile is comparable to previous observations at ocean beaches (Inman et al., 1993).", "For the Dutch coast without groynes however, it is more common to find multiple barred beaches (Ruessink and Kroon, 1994; Wijnberg and Terwindt, 1995).", "Whether a multiple bar will eventually form, can be assessed only from the continuation of our monitoring on longer timescales.", "The results displayed in Fig. 16a show that the net volume change between consecutive surveys varied widely over the investigated period from ~ 1300 m3 to ~ 550.000 m3.", "Especially the first months showed large volume changes, while for some months in early 2012 hardly any net change was observed.", "Gross changes show far less variation (from ~ 160.000 m3 to ~ 770.000 m3, i.e. a factor 5) yet still substantial gross volume changes took place within the profile.", "R values varied between survey periods (Fig. 16b), with the temporal fluctuation highlighting the difference in feeding behaviour in these months.", "The parameters were extracted for each survey period and are displayed in Fig. 16c, d and e. Relationships between the volumetric feeding parameters (ΔVnet, ΔVgross, R) and the potential controls were quantified using least squares linear regression, with the resulting correlation coefficients between individual pairs given in Table 1.", "A large change in cross-shore profile slope was observed, with the average slope over the outer edge decreasing from 1:44 to 1:58.", "The slope adjustment took place in a episodic manner coinciding with the energetic winter period.", "This temporal behaviour provided no correlation with the observed volume changes on the timescale of months.", "Similar findings were found for the coastline angle difference.", "The difference between these coastline angles at either side of the peninsula was large (~ 100°) in the first surveys.", "Over time the shape became smoother and the coastline difference angles reduced to 65–75° (Fig. 16e).", "Although the curvature of the planform shape plays an important role for the erosional volumes in the conceptual view of dispersion of blunt nourishments shapes (e.g. Elko and Wang, 2007), this was not reflected in a significant correlation for the first 18 months of the Sand Engine evolution.", "The relatively static behaviour of the cross-shore slope and coastline curvature in the 17 months and the large dependence of volume changes on the wave power obscures a correlation between the secondary effects i.e. the effect of the slope and curvature on erosional volumes.", "A partial correlation analysis for three variables was performed to test the dependency of volume changes on the wave power was computed while controlling for the profile slope (or coastline angle).", "The partial correlation analysis confirmed the dependency of volume losses on wave power but did not improve the correlations nor reveal a significant effect of the profile slope (coastline curvature) due to the small correlation between the wave power and the profile slope (coastline curvature).", "It was however noticed that the first two periods after construction (with steep slopes and strong curvature) experienced more erosion than other months with similar wave forcing (Fig. 16a).", "Summarizing, the data reveal that the differences in feeding behaviour on a monthly timescale were primarily connected to the magnitude of the wave forcing.", "An effect of the morphology (planform curvature and cross-shore slope) might be present, but cannot be substantiated with this dataset.", "Mega-scale nourishments feeding larger sections of coasts have been proposed as an alternative for regular nourishments to increase beach width and coastal safety.", "This paper presents the analysis of the morphological evolution of a 17 million m3 mega nourishment during the first 18 months after implementation.", "The observations show a planform adjustment of the mean-sea-level contour on the timescale of months as the original asymmetric outline was reworked to a nearly symmetrical shape.", "Along the outer perimeter of the peninsula the shoreline retreated O (150 m) in these 18 months, with some locations showing a retreat up to 300 m. Simultaneously, the shoreline prograded by up to 200 m in the adjacent coastal sections, resulting in an increase of the alongshore extent of 1200 m (50% increase).", "Within the measured 18 months after implementation the profile steepness in the region + 1 to − 4 m around mean sea level adjusted from a steep (~ 1:45) man-made slope to a 1:55 to 1:60 slope, which is similar to the natural slope steepness in this region, thus highlighting the different timescales of the cross-shore and alongshore adjustments.", "The surveys show that the volumetric losses on the nourished peninsula were 1.8 106 m3, i.e. about 10% of the added volume.", "The majority (70%) of the volumetric losses in sediment on the peninsula were found to be compensated by accretion on adjacent coastal sections and dunes, confirming the feeding property of the nourishment.", "Further analysis shows that the morphological response was strongest in the first 6 months while the planform curvature and the surf zone slope reduced.", "In the following 12 months changes were less pronounced.", "Overall, the feeding property was related to incident wave power, such that months with high-energy waves result in more alongshore spreading.", "Months with small wave heights resulted mostly in cross-shore movement of the nourished sediment." ]
[ "The initial spreading of a large (17 Mm3), concentrated nourishment was examined.", "About 1.8 Mm3 of sediment is eroded from the nourishment in the first 18 months.", "The blunt outline is found to deform quickly into a near Gaussian shape.", "Profile adaptation is occurring on a timescale of one year.", "Adjacent coastal sections are primarily fed during the energetic winter months." ]
Advanced Oxidation Process for DNAN Using UV/H2O2
2,4-Dinitroanisole (DNAN) is an important component of insensitive munitions that is anticipated to replace 2,4,6-trinitrotoluene (TNT) in munitions formulations. Photocatalyzed hydrogen peroxide (H2O2) oxidation experiments and chemical analyses were conducted to study the effect of initial pH and H2O2 dosage on the kinetics of DNAN decomposition and the reaction pathways. The results show that DNAN degradation followed zero-order kinetics when a 250 ppm DNAN solution was treated with ultraviolet (UV) light and 1500–4500 ppm H2O2 in an initial pH range of 4–7. However, when the H2O2 concentration was 750 ppm, DNAN degradation followed pseudo-first-order kinetics. The results indicate that DNAN can easily be oxidized by UV/H2O2 treatment. When the H2O2 dosage was 1500 ppm and the initial pH was 7, DNAN was reduced from 250 ppm to less than 1 ppm in 3 h. However, the total organic carbon (TOC) and total carbon (TC) concentrations were reduced slowly from 100 to less than 70 ppm carbon (C) in 3 h, and decreased to about 5 ppm after 9 h of treatment, suggesting the formation of other organic compounds. Those reaction intermediates were oxidized to carbon dioxide (CO2) at a slower rate than the oxidation of DNAN. CO2 was emitted from the solution because the solution pH decreased rapidly to about 3 during the UV/H2O2 oxidation. Most of the nitrogen in DNAN was converted to nitrate by UV/H2O2 oxidation after 9 h of treatment. The research results indicate that UV/H2O2 oxidation is a promising technique for the treatment of DNAN in wastewater.
[ "CENG", "COMP", "ENER", "ENGI", "ENVI", "MATE" ]
[ "2,4-Dinitroanisole", "Advanced oxidation processes", "Photocatalysis", "Wastewater treatment" ]
[ "1500", "1700", "2102", "2200", "2305", "2501" ]
[ "2,4-Dinitroanisole (DNAN) is an important constituent of insensitive munitions that is intended to replace 2,4,6-trinitrotoluene (TNT) in munitions formulations.", "It is also used for the synthesis of dyes and insecticides [1].", "Therefore, the production, transport, storage, and use of DNAN is expected to increase, which will result in large amounts of wastewater containing DNAN.", "Accordingly, increased concern has been raised regarding the environmental fate and impacts of DNAN.", "DNAN is poorly soluble in water (about 276 mg·L−1 at room temperature), and produces rod-shaped crystals at higher concentrations, with adsorption to clay and organic soils [2].", "DNAN has been found to be toxic to aquatic organisms, such as algae, cladocerans, and fish, and lethal to earthworms in soil [3–5].", "Studies on rats have indicated that the acute toxicity of DNAN (lethal dose (LD50) = 199 mg·kg−1) could be even higher than that of TNT (LD50 = 794–1320 mg·kg−1) [6].", "Moreover, the degradation products of DNAN, such as 2,4-dinitrophenol and nitrite, are even more toxic than DNAN itself.", "Furthermore, DNAN can result in a persistent yellow color in wastewater, which must be removed by a pretreatment process.", "Only a few studies have been conducted to characterize the environmental fate and behavior of DNAN, and to examine the biological transformation of DNAN under aerobic and anaerobic conditions.", "In this research, the relevant sensitivity, chemical compatibility, and thermal properties of DNAN have been determined through a literature review and experimental work [6].", "Advanced oxidation processes (AOPs) appear to be some of the most promising methods for the treatment of wastewater containing nitroaromatic compounds.", "AOPs are associated with the generation of radical species (e.g., hydroxyl radical (OH)), which, given sufficient contact time and proper conditions, can mineralize the target pollutants to carbon dioxide (CO2) [7].", "Ultraviolet (UV) photolysis in combination with hydrogen peroxide (H2O2) oxidation is an important AOP technology that has been used to treat wastewater contaminated with nitroaromatic compounds [8].", "H2O2 is readily available and relatively inexpensive, and the photolysis of H2O2 results in the generation of OH, which may degrade contaminants.", "UV/H2O2 has been shown to be an effective degradation and mineralization treatment for most organic compounds [9–13].", "Thus far, studies have examined the degradation of DNAN using the Fenton oxidation process [1], biotic or abiotic transformation [14–18], alkaline hydrolysis [19,20], and bimetal reductive treatment [21].", "Only a few studies have been conducted on the oxidation of DNAN with UV/H2O2 [22], and the results have shown that photo-oxidation is the main mechanism for DNAN degradation, with the formation of nitrite and nitrate as major nitrogen (N) species and 2,4-dinitrophenol as a minor species [22].", "An evaluation of treatment technologies for industrial process wastewater containing insensitive munitions (i.e., DNAN) has been summarized [23], but the optimal conditions for the design and operating parameters for a UV/H2O2 degradation process need to be determined.", "The objective of this study was to investigate the feasibility of treating DNAN-contaminated water by means of UV/H2O2 advanced oxidation, and to determine the optimal oxidation conditions for DNAN degradation, including H2O2 dosage and initial pH. This study has fundamental implications for the treatment of wastewater containing DNAN.", "Aqueous DNAN stock solutions were prepared by dissolving reagent-grade DNAN (C7H6N2O5; 98% purity, Sigma Aldrich LLC., USA) in tap water.", "Considering that the solubility of DNAN in water at room temperature is about 260 ppm, the initial concentration of the DNAN solutions prepared in this study was about 250 ppm.", "The UV lamp used for the treatment was an LSE Lighting UV Bulb (254 nm, 13 W for Pura 1GPM 10–212 UV10 UV11).", "H2O2 solution (30%) was used in the oxidation process (reagent grade, Thermo Fisher Scientific Inc., USA).", "Sodium hydroxide (NaOH) was used to adjust the initial pH of the DNAN solutions (reagent grade, Thermo Fisher Scientific Inc., USA).", "Oxidation experiments of DNAN by UV/H2O2 were conducted using a 1000 mL glass beaker.", "The 1000 mL beaker, which had a diameter of 8.9 cm and a height of 18.7 cm, was used as the reactor.", "The UV lamp was fixed in the center of the beaker, with most of it under the surface.", "A magnetic mixer with a length of 3 cm was used to mix the solutions.", "The UV intensity measured during the oxidation process was about 4500 lx.", "In order to determine the optimal conditions for the treatment of DNAN, batch experiments were conducted with different H2O2 dosages (1500, 2250, 3000, and 4500 ppm) at a fixed pH of 7 and at different initial pH (4, 5, 6, and 7) using a fixed H2O2 dosage (1500 ppm).", "For each experiment, 800 mL DNAN solution was used, and the initial concentration of DNAN was about 250 ppm, or 35 ppm as DNAN-N.", "Next, the solutions were exposed to the UV light for a total time of 9 h while being continuously mixed by magnetic mixers.", "Samples were collected (25 mL) from the reactor at specific time intervals during the degradation process for analysis.", "Data were checked for deviations from normality and homogeneity of variance before performing statistical analyses.", "Pearson pair-wise correlation coefficients between the N-concentrations and carbon (C)-concentrations were determined using SPSS software (Statistics 21, IBM, USA).", "The analysis of DNAN was performed with a high-performance liquid chromatography (HPLC) system (Varian Inc., USA; equipped with a ProStar 410 Auto-sampler and a 330 UV–Vis PDA detector).", "A Dionex Acclaim™ 120 C18 column (15 μm, 4.6 mm × 250 mm, Thermo Fisher Scientific Inc., USA) was used.", "The eluent was an isocratic methanol–water mixture at a ratio of 70:30 (v/v).", "It was pumped at a flow rate of 1 mL·min−1.", "The analytical wavelength was 284 nm and the sample injection volume of was 10 μL.", "Under these conditions, DNAN elutes at 4.9 min.", "The total-N and nitrite-nitrogen (NO2-N) of the samples were analyzed using UV spectrophotometry (DR2800, Type LPG422.99.00012, 15 V, 30 VA, HACH, USA).", "The concentrations of H2O2 were measured using a H2O2 test kit (Model HYP-1, HACH, USA).", "The nitrate-nitrogen (NO3-N) concentrations of the samples were measured by means of an ion chromatography system (Dionex, Thermo Fisher Scientific Inc., USA) equipped with an anion separation column (AS16).", "Total organic carbon (TOC) and total carbon (TC) were measured using a Phoenix 8000 UV–Persulfate TOC analyzer, equipped with a TOC boat sampler (Rosemount Dohrmann Model 183), and TOC Talk software (Teledyne Tekmar Company, USA).", "A TOC and TC concentration range of 0.1–20 ppm C were used to calibrate the instrument.", "The kinetics of DNAN removal under four different H2O2 dosages is shown in Fig. 1.", "The DNAN removal rate depends markedly on H2O2 dosage, indicating that H2O2 dosage has a strong effect on the degradation rate of DNAN.", "For dosages of 1500–3000 ppm, the degradation rate increased with increasing dosage.", "This is because the increasing H2O2 concentration can promote the formation of OH, which can accelerate the DNAN degradation process.", "However, studies on the photo-degradation of some organics have shown that excessive H2O2 may reduce the degradation rate because it acts as a scavenger for OH [24–26].", "From Fig. 1, it can be seen that the DNAN removal rate at 4500 ppm H2O2 is slower than that at 3000 ppm, suggesting that excessive H2O2 reduces the DNAN degradation rate.", "It can be concluded that excessive H2O2 may compete with DNAN for reaction with OH, which would decrease the degradation rate of DNAN.", "The results show that the optimal H2O2 dose for the oxidation of a DNAN solution with an initial concentration of 250 ppm is 3000 ppm.", "However, a higher H2O2 dosage would result in higher treatment costs in engineering applications, and the H2O2 remaining in the treated solution must be removed for environmental protection.", "Thus, it is of great importance to optimize the H2O2 dose for the treatment of DNAN solutions.", "The results show that DNAN degradation follows zero-order kinetics under our experimental conditions; the rate constants are summarized in Table 1.", "The zero-order kinetics were different from the reported pseudo-first-order decay kinetics of DNAN under sunlight and UV only, under which the degradation process was much slower [22].", "This difference was probably due to the presence of H2O2, which can accelerate the DNAN degradation process.", "Linear regression was developed for the DNAN degradation kinetics; the results are shown in Fig. 1.", "The points after the degradation was finished were not used in the linear regression.", "A lower H2O2 dosage of 750 ppm was used to treat the DNAN solution; the DNAN removal kinetics are shown in Fig. 2.", "It was clear that the kinetics of DNAN degradation were pseudo-first-order decay kinetics.", "The results in Figs. 1 and 2 indicate that DNAN can easily be oxidized by UV/H2O2 in the presence of sufficient H2O2, and that the oxidation rate of DNAN is not affected by decreasing DNAN concentration during the treatment.", "Zero-order decay kinetics for the advanced oxidation process for other organic contaminates, such as nitroguanidine, have also been reported [27].", "Assuming that all the H2O2 was used to oxidize DNAN to nitrate (HNO3) and CO2, 730 ppm H2O2 was needed to completely oxidize 250 ppm DNAN, as calculated based on Eq. (1).", "Under experimental conditions, only part of the H2O2 was consumed to oxidize the DNAN and its degradation products, and the actual demand for H2O2 was greater than 730 ppm.", "In addition to H2O2 dosage, the effect of initial pH on DNAN degradation was studied.", "Batch experiments were conducted to treat DNAN solutions at an initial pH of 4, 5, 6, and 7, respectively, with the same H2O2 dosage of 1500 ppm.", "The kinetics of DNAN removal at four different initial pH values are shown in Fig. 4.", "The results show that DNAN degradation follows zero-order kinetics under the experimental conditions used here; the rate constants are shown in Table 1.", "Linear regression was developed for the DNAN degradation kinetics, and the results are shown in Fig. 4.", "The points after the degradation finished were not used in the linear regression.", "Fig. 4 shows that the initial pH can affect the DNAN degradation processes to some extent: The higher the pH, the faster the degradation rate within the pH range of 4–7.", "When the pH is higher than 7, alkaline hydrolysis of DNAN will take place, which is a different degradation process [19,20].", "However, the effect of initial pH on the degradation rate of DNAN is modest compared with the effect of the H2O2 dosage.", "Studies on the photo-transformation rate of DNAN in water showed that a variation in the solution pH (pH 6.5–8.0) had only slight effects on the degradation rate of DNAN [22].", "Considering that the initial pH of the DNAN solution is about 7, the most suitable pH is 7 for DNAN oxidation, as no pH adjustment is needed and the degradation efficiency is not affected.", "Tables 2 and 3 show the changes in pH during the oxidation processes under different initial pH and H2O2 dosages.", "The results show that the pH decreased quickly when the oxidation process of DNAN began, regardless of the initial pH and H2O2 dosage, and remained constant at about 2 after the DNAN degradation processes finished.", "This finding indicates that some acid substances—mostly HNO3—were formed as a result of DNAN degradation.", "In order to determine the oxidation products of DNAN, concentrations of the various kinds of N species—including total-N, ammonia, nitrate, and nitrite—formed during the oxidation processes of DNAN were measured in addition to the analysis of DNAN-N.", "The results (Fig. 5) show that during the DNAN degradation processes, nitrate and nitrite were formed and no ammonia was detected.", "The concentration of NO2-N was very low and disappeared after the DNAN degradation processes finished, indicating that NO2-N is one of the major intermediates.", "Nitrite was oxidized to nitrate immediately after being generated from the decomposition of DNAN.", "The concentration of NO3-N continued to increase during the degradation processes, and the final concentration of NO3-N was similar to the initial DNAN-N concentration.", "This result suggests that NO3-N was the end product, and that almost all of the DNAN-N was transformed into NO3-N, similar to the other nitroaromatics treated with UV/H2O2 [8,28].", "These results are consistent with those of other studies that have been conducted on the UV photolysis of DNAN [22], and suggest that photo-oxidation is a promising technique for the treatment of DNAN in wastewater.", "The total-N remained constant during the DNAN degradation process, indicating that no gaseous N was formed (Fig. 6).", "Furthermore, the N compounds showed similar change trends at a different initial pH within the range of 4–7.", "However, the DNAN-N decreased faster and the nitrate increased faster with a higher initial pH, as a higher pH increases the rate of the DNAN degradation progress.", "After 9 h of treatment, the NO3-N was still a little lower than the total-N, suggesting that there was a small portion of N remaining in the form of nitro-organics.", "Similarly, Fig. 7 shows that the TOC was about 5 ppm after 9 h of treatment, indicating that some organics containing N were present in the reacted solutions.", "This probably occurred because the very low concentration of H2O2 that remained after 9 h (Fig. 3) was unable to oxidize the residual nitro-organics into nitrate and CO2.", "TOC and TC were measured during the degradation process of DNAN in order to investigate the changes of carbon species; the results are shown in Fig. 7.", "The TOC concentrations are almost the same as the TC concentrations, and both the TOC and TC decrease during the degradation process of DNAN.", "This result indicates that gaseous carbon (probably CO2) was generated during the DNAN degradation process, and that no carbonate or bicarbonate was present in the treated water.", "As discussed before, the pH decreased quickly during the oxidation processes when the oxidation process of DNAN began, regardless of the initial pH and the H2O2 dosage, and remained constant at about 2 after the DNAN degradation processes finished.", "This result indicates that some acid substances—mostly HNO3—were formed as a result of DNAN degradation, which also explains why no carbonate or bicarbonate was present in the treated water.", "The results show that complete mineralization of DNAN can be achieved with UV/H2O2, as the TC and TOC decreased to about 5 ppm after 9 h of treatment.", "As the DNAN degradation finished in about 4 h, DNAN was converted to other organic compounds, which were subsequently degraded.", "The degradation process of DNAN may follow parts of the decomposition pathway of 2,4-dinitrotoluene by UV/H2O2, in which some lower molecular weight carboxylic acids are produced and then oxidized into CO2 [29].", "This would explain why the pH increased slightly after the DNAN degradation was complete.", "Fig. 6 shows that the rate of increase of nitrate was faster during the degradation process of DNAN than after the DNAN degradation finished, indicating that the cleavage of nitro groups took place in the early stages of DNAN decomposition.", "When the DNAN degradation was complete, about 77% of the N had been transformed into nitrate, suggesting that 23% of the N remained in the form of nitro-organics.", "Meanwhile, Fig. 7 shows that about 65% of the TOC remained at that time, which indicates that the nitrification process is faster than mineralization during DNAN degradation.", "The research results indicate that UV/H2O2 oxidation is a promising technique for the treatment of DNAN in solutions, as most of the DNAN can be oxidized to CO2 and nitrate.", "During the oxidation treatment, the total-N concentration remained almost constant, suggesting that no gaseous N compounds (i.e., nitric oxide, nitrous oxide, nitrogen gas, and ammonia) were formed.", "Nitrite was one of the major oxidation intermediates.", "The rate of DNAN oxidation was affected by the H2O2 dosage and pH. An H2O2 dosage of 1500 ppm and an initial pH of 7 were the optimal conditions for the treatment of 250 ppm DNAN solution." ]
[]
Understanding social media and identity work in young people transitioning to university
Social media (SM) are a core component of young people's lives and have been researched in relation to relationship building and maintenance. While SM are known to be useful in supporting life transitions for young people, we know little about the specific use patterns or activities associated with social adjustment during the specific transition to university. We explore the use of social media during the student transition to university in relation to theories of social comparison and community building and describe a three stage process which accounts for this transition. Participants move through the stages of affirmation, assimilation and integration similar to other life transitions, but in doing so, we reveal the importance of the intersection between offline and online activities and highlight the benefits and limitations of SM use in this transitional period.
[ "ARTS", "COMP", "PSYC" ]
[ "Identity", "Social media", "Students", "Transition", "University" ]
[ "1201", "1709", "3200" ]
[ "Adolescents rely upon social media (SM) as a core component of their social lives (Boyd, 2014; Yang & Brown, 2013) and will typically use SM to build new peer affiliations, manage existing relationships and stay informed about social activities within their network (Lampe, Ellison, & Steinfield, 2006; Pempek, Yermolayeva, & Calvert, 2009).", "It is not surprising, then, that young people can find SM especially useful when moving from home to a new university environment.", "These ‘emerging adults’ experience marked change at this time, and use online services to bridge these kinds of life transitions (Orzech, Moncur, Durrant, & Trujillo-Pisanty, 2016).", "University students have demonstrated that they use SM when searching for new relationships or maintaining existing friendships (Ellison, Steinfield, & Lampe, 2011; Lampe et al., 2006; Pempek et al., 2009) and SM resources are particularly important for first year students seeking to build new relationships and establish a sense of community at college (DeAndrea, Ellison, LaRose, Steinfield, & Fiore, 2012).", "Yet, little is known about how specific use patterns or activities are associated with social adjustment to university; how students use SM and digital communication to establish new communities, as well as maintain existing ones; how students balance their online and offline identities during this process; and finally the possible drawbacks of SM use during these transitional periods.", "This study addresses these questions.", "This empirical paper reports on a study of first year undergraduate students in the UK in relation to their use of SM when transitioning from home to university in order to understand the role played by SM in their transition.", "We contribute to this discussion by identifying similarities between our work and the theoretical stages identified in previous work exploring when people cross borders or cultures, as in the example of expatriates living abroad (Mao & Shen, 2015), and literature on people coping with acculturation.", "The paper is structured as follows: we describe related work in terms of SM use in young adults, with a particular emphasis on the literature on social comparison and identity-management within SM, and a review of research surrounding the role of SM in community building and the much smaller literature on SM use when making the transition to university.", "We then outline and justify our own study, which uses novel methodology to capture and interpret SM use during times of transition.", "There has been a rapidly growing body of research which seeks to understand young people's use of SM technologies and their participation in online communities (Boyd, 2007; Schoenebeck, Ellison, Blackwell, Bayer, & Falk, 2016).", "Many of these studies have focused on patterns of Facebook use in large part because it is the most widely used of the social networks in a Western context, but we should note at the outset, that SM use evolves quickly across different platforms and that, while Facebook remains popular, other sites such as Instagram, WhatsApp and Pinterest are seeing an increase in their user base (Duggan, Ellison, Lampe, Lenhart, & Madden, 2015).", "Young people typically use SM to maintain and regain social connections and for ‘identity work’ that includes the sharing and tagging of photographs, the creation of ‘status updates’ and associated forms of social approbation such as ‘liking’ posts (Ellison, Steinfield, & Lampe, 2007; Pempek et al., 2009; Sosik & Bazarova, 2014; Subrahmanyam, Reich, Waechter, & Espinoza, 2008).", "Such exchanges can help to bind a community together, but can also induce various forms of social anxiety.", "This is particularly true for young people who seek social approval to reinforce their own sense of self, and who can sometimes be negatively affected by the social comparison processes that accompany SM exchange (Feinstein et al., 2013).", "These two elements: social comparison and community building are explored further, below.", "The social comparison processes invited by SM are well recognised.", "For example, selfies are common currency on many SM sites, but selfie posting is also associated with certain social pressures such as the need to post positive ‘fun’ selfies and to try to gain sufficient ‘likes’ or risk damaging self-esteem (Pounders, Kowalczyk, & Stowers, 2016).", "Such comparison processes are far more likely to take place when the other members of a SM community are similar to the self, whether by age, sex, or other dimensions of identity (Knobloch-Westerwick & Hastall, 2006; 2010).", "Young people spend many hours viewing the posts of similar others (Pempek et al., 2009) and inevitably are drawn into the process of social comparison (Johnson & Knobloch-Westerwick, 2014).", "This process, first described by Festinger (1954), involves two possible acts: people can either compare themselves unfavourably to others – making an upward comparison (Gibbons & Gerrard, 1989; Lockwood & Kunda, 1997) said to form part of the drive to self-improvement (Collins, 1996), or they can compare themselves favourably to others, making downward comparisons that can be used to restore threatened self-esteem (Wills, 1981) and create positive affect (Gibbons & Gerrard, 1989).", "These upward and downward comparisons are part of self-presentation (Marder, Joinson, Shankar, & Houghton, 2016).", "They affect the creation of a desired self (Leary & Kowalski, 1990) and inform personal choices about how to ‘look good’ (Wang, Hinsberger, & Kraut, 2016).", "Goffman (1959) has influenced our understanding of the ways that people might use self-presentation to claim membership of a group or community.", "Goffman argues that, during periods of transition, the ‘performers’ withdraw until they have established new social norms and rules of interaction.", "In SM terms, this withdrawal can take the form of self-censoring or editing elements of the digital persona in order to avoid projecting an undesired image to a new online audience (Lang & Barton, 2015; Marwick & Boyd, 2011).", "When this fails, or when SM users are tasked to ‘perform’ to multiple and diverse audiences simultaneously, then we talk about this in terms of ‘context collapse’ (Marwick & Boyd, 2010).", "While self-presentation in the presence of multiple audiences can have positive effects (Leonardi, 2014), context collapse can have negative implications since users find it difficult to meet the expectations of multiple audiences simultaneously (Xie & Kang, 2015).", "For example, Lang and Barton (2015) report that 84% of users have experienced being tagged in undesirable photos and subsequently taken defensive action.", "According to Marwick and Boyd (2010: 122), this creates “a lowest-common denominator effect, as individuals only post things they believe their broadest group of acquaintances will find non-offensive.”", "Furthermore, recent research has found that users self-censor and edit their offline behaviour in fear that content will be communicated online causing self-presentational predicaments (Marder et al., 2016).", "Kerrigan and Hart (2016) illustrated the importance of a temporal approach to understanding performance on SM, as older online selves may be seen as incompatible with new lifestyle or career developments, and users have not yet developed practices for dealing with past selves.", "Ackerman et al. (2004) discussed the ways that an effective ICT infrastructure can be used to bridge relationships within communities, providing the kind of ‘social capital’ platform that “enables joint activities and cooperation for mutual benefit” (Ackerman et al., 2004).", "SM systems can support the development of social capital and community cohesion (Malinen, 2015) while online social networking can also promote psychological well-being (Ellison et al., 2007; Wellman, Haase, Witte, & Hampton, 2001).", "When we explore the creation of new communities, or focus upon communities in transition, then we can see that SM also has a role in both creating and enhancing community relationships.", "Across a wide range of contexts, including disenfranchised youth in rural Sweden (Svensson, 2015) or new mothers in Canada (Valtchanov, Parry, Glover, & Mulcahy, 2014), SM has been shown to both mobilise and unite individuals.", "For new students wishing to make an adjustment to university, SM offers a way to support the transition from adolescence to adulthood and presents individuals with the opportunity to tentatively build new relationships ‘at a safe distance’ (Ferguson et al., 2016).", "Many students relocate when starting university, and so become physically removed from their close friends and family.", "They are then faced with the task of maintaining existing connections whilst being surrounded by thousands of unknown contacts (Scanlon, Rowling, & Weber, 2007) from whom they need to forge new intimate bonds.", "Thus, both relationship maintenance and establishment have been found to be crucial to successful adjustment during the transition to university (Buote et al., 2007; Swenson, Nordstrom, & Hiester, 2008) and, to a certain extent, SM can support both (Ferguson et al., 2016), although it has been argued that Facebook is more effective in the maintenance as opposed to the creation of new student relationships (Lampe et al., 2006).", "Ellison et al. (2007) argued that students use Facebook primarily for keeping in touch with high school acquaintances and classmates, although in a later survey of undergraduates, Ellison et al. (2011) identified three SM strategies: 1) initiating strategies, which allow for building connections with strangers that have never been met offline; 2) maintaining strategies, used to maintain existing relationships with close friends/ties; and 3) social information seeking, to find out information with newly connected acquaintances.", "In Ferguson et al.'s (2016) small-scale study, the use of Facebook to support social cohesion and academic communication in a small group of student nurses was clear, but the challenges of SM use in relation to building and supporting a new professional identity were also outlined.", "These two processes of social comparison and community building are important when we consider how young people make the transition to university, although we find more limited and somewhat contradictory research in this context.", "Yang and Brown (2013) examined associations between Facebook activity and social adjustment to college and found peer interaction to be positively associated with adjustment.", "Yet, those who were motivated to use Facebook to pursue new relationships reported greater levels of loneliness and those who intended to maintain existing friendships reported less loneliness.", "Consequently, participants more often planned to use SM sites to stay in touch with existing friends, as opposed to making new connections.", "To a certain extent this ‘staying in touch’ via SM then became a key factor in determining which of the old and new relationships survived.", "In their more recent work, Yang and Brown (2016) assessed SM use during the transition to college, and found that deeper, positive, more authentic self-presentation on Facebook was positively associated with perceived support from the audience, thus raising self-esteem for those students.", "This same point was demonstrated by Sosik and Bazarova (2014: 130) who measured SM use in a sample of 256 students enrolled in a large northeast US university, using an app that collected Facebook communication between close friends in order to explore ‘relationship escalation’.", "They noted the importance of a maintained presence on SM arguing that: “frequent and recent communication through multiple Facebook channels can help partners convey a feeling of closeness … while communication absence from each other's life corresponds to alienation and relational de-escalation”.", "We know less about the development of new relationships at university, although many university programs explicitly seek to promote SM networks for new students arriving on campus (Swenson et al., 2008; Ye, 2006).", "This is understandable, as new peer relationships and interactions have been shown to influence students' adaptation to and retention in universities (Tinto, 1975; Yang & Brown, 2013).", "For example, overseas students were able to use SM to build new relationships with those from their host country, rather than focusing on maintaining relationships from home (Fraiberg & Cui, 2016; Saw, Abbott, Donaghey, & McDonald, 2013).", "DeAndrea et al. (2012) designed a student-centred SM site to give students advanced information about other students and staff.", "It was found that increased use of the website correlated with increased perceptions of a diverse social support network, with increased website activity also predicting successful integration in college life.", "The activities engaged in prior to the start of term included signposting to fresher's events and how to get the most out of welcome activities.", "This kind of niche design of SM platforms is helpful to facilitate new interactions at university, but says little about how students manage to maintain the balance between their community building and social comparison activities, nor does it tell us anything about the balance between online and offline communities.", "A limited body of work has demonstrated that SM resources can be beneficial when building new relationships at university.", "However, it also suggests that student concerns about the way they self-present online can cause anxiety.", "We sought to explore this tension, developing a research design which allows for a more holistic consideration of SM usage, moving beyond a simple consideration of Facebook as the dominant SM platform and taking into account social activities before, during and after the transition to university.", "The limited research that focuses on students forming new connections (e.g. Ellison et al., 2011; Ferguson et al., 2016) fails to fully explore how they balance their new vs old, online vs offline communities to best effect.", "We need to develop greater theoretical insight into how students edit and adapt their online and offline identity during these transitional periods.", "Despite the frequent social comparison and self-management behaviour of younger users, no work directly examines these behaviours in relation to adjusting to university.", "Goffman's (1959) self-presentation theories are relevant here, specifically the ‘adjusted’ self in times of transition, in which performers restrain their behaviour until they have established the new rules of interaction.", "Finally, many studies focus on the positives of SM in terms of increased social connectivity when establishing new communities but fail to fully explore the possible drawbacks of SM, such as over-disclosure to untrusted individuals (Lee, Park, & Kim, 2013; Sleeper et al., 2016).", "Our study focuses on the use of SM and digital data in a sample of first year undergraduate students during their first six weeks at the start of university.", "We use a novel methodology that asks the students to gather their own SM and other digital communications interactions week by week, using the platform ‘Pinterest’ 1 to collect items of interest, effectively creating a pin-board of SM activity for each week.", "This was used as a kind of ‘cultural probe’ (Gaver, Dunne, & Pacenti, 1999) to elicit rich discussion at interview.", "In this way, we hoped to assess the role of SM during the early few weeks as an undergraduate and better understand the role played by SM in transitioning from home to university and understand that in relation to more personal forms of communications used.", "The following research questions were developed to inform this study: How do young people use social media in times of transition to university?", "How do young people balance the demands of their online and offline communities?", "How do young people manage the ‘context collapse’ when their old and new SM communities collide?", "We recruited 25 university students (aged 18 or over) from two UK institutions.", "In order to understand the transition to university, respondents needed to be first year undergraduates commencing university in September 2015 and who regularly used a broad range of SM platforms.", "Six male and nineteen female participants, aged 18–22, took part.", "We recruited 13 British participants, with the remaining 12 originating from Tanzania, Germany, Romania, Italy, Lithuania, Bulgaria, Hong Kong, Indonesia, the Philippines, India and Singapore.", "Participants were recruited through campus networks, advertisements, SM postings and in class announcements.", "Once they had agreed to participate, the students were invited to a project briefing where the data collection process was explained in more detail and participants were introduced to the first phase of data collection.", "This required them to set up a Pinterest board where they would post a range of broadly specified data documenting their use of SM (e.g. Facebook, Twitter, Instagram, etc.) and digital data (e-mail, text messages, instant messaging etc.) over a 6-week period.", "Board 1 covered the week before leaving for university, board 2 covered the first introductory week of university (‘fresher's week’), board 3 covered the first week of lectures, board 4 the second week of lectures, board 5 the third week of lectures, and finally board 6 the fourth week of lectures.", "These boards were shared with the researchers and analysis of the boards informed the design of the interview guide used for the second stage of data collection.", "In the following stage, we conducted in-depth semi-structured interviews with individual participants after a two-week phase of self-directed collation of their data, utilising the concept of a digital scrapbook.", "Participants only had access to their own scrapbook which consisted of their own data and that shared with them by their friends and families.", "We wanted to understand what sorts of interactions the students had during this time period, and they were encouraged to ‘pin’ content that was solely their own, as well as interactions with other people or groups.", "An average of at least 20 pins (i.e. postings) on each weekly board was requested.", "These data were created by the participants in the period prior to coming to university and immediately on arrival, but added to the Pinterest boards retrospectively for the purpose of this study.", "An example board can be seen in Fig. 1.", "After data collection was completed, the boards were digitally shared with the researchers (by the participant granting access via the Pinterest platform).", "The subsequent interviews helped us understand the participants' early experiences of starting university, and set the context for their use of media during this time, using the boards as a cultural probe around which to focus the interviews.", "This study was carried out in accordance with the recommendations of [anon] University Ethics Committee, with informed consent from all participants.", "Electronic permission to access Pinterest boards was revoked once interviews had taken place, but participants gave permission to use anonymised screenshots of the resulting boards, as below.", "Pseudonyms have been used throughout to protect participant's identities.", "All interviews were audio recorded, transcribed, and a thematic analysis was conducted.", "As a significant amount of data were in the form of “free-flowing text”, they were analysed by finding meaning in large segments of the texts rather than in isolated words, hence coding the data (Ryan & Bernard, 2000).", "Each data corpus was coded by all four members of the four-person research team, working in pairs and then coming together to reach agreement regarding the codes.", "Following Braun and Clarke's (2006) stages of coding, it was first important to familiarise with the data, which meant reading and re-reading the transcripts.", "This generated initial descriptive codes through constant comparisons between the data, and allowed us to search for key themes whereby patterns and repetition emerged in the data.", "We then reviewed each emergent theme against the overall data set, before developing higher order codes; our final themes.", "Once the final themes were agreed, the findings were prepared for write-up.", "The focus of this analysis was on how the students utilised SM during the first six weeks of university, in particular the role of SM to enhance community building and social comparison processes.", "Analysis led to the development of our key findings explained below and our analysis resulted in the identification of the following practices; affirmation, assimilation, integration and social media use and barriers to integration as discussed below.", "Our analysis revealed a number of important ways in which students used SM during the transition to university, but more significantly, showed us how online and offline activities intersected as part of this transition.", "We present our key findings within four sections.", "Firstly, we discuss how students use SM to support a process of affirmation - reflecting and celebrating the community ties in existence prior to starting university.", "Secondly, we show how they use SM to engage in the process of assimilation to the new community, i.e. establishing a new university identity and making new online and offline friendships during the transition from home to university.", "Thirdly, we consider the act of integration – exploring the kinds of ‘juggling’ that goes on to maintain identity ties with both home and university communities.", "Finally, our interview data revealed some of the ways in which SM acted as a barrier to integration, and we highlight the limitations of social media use in this context.", "We describe our findings under these subheadings to provide structure, yet we acknowledge the recursive nature of the practises we describe, and the overlap of themes in places.", "In instances where there is overlap, we have selected the stage we felt was best fit for the example.", "We found that in the early stages of transitioning to university, SM played a large role in affirming the ‘home’ identity for our participants.", "SM posts in the early weeks had a strong focus on the process of saying goodbye to friends and family, but there was a celebratory or appreciative tone to this, evidenced by family meals, nights out with friends from home and so on, but also these posts played an important role in reassuring those left behind:", "My boyfriend was worried that me going to university was going to alienate him so I was trying to put nice stuff about him on social media.", "So he'd bought the Chinese take-away that we were having that night and that's a fortune cookie from the Chinese take-away.", "(Samantha)", "While the posts did reveal a sense of excitement about going to university, the need to represent the importance of relationships being left behind was apparent:", "Me and my best friend from home used to play the piano for a very long time when we were younger, and I came across an old video of us both playing at a competition, and I just decided to post it on Instagram.", "I was feeling quite sad that week about leaving home as I knew my life would change, new friends, new environment, new language, everything.", "So I guess I was feeling quite nostalgic and wanted to post something that would remind me of happier times and something that symbolises my friendship with my best friend from home.", "(Michelle)", "It was important to our participants that they were still connected to home through being kept informed and connected to family life:", "That's my dad sending me pictures of my dog.", "He's just showing me that he took him to the pub, and he's all muddy.", "I love being filled in on stuff about my dog.", "(Abigail)", "We see from the extract above, that as well as generating content themselves, communication was not unidirectional, as participants would often get prompts of home in the weeks after moving to university.", "This was especially true for international students who would get reminders of the cultural differences back home, such as the native food and weather:", "There are lots of pictures of [Italian] food and coffee from back home, they just seem to be everywhere.", "And I seemed to be complaining about the weather again as its 3 degrees here, which in my world is freezing cold … It's such a cultural change, it's only natural to miss home.", "(Luis)", "These practices were documented as a way to remind the participant, as well as their families, of their ties to home.", "It can be seen as a means of strengthening home ties and affirming connectedness with a former life and place.", "It resonates with Lingel, Naaman, and Boyd's (2014) work on migrants to New York city both in the sense of affirming ties to friends and family, but also in the sense of celebrating an identity that is tied to place.", "For the majority of our participants, the first Pinterest board reflected ties to home, to people that were going to be missed, and the leaving parties or meals that were planned.", "Homesickness was common for our participants, but for some, the distance to travel home was much greater, and therefore the leaving rituals were more notable:", "This is my best friend.", "We spent my last day in Romania and we spent it together.", "Because it was like the last week spending with my family and my friends.", "So it was kind of important, because I haven't been home for five months or so.", "(Lauryn)", "For international students, the distance from home made the use of SM and digital technology even more important as a means of keeping in touch:", "I was using Skype literally every evening so I was a bit homesick.", "I'm used to a completely different life where mom could cook something for me to eat.", "That transition was really hard.", "Thanks to social media, communicating with my father on Facebook or using Skype to video chat, all these were extremely useful.", "And I think for every international student, these inventions are probably number one.", "(Nathan)", "Participants said they would make attempts to retain a tradition from home to comfort them whilst in their new environment.", "This served as a comfort for them but also reassured their families that they were settling in, and that family ritual was still important:", "My mum she always used to cook me a lot of Indian food before I came to uni, but then on a Tuesday she would also make pasta.", "So now since I've started uni, I always make Indian pasta every Tuesday and Thursday as it provides me with some home comfort and some kind of routine I had when I was at home.", "I would also Snapchat a picture of the food and send it to my mum.", "(Kaden)", "Others have written about the importance of family rituals as a means of celebrating the connectivity and shared values of family life (e.g. Bell, 2009) and, more recently, the role that SM can come to play in shared affirmation of these family rituals (Petrelli & Light, 2014).", "What we see here is the role of the ritual in preparing the way for transition, acknowledging, documenting and sharing thoughts and images about what will be missed.", "This is part of the process of ‘bridging the gap’, between home and the first few days at university.", "Students also took selfies with family, personalised their accommodation with artifacts as reminders of home and posted the pictures online as part of the process of affirmation, and joined societies or clubs that were part of previous home life to help them settle:", "I'm the type of person who has to feel at home.", "You want it to feel safe and homely.", "So I remember putting up my photo wall in my room next to my bed, so that it felt like everyone was closer to me even though they are all the way back in Germany.", "(Anna)", "Here, in the printing and pinning of photographs, drawn from SM, we can also see the need to make these memories into more tangible artifacts i.e. to become a touchpoint to home that exists in the physical, offline world.", "This physicality can be important – something noted by those writing about the value of ‘slow technology’, who have observed that virtual forms seem to be less valuable that physical forms (Gerritsen et al., 2016).", "So, while we recognise the importance of using SM in its digital form as means to reconnect with an earlier life (Zhao et al., 2013) what we see here is the importance of offering people from the old home some kind of physical space in the new one.", "All of our students were involved in making a major life transition and most were setting up home for the first time, some in a new country.", "The two universities involved in our study gave all students the opportunity to use SM to help establish new peer networks prior to their arrival on campus in order to better manage the transition to university (a process supported by the findings of Swenson et al., 2008; Ye, 2006; DeAndrea et al., 2012).", "In this section, then, we report on the uptake of this opportunity, describing the ways that students took advantage of SM, but also the ways in which they were cautious about disclosure, particularly during the first two or three weeks, highlighting some of the ways that they chose to ‘edit’ themselves or announce their new identities.", "This meant that students were engaged in a considerable amount of identity work in the weeks before they felt comfortable with their new ‘friends’.", "In our sample, we found good uptake of the SM opportunities created by universities to aid students in finding their flatmates – with a typical example being the creation of Facebook groups organised around accommodation sharing.", "Our respondents were thus able to connect with prospective housemates in advance and used this to get to know each other:", "My accommodation at [location] had its own Facebook page, and people were posting their flat numbers when they got their flat numbers.", "That's how I managed to find my housemates, and then we started a Messenger group after that.", "(Rachel)", "This Facebook group also allowed future housemates to find those who had been offered accommodation in nearby flats, thereby building up a wider virtual community around the physical location of their new homes:", "We would all post our flat details so we would get to meet other flatmates or people staying near you, so yea that was really good because you get to know people a little beforehand.", "(Vanessa)", "This allowed the students to engage in some practical pre-arrival activities to make the transition easier:", "We talked about what I was bringing and what she was bringing, so we could help each other out and share certain stuff.", "And in general it made it a lot less awkward before actually meeting face-to-face.", "(Brittany)", "Importantly, the pre-arrival contact helped new students to reduce some of the uncertainties about their forthcoming experience and helped them gain a sense that they would meet compatible people with similar interests:", "The girl I met was quite similar to me, so when we got speaking it felt like we'd known each other for a long time.", "So for me it was good to know that I felt like I had already known someone before I had even moved to uni … I didn't expect to be best friends, but we just hit it off straight away and we're moving in together next year.", "So Facebook really helped our relationship grow.", "(Vanessa)", "We should note, however, that SM exchanges prior to actually meeting face-to-face were often problematic.", "We know that people will pre-judge new acquaintances on the basis of their online profiles, a process that can result in false or inaccurate misconceptions (Tuch, Presslaber, Stöcklin, Opwis, & Bargas-Avila, 2012), and this was apparent in our findings:", "I was really keen to know who I was living with, but then I think you can prejudge people a bit too much from it.", "So with [friends name], I saw that she was from a private school down south, she's well-travelled, so I was a bit nervous that we won't have much in common.", "But actually we just clicked and we get on really well.", "(Maria)", "We found plenty of evidence that the students were using SM to make very early social comparisons.", "In keeping with the findings of Pempek et al. (2009) and Subrahmanyam et al. (2008), we heard narratives from participants about their investigations of other students before joining university and continuing during the next five weeks at university.", "As Trottier (2012) points out, on SM we have the opportunity to be a subject, as well as an agent of surveillance.", "Most of our students recognised that the SM personas they encountered prior to coming to university might be misleading.", "Indeed, our participants had sometimes ‘edited’ their own posts and acknowledged that others might do the same, recognizing that self-presentation is important (Chua & Chang, 2016; Marder et al., 2016) and that people generally like to make themselves look good (Leary & Kowalski, 1990; Wang et al., 2016).", "Participant's ‘cleaned’ their SM profiles in various ways:", "Social media gives you the ability to edit everything you post and make everything seem amazing.", "I remember I spent a day when I just came to university where I deleted all the photos that I'm not pretty in.", "I'm pretty conscious about what I post on social media, because I do care how I come across, especially new people at uni.", "(Nicole)", "I think I manage my social media quite well anyway, but I did clear every ugly photo of myself before I came to university … You just never know how judgmental people can be and you can soon feel exposed and vulnerable if you let people see every photo of you or all those childhood photos of you.", "They might make a judgment of you before they even get a chance to meet you.", "(Patience)", "It was clear that SM meant that the process of ‘meeting for the first time’ involved preparation over days or weeks rather than over a matter of hours as is typical with a face-to-face meeting.", "This then led to further tensions as students had to live up to the promises they had made online:", "I've definitely become more vain as I have come to university, so I make sure every post of myself shows the best side of myself … Because that's how they [new university friends] get to know you.", "(Patience)", "I was a bit worried about who was in the flat, and things like that, because you've never seen them before, and all you can see is what they look like on Facebook, and obviously that's a really one-sided view to take of someone.", "(Alexis)", "Facebook was seen as a SM platform where self management was imperative, particularly in this adjustment stage.", "Consequently, some of our participants were very cautious in the use of SM during these early transitional weeks as they preferred to wait and see what the offline, face-to-face interaction would bring:", "It took me time to establish my university friends … I don't think social media is the best platform to form new friendships.", "I think that takes time and you need face-to-face interaction.", "If you rely solely on social media, then I think it becomes false way to form a new friendship.", "I much preferred doing that in person and going to the pub to watch the rugby or meeting up after games and things like that.", "(Henry)", "Many of our participants expressed reservations regarding their use of SM, recognizing that others, like themselves, may be setting out a poor foundation for a friendship by highlighting only the most attractive elements of themselves.", "The majority of the participants showed signs of what Goffman (1959) referred to as the ‘adjusted self’ during times of transition by holding back their typical online behaviour until the new rules of interaction have been established.", "Inevitably, this meant that in their early interactions, the students were sometimes uncomfortable in their new selves – believing that in their offline behaviours, they were being forced to live up to the promise of their online personas:", "I don't really post loads of stuff, but when I do I want people to know that I have a social life, and I don't stay in my room all the time, even though I do … We want to paint this perfect picture of our life online, because it's not so perfect in the real world.", "So we feel the need to show-off online to cover up the not so perfect life away from social media.", "(Yolanda)", "But also they felt the need to modify their online behaviours so as not to be misunderstood:", "It's a bit difficult to talk to new people who you don't really know on social media.", "I think you have to try harder because stuff can come across in the wrong way.", "My best friend from back home on Facebook would know the way I mean stuff because we have been friends for 7 years.", "But with somebody I didn't know, you have to be really careful how you say stuff.", "(Maria)", "This was important to our student participants.", "Their SM activities at this time were very typical of what one would expect in the early stages of relationship building, where some self-disclosure plays an important part.", "As Altman and Taylor (1973) have noted, at the start of a relationship, people are more cautious and typically only talk about one or two areas of their life – sticking to fairly superficial conversations until the relationship develops.", "Of course, in these early stages, the opportunities for misunderstanding or misinterpretation are manifold and this impacts on SM usage:", "I felt like in the first couple of weeks you had to sort of justify yourself to people more, and put on more of an act for people.", "And so because of that you are always conscious whether these people actually like you, or whether they're just hanging around with you because you're in the same friendship group.", "(Brooke)", "The students were clearly conscious of the lack of any real intimacy between friends and recognised the need to be careful in their posting.", "Lee et al. (2013) have written about the perceived risks of sharing information on SM in a life transitional context, and we see the same effects in our study, particularly when sharing new personal content on SM at the start of the university year (see also Sleeper et al., 2016):", "Back at my old school, I wasn't really conscious about what I put on social media, because everyone knows me for what I am.", "But since coming to uni I feel I am very conscious about what I now put on social media because of all these new people following me.", "(Kaden)", "There was a clear evidence that participants were biding their time before they felt comfortable sharing personal SM content online – a finding entirely in keeping with the literature on friendship formation and the social penetration model (e.g. Altman & Taylor, 1973; Hsu, Ju, Yen, & Chang, 2007; Utz, 2015), which suggests some hesitation about making important disclosures in the early stages of friendship.", "In the later weeks, we began to see new patterns of disclosure, as the students began to share more about themselves.", "I was just so much more relaxed [in the later weeks] and enjoying my time at uni.", "And this was also the first week where I uploaded uni photos on Facebook, which was quite a big thing for me, and the fact that it took me until week 5 to upload anything, I think just shows how difficult my journey has been so far and how long it has taken me to settle. [", "So] it definitely shifts to more university related stuff as you work through the boards.", "So at the start I put up a Instagram of all my friends from back home, but now or in the later weeks, it would be an Instagram of my new flat or my new university friends.", "(Maria)", "The kinds of identity work undertaken by our participants changed over time and some forms of open self-disclosure were slower to emerge.", "This was clearly the case for a student who suffered from anxiety and depression:", "I wasn't that bothered about my flatmates knowing that I had depression, but I didn't want them to feel that I was a burden.", "I didn't want them to be like, “Are you okay?”", "and making me food and stuff.", "So I was putting it on [Tumblr and my blog] instead so that they thought that I was okay. [", "But more recently] there is a lot more stuff on Facebook about depression and anxiety.", "Like I feel a lot more comfortable posting about it on there now.", "(Samantha)", "Samantha's use of different forms of SM for different purposes illustrates the transitional period.", "Before forming strong offline relationships, she restricted her posts about her struggles to a less visible platform, but once she had assimilated more into her new environment, she was more comfortable using Facebook to do this.", "Throughout the study, Facebook was seen as a performance zone.", "Thus, it was important to announce one's new student identity on SM.", "The delay in many of our respondents fully expressing their new life through SM platforms was due to this process of assimilation where they were managing their transition from their old to new lives.", "Facebook seemed to be used less as a tool to aid transitioning, but more of a signal of assimilation, while some other SM platforms were used as aids to support students through a more difficult transition (such as Twitter, Tumblr, or WhatsApp).", "Other identity changes were more commonplace.", "For example, many of the students recognised that the transition to university was, in part, about creating a new ‘student identity’.", "For many, this was an important status change that should be reflected in their SM profile:", "If you list something on your Facebook page, like your relationship status then it definitely feels more official, and it's the same when you list your educational status.", "So saying I am a studying at the University of [anon] felt more real because you are like telling the whole world ‘I'm officially a student now’.", "(Yolanda)", "However, participants had different views on what it meant to be a student and also these perceptions were subject to change during these early weeks.", "For some, being a student required a strong responsibility to focus on their academic studies:", "Having that first assignment, making you going to the library and search for articles and see how it was the student life and everything.", "I think the third week was the week that I started to feel like a student.", "(Lauryn)", "The first time I entered a lecture room I was like, “This is serious.", "You're part of this life now”.", "(Alexis)", "Others saw the value in joining or creating new university societies in order to feel included within university life:", "I started my own society in the fifth week with my flatmate, which is basically a new dance group society, where people can learn how to dance Hip Hop.", "So we organise classes and socials and nights out and stuff.", "We also had to create a Facebook page for it, a Twitter page, and just generally advertise what we do.", "So yea it was pretty cool to run my own society and get involved with university life that way.", "(Kaden)", "Interestingly, however, for some respondents, joining the ‘student’ community required demonstrating signs of maturity and gaining independence as an individual.", "For example, eating healthy and preparing your own food was indicative of being successful at university as well as showing signs of integration:", "When I started cooking on my own, I wasn't that good and I didn't know what to do or what to put in food.", "But I did get better and I was really proud of the meals I cooked here, so I was taking pictures to send to my mother back home.", "I wanted to show her that I am keeping healthy.", "I think it's a sign on my independence and me growing up as a person, saying to back home that I can survive on my own.", "(Nicole)", "When you think of food and uni, you think of kebabs and chips at three in the morning.", "But we were all very keen to cook food properly, discussing [on SM] what recipes would work.", "When you post about it, you're saying to your parents ‘I can cook, I am doing it, I am adulting properly, I am settling in at university, and I can take care of myself’.", "(Brooke)", "Finally, some of the ‘identity work’ was about the transition to a new city or even new country.", "I think everyone has this ceremonious “I have moved to my new city” on their Facebook.", "I think it is a nice thing to do to say, “I have moved and be proud of me.”", "(Charlie)", "For the international students, this transition had its own particular challenges.", "For example, they would often face communication barriers in real time and many felt they could be more articulate on SM, where the asynchronous nature of the communication allowed them to take care in self-expression:", "Social media is one of the easiest things where you can find new friends, especially for foreign students.", "Because you can call people, message them, and you can make things grammatically correct before you send them stuff.", "(Michelle)", "Thus SM was seen as offering the opportunity to alleviate potential awkwardness in future face-to-face situations (Indian & Grieve, 2014).", "For non-UK students, it was very much about trying to adapt as quickly as possible, which meant learning to adopt new SM practices commonly used in the UK to integrate into a new community:", "I was quite wary about using Facebook because I'm not used to it and I didn't really know what I was doing.", "So I was quite conscious as to what people are going to think about me if I share something that's perhaps not funny even though I find it funny.", "(Ruby)", "[SM usage] definitely increased since I have moved to university, especially Facebook.", "Back home in Germany all my friends didn't really use Facebook, we would just text each other, but here it's just the main source of communication.", "When I joined the different sports clubs all the detail was posted online and you had to sign up for everything online.", "And because of that everyone else is also on Facebook connected to these groups, so it became a very good channel of communicating, meeting up with people, knowing when classes are on, stuff like that.", "(Anna)", "Some of the students claimed they still posted in their native language, but others quickly realized that in order to integrate, they would need to post to SM in English:", "The language also changed because I used to post in Chinese but when I moved to the UK, I posted in English.", "(Patience)", "Yes, and the thing is, that we are talking in Lithuanian and Polish language and you would actually not understand anything so it was pointless to post it.", "(Nathan)", "These overseas students identified Facebook as an important means of adapting to UK university community culture.", "Both home and overseas students recognised that they still had two audiences, but overseas students were more likely to use different SM platforms for these different audiences so that they could target their communications more effectively:", "I used Path as a way to keep in touch with friends back home, but nobody here knows what it is … It's a bit like Instagram and Foursquare- it's really big in Asia.", "It was a great way to keep in touch with my Indonesian friends, particularly early on when I first moved to [university] and I wasn't quite settled.", "But gradually I found myself using more Facebook, Instagram, and Snapchat to communicate with my friends here.", "It was pointless to use Path because nobody knows what it is.", "(Ruby)", "As we have seen, the students spend a significant amount of time adjusting to their new lives at university, utilising on and offline resources in order to establish a new identity that would reflect who they wanted to be in this new environment.", "In this section we consider how the students began to think about how to integrate their old and new lives.", "The data presented here speaks of the students' need to disclose their ‘true’ identity, and their desire to reconcile the needs of people at university and those left at home.", "This results not in one single identity, but two- and the following quotes demonstrate how students come to be more comfortable with managing the two identities across audiences at home and university.", "Sometimes this was simply about addressing the pragmatics of things like time-differences.", "International students, for example, realized that they needed to think about more than which platform to use to connect with friends and family, but that different time zones also posed a challenge when trying to integrate old and new lives:", "I have to think about the time difference between here and Hong Kong.", "So I want to make sure that if I do post something that my friends back home can see it and its not lost at the bottom of a newsfeed, where it won't get many ‘likes’.", "So coming to the UK did make me think about what I should post and when.", "(Patience)", "At other times this process was about selectively sharing aspects of the new student identity.", "As we've seen, using SM to showcase their new academic life, acquiring a job, forming part of a university society, the ability to cook, signs of maturity and independence, and so on, was crucial to inheriting a student identity, and indicative of maturing as an individual.", "It was also important to communicate this new transformed identity with connections back home, so as to reassure them about successful integration:", "I'm from Manchester so my scenery changed completely and that was a photo [of the Quayside] to summarise where I live now and to show everyone back home … My mum was distraught for a good month so I tried to post as many pictures as I could so she could see I was having a good time because her worst thought was me just sat in my room by myself crying.", "I think my mum enjoyed seeing stuff like this because she realised that I was okay and I was going to be okay.", "(Lena)", "We also see evidence that the students will edit their SM in order to show more conservative posts to those back home:", "I don't like posting pictures from nights out, because I don't like my family seeing them.", "Because I don't like them seeing me in states like that, so I keep those things on my phone.", "I will post a picture if it's family friendly, on Instagram.", "A lot of my family doesn't approve of drinking.", "(Rachel)", "This presentation of different elements of self to different audiences is, as we've seen, well rehearsed in Goffman's work, but it also reflects the way that disclosure loyalties shift from family to peers in adolescence (Guerrero & Afifi, 1995).", "While many of our participants were performing happy, healthy adulthood to reassure friends and family at home, other participants adopted different strategies in order to engage with the new student community.", "One student in particular deliberately performed a change of persona on their SM profiles to construct their imagined student identity:", "Before this first week, I was just a normal person on social media.", "I posted pictures of my cats and stuff, but then when I came to university that died down quite a lot and I sort of dropped that image of me.", "I wanted people to think ‘oh she is someone I want to go out on a night out with’.", "You can see it's all nights out, pre-drinks I went to and partying in clubs and stuff.", "I wanted people to think that I was the life and soul of the party.", "I don't think I posted anything on Facebook that was about home because I didn't want to come across as boring.", "(Sara)", "Here, Sara imagines a successful student community in conjunction with partying, socialising, and utilising SM to increase her social capital (e.g. Ellison et al., 2007; Wellman et al., 2001).", "In doing so, she drops her family side completely, and utilises SM technologies to keep her new and old communities separate, which also resonated with another participant:", "For the first week my parents said, “For the first two or three weeks you shouldn't call us, you should try your best to get away from home life.”", "I have completely gone against that now, but back then I did spend the first few weeks not really talking to people from home.", "(Charlie)", "These forms of self-censorship are understandable, but they are conducted at some psychological cost and we can see in the later weeks of our study that participants then struggled to reconcile different versions of themselves as they became more comfortable with friends.", "We only really began to see hints of successful integration when there was offline integration – e.g. when participants talked about uniting their friends from home with newly made housemates- usually by inviting old friends to stay at university:", "One of the main things that happened that week was my friend from Edinburgh came down for the weekend.", "It had only been two weeks and we met again.", "We went out to a few bars.", "It was just nice to see an old friend again.", "I put those on Facebook just to show that I am still friends with my old friends.", "I like doing that old and new thing.", "(Charlie)", "That fourth post is about my best friend coming up to see me for the first time.", "We're just saying we can't wait to see each other, because we were texting every day and ringing each other every single day, but it's not the same as being with them.", "(Abigail)", "Very occasionally, we would see our students thinking ahead about the need for integration in the longer-term.", "One example is particularly salient – and concerns a student who was struggling to deal with the death of a sister and suffered from anxiety and depressive thoughts.", "She found the opportunity for honest SM disclosure in advance of meeting new people to be a huge positive:", "My sister passed away a couple of years ago and I always find it's best to tell people before I meet them.", "But sometimes people find it really awkward when I just randomly bring it up.", "So I try and tell people beforehand, because then it gives them a chance to process it themselves.", "I find that I'm a lot more eloquent online.", "I might have found it hard to say to people in person … “Oh, yes, my sister passed away a couple of years ago.”", "Whereas online it gives people time to reflect on it and stuff.", "(Samantha)", "Most students welcomed the convenience of SM in making introductions, but also worried about some of the social comparison processes taking place online.", "Here we found a very damaging cycle for any student who was struggling in the early weeks as they felt both the need to ‘look happy’ in their own posts, but were then inundated with similarly ‘happy’ posts from others.", "Particularly notable was the feeling that they couldn't share their true feelings online:", "For me social media was really really helpful during the transition and meeting new people beforehand and getting rid of that awkwardness when you first meet one another.", "But you do have to make sure you're not totally wrapped up in social media, because I felt for quite a lot of people, they were trying to maintain this pretense of looking like they're having fun, but actually they're not.", "(Brooke)", "So usually I just post things where I'm feeling happy, like I'm going out for dinner, I'm hanging out with my friends, but I don't really post stuff when I'm feeling down, because I don't want people back home seeing that … I felt that I needed to show-off to my friends back home to show that I'm having an amazing time.", "(Ruby).", "It is interesting to note such pressures, particularly in light of research that suggests that upward social comparisons (the perception that other people are doing better than oneself) might be associated with depression and feelings of failure (Feinstein et al., 2013; Jang, Park, & Song, 2016; Steers, Wickham, & Acitelli, 2014).", "Almost without exception our students felt the need to pretend that they were settling in well, even when they were experiencing difficulties including homesickness and anxiety about making new friendships, dealing with difficult flatmates and coping with the demands of their course.", "Some specific examples will illustrate this point.", "Firstly, let us consider Maria, who had briefly dropped out of university due to problems with her new housemates, before re-registering and moving into different accommodation:", "I was very reserved about putting stuff up about missing home.", "I did occasionally, but on the whole I refrained from doing that, especially in fresher's week even though I was missing home like mad … I just didn't want my family back home to be worried I guess, but then not being able to talk about it on social media made it worse and you felt even more isolated.", "I momentarily dropped out, pretty much stopped using social media altogether.", "I didn't want this sort of stuff being seen on Twitter or social media generally for that matter.", "(Maria)", "We see a similar story with Sara, who also experienced accommodation struggles and experienced a number of anxieties about being judged by her close peers:", "I represented myself, especially on the Facebook posts, as someone who I'm maybe not when you get to know me as a person.", "The majority of my boards are from posts on Facebook and me going on nights out and partying and portraying myself as that sort of person.", "It didn't show that I didn't get into any accommodation on campus, and it didn't show that bad side and that my accommodation was awful.", "I don't think I wanted other people to see me struggle and I didn't want other people to think that of me ….", "Especially to distant family members and even my parents, I didn't want them to see me struggle.", "I just wanted them to check on Facebook and see that I'm having fun.", "(Sara)", "Not all of the struggles were tied to accommodation.", "In some cases students experienced problems with the academic aspects of their work, or struggled to make friends with others on their same course.", "Two students provide a case in point - Rachel and Brittany, both of whom struggled to integrate with coursemates, although for different reasons, Rachel because of the lack of an offline group that was course specific and Brittany because she'd changed course after the first week at university and struggled to integrate subsequently:", "I didn't really feel connected to my course mates, because we didn't have a society, like the Law Society, so I didn't feel like there was any way for us to meet.", "I wished that we had a society at the start of fresher's, because I think I would have felt more comfortable with my course mates.", "(Rachel)", "It was really rushed to find a new course.", "I was also quite sad about leaving some of the friends I'd made on the [old] course.", "So I remember that week being really stressful and kind of tainted my time at [university].", "I think I was ringing my parents at like three in the morning … So I decided to go home the weekend after to talk to my parents … [But] I still don't think I've settled from an academic perspective … If I'd done another [Pinterest] board of the last week, I think it would be all of home … I don't know many people because I joined the course late and everyone has formed their own little friendship groups.", "(Brittany)", "This raises some interesting questions about the extent to which SM can only really support assimilation and integration processes if the offline processes of community building are working well.", "When we consider both accommodation and course integration, it becomes clear that those students who are lucky and placed with a compatible group (liking those they live with or those they sit with in class) generally do well.", "The others often struggle to repair an early setback and SM doesn't seem like the right tool to fix these problems.", "In many cases some kind of face-to-face intervention is required, but not always available.", "In addition, for some students, SM can actually act to hold back the process of assimilation:", "Social media has actually made it worse at times.", "My friendship group at home has a big Facebook group chat so they are all always chatting away on this group about what they're getting up to in [city].", "So when they post pictures of being all together it's just a constant reminder of what you're missing at home.", "(Brittany)", "This is when my boyfriend came up to visit me for the first time.", "So he was really wanting me to hammer home to people on social media that I had a boyfriend.", "So I put quite a lot of pictures up.", "I was trying to do well in university and still maintain my social relationships back home and it was really hard to balance.", "(Samantha)", "Clearly, in times of transition and particularly for those experiencing difficulties with that transition, SM has proved to be a double-edged sword.", "That said, we should note that not all forms of SM were viewed in the same way.", "For example, while Facebook could sometimes exacerbate feelings of loneliness and isolation, some students found that Twitter offered an outlet that allowed people to be more open and honest:", "I was struggling with my emotions quite a lot- whether I wanted to be here or not.", "I tweeted it.", "Most of my closest friends are on Twitter.", "They are having the same struggles as you.", "So I think being able to look and see people tweeting, seeing people say the same sort of things as you, you know you're not the only one struggling.", "(Alexis)", "Interestingly, Facebook and Instagram provided greater challenges in terms of the way they collapsed audiences and blurred the boundaries between family, friends, work colleagues, and so forth.", "We found that the students were more careful disclosing private matters or personal worries and concerns on those platforms.", "However, for more serious matters and potential emergencies, our students inevitably turned to offline interactions for immediate support.", "We began our study with a focus on two ways in which SM may have a strong role to play for students making the transition to university.", "Firstly, we talked of the kinds of identity work at play in SM and the comparison processes that can undermine or support an individual who is judging their place in the new social order.", "Secondly, we talked about the role of technology in community building and reflected on the ways that students are encouraged to develop and share a SM presence in the weeks prior to coming to university and in the period thereafter.", "Our study also set out to explore the demands of balancing online and offline communities, and the management of ‘context collapse’ on SM for new university students.", "In pulling together the thematic analysis we were struck by the importance of different stages in the transition to a new community and described in some detail the way SM was used to support three different stages of affirmation, assimilation, and integration.", "These phases have been identified as prevalent when people cross borders or cultures, as in the example of expatriates living abroad (Mao & Shen, 2015), and we can see similarities between themes in the literature on people coping with acculturation- the maintenance of connections to the past, as well as assimilation with new environments (see Lingel et al., 2014; Zhang, Jiang, & Carroll, 2012).", "In the following section, we will say a little more about how identity work and community building plays out across the three stages of affirmation, assimilation, and integration.", "Firstly, we can see clearly that the need to both affirm identity with a previous community and to create a new identity and loyalties to a new community can create a number of tensions.", "In related work, young adults expressed concern with building and adding to an online persona, and the implications for shaping digital identity (Orzech et al., 2016).", "We are reminded here of the work of Goffman (1959) in articulating the need for an individual to perform on different and discrete social stages and the way that this then translates in SM terms into the problems we experience in managing different aspects of self when different SM worlds collide (Marwick & Boyd, 2010).", "Indeed, impression management was evident for our new students, and caused them to think about their use of SM in the lead up to, and the first few weeks of student life, to ensure they posted or tailored their details to convey a particular impression to others- even if this impression was not of their authentic self (Schoenebeck et al., 2016).", "However, the motivations for such impression management were complex as our participants wanted to affirm connections to their previous life while assimilating into their new life.", "They carefully managed their belonging to their new place through photographs of their new city or university as well as changing social media to confirm their student status, while taking care over their posts involving other people.", "One thing that emerges from our own study is the way that the temporal dimensions of context collapse are changing.", "So, for example, it is relatively new for universities to encourage fresher's to enroll on their SM platform to learn about their new housemates or course colleagues in advance.", "Does this ease the transition to university?", "In some ways it may help to reduce uncertainty, but it also brings social comparison processes forward, beginning before the university term starts.", "We can see clear evidence, in our own data, that these comparison processes are a little fraught.", "At a time when individuals should be celebrating and affirming the life they currently lead, they are also trying to ‘tweak’ their SM profiles such that they look a little better to an outsider.", "A single identity is no longer feasible or desirable (Belk, 2014) and the celebration of an established life is tainted with the thought that this might not stand up to scrutiny.", "This can result in anxieties tied to the presentation of self - a phenomenon already seen in offline communication (Baumeister, Tice, & Hutton, 1989), but now emerging online (Grieve & Watkinson, 2016).", "Note that this process is similar to, but also markedly different from the process of online dating, whereby people can review the SM profiles of other people before meeting face to face.", "Online dating is also fraught with identity management issues (Zytko, Grandhi, & Jones, 2014), but at least it gives participants the option to ‘opt-out’.", "However, our students cannot alter the outcome of these early peer ‘previews’ - their course mates and the roommates are already fixed and an ultimate face-to-face relationship is inevitable.", "In the examples where we do see tensions arise with living situations, the result may be at best a change of residence, and at worst, a factor in the decision to leave university.", "In subsequent weeks, where students are seeking to integrate with their new communities, the management of two disparate SM identities takes on a different challenge – as people try to play to at least two audiences upon one stage.", "Again, there is a temporal component to this.", "For international students, the challenge of balancing home and university identities is helped by the fact that they are acting in two different time-zones and we found many examples in which students would carefully consider the time of day that posts were made in order to play first to one audience and then the other- something which has caused exhaustion for others in studies of cultural transitions (Lingel et al., 2014).", "We note that whilst we use the term ‘social media’ to describe the kind of platforms students use to manage their online identities, it is clear from our data that different SM sites are employed strategically in different ways.", "As we have highlighted, Facebook was often used as the broader platform to reach out to a wider audience once comfortable with new friends, whereas more nuanced SM such as Tumblr and Twitter allowed students to ensure different audiences received different information in times of crisis.", "For all students, the transition to university involved some self-editing and this was accompanied by a range of anxieties about whether or not they would fit in.", "Early impressions did not always reflect the ‘authentic self’ and the pressure to appear successful and happy brought its own pressures.", "Recent work exploring self presentation on Facebook has highlighted there are psychological benefits to being authentic on Facebook (Grieve & Watkinson, 2016; Reinecke & Trepte, 2014), yet we saw a number of disparities between participants' true selves and their Facebook selves, possibly affecting psychological health.", "Many of our participants were lonely or homesick at points during these early weeks and for some, particularly those who had unsuccessful accommodation or course experiences, these feelings were acute.", "Yet few felt that they could meaningfully share this with others.", "There is an emerging body of research which focuses on transient loneliness (e.g. Lawson, Vines, Wilson, Barnett, & Barreto, 2014) dealing with those, such as university students, who are temporarily separated from their home community.", "We feel that further work in this space would be worthwhile as the success of such a transition may contribute to feelings of loneliness in young people at a time when they are at their most vulnerable.", "From our work we are now beginning to question whether the university induction model, as it stands, is the most appropriate for young people who heavily rely on SM for guidance and making friends.", "We recognise there are some limitations to our study, and also avenues for future research.", "Our method to engage with participants by firstly asking them to curate their experiences on a Pinterest platform was novel, and ensured that our data were not tied to any one specific SM account or type of action and that our interviews were based on exchanges that genuinely took place during the six week transition period (i.e. were not entirely retrospective).", "However, the interviews themselves did take place 4 months after the start of university, and relied on students own curation of their most ‘valuable’ social media posts’- it may be helpful in future studies of this type to recruit participants to ‘pin’ their SM content at the start of the period of interest rather than ask them to pin retrospectively.", "In the next stage of our work we will use this analysis to design a set of near-future university induction scenarios.", "We plan to use these in a series of workshops that will help us to further explore digital solutions to the issues facing new students.", "We also plan to collate SM use in the final six weeks of university where students may be preparing for yet another transition – to the world of work or further study.", "In conclusion, this study contributes to a growing body of work on the ways that young people use SM to manage their identities- at a stage where they are undergoing a period of adjustment and identity management.", "In recent years, this work has started to show that young people are quite accomplished at juggling identities and maintaining their privacy, although they are still vulnerable when there is context collapse between online and offline selves or between different versions of self on different SM platforms.", "We found particular social pressures at play in the presentation of self during times of transition- most worrying of these was the sense that the ‘authentic self’ could not emerge too early in the process of transition.", "We found a number of young people struggling to make the transition work, but not having the personal resources available (online or offline) to seek help within the new community." ]
[ "We explored the experiences of first year students transitioning to university.", "We assessed their self comparison and community building via social media use.", "Participants collated social media data using Pinterest.", "We identified three phases of transition- affirmation, assimilation, integration.", "We recommend further work to explore loneliness in this unique population." ]
Survey data on e-Procurement adoption in the Nigerian building industry
This article is a description of data related to the research article entitled “Factors influencing e-Procurement adoption in the Nigerian Building Industry” (Ibem et al., 2016) [1]. The data were derived via a questionnaire survey involving 213 participants comprising architects, quantity surveyors, builders, construction/project managers, and engineers working in consulting and contracting firms, private client organisations and government agencies. The survey was conducted in Nigeria between June and November 2015. The data set contains responses on the levels of awareness and extent of use of the different e-Procurement technologies, the barriers to the uptake as well as the factors influencing e-Procurement adoption by organizations in the Nigerian building industry. The survey data and results of the analysis are made available in this article for further use by researchers; and for the purpose of improving understanding of the key findings of the survey.
[ "MULT" ]
[ "Building industry", "Nigeria", "Survey", "e-Procurement" ]
[ "1000" ]
[ "The data set contains responses obtained from a questionnaire survey of architects, builders, quantity surveyors, engineers, construction/project managers and procurement officers working in consulting and contracting firms, client organisations and government agencies who are directly involved in the procurement of building, materials, equipment/machineries, works and services in Nigeria.", "The questionnaire used was designed to elicit responses from the aforementioned stakeholders on a number of key issues related to e-Procurement use in the Nigerian building industry.", "Specifically, the data set shared in this article captures the responses of the participants on several issues, including (i) the respondents’ profiles and that of their organisations (ii) the respondents’ level of awareness of e-Procurement in construction (iii) the proportion of users to non-users of e-Procurement amongst the respondents in the survey (iv) the extent of use of the different e-Procurement technologies at the design, tendering and construction phases of building projects (v) barriers to the uptake of e-Procurement; and (vi) the technological, environmental and organisational factors that influenced the extent of adoption of e-Procurement in the Nigerian building industry.", "Analysis of the data (responses provided by participants in the questionnaire survey) can provide fresh insight into the aforementioned six key aspects covered in the survey of key stakeholders in the Nigerian building industry.", "Appendix A contains the sample of the questionnaire used.", "The SPSS file and results of the analyses of the responses are also shared in this article.", "As stated earlier the data set shared in this article was sourced via a cross-sectional survey of key stakeholders in the Nigerian building industry.", "Previous studies [2–6] have adopted similar approach in obtaining empirical data from construction industry stakeholders.", "A structured and pre-tested questionnaire was used to elicit responses from architects, builders, quantity surveyors, engineers, construction/project managers and procurement officers in consulting and contracting firms, client organisations and government agencies who are directly involved in the procurement of building projects in Nigeria [1].", "A sample of the questionnaire used is presented in Appendix A.", "A careful examination of the questionnaire reveals that it has three main sections.", "Section A was used to extract data on the professional roles of the respondents and their organizations, their levels of awareness of e-Procurement in construction, and the extent of use of the different categories of e-Procurement.", "Section B had questions on 29 different factors considered important in the decision to adopt e-Procurement by the respondents’ organizations.", "The data were extracted using 5-Likert type scale, where 1 is for “Not Important”; 2 is for “Least Important”; 3 represents “Undecided”; 4 is for “Important”; and 5 represents “Most’.", "The last part of the questionnaire -Section C, was used to gather data on 26 factors that have adverse impact or are barriers to e-Procurement adoption in the organisations.", "Similarly, data on the barriers were derived using 5-Likert type scale, where 1= ”Has No Significant Effect”; 2 = “Has Very Little Effect”; 3= “Undecided” 4 = “ Has Significant Effect” 5 = “ Has the Most Significant Effect”.", "Before the main survey the questionnaire was pre-tested among 30 purposively selected respondents in architecture and quantity surveying firms, as well as client organizations in Lagos southwest Nigeria.", "Feedback from the pilot survey was used to fine-tune the final version of the questionnaire used in the main survey.", "In addition, the reliability of the scale of measurements used in Sections (B and C) of the questionnaire was investigated using Cronbach's alpha coefficient test.", "The tests returned Cronbach's alpha values of 0.851 and 0.834 for items in Section B and Section C, respectively.", "Since these values are more than 0.7 recommended in the research literature [7], the questionnaire instrument was considered reliable and consistent in measuring what it was designed for.", "Part of the data set shared in this article was extracted from respondents in architectural firms in June 2015 during the 2015 Architects’ Colloquium in Abuja.", "The data from the builders were collected at Annual Builders’ National Conference and Meeting at the University of Ibadan in August 2015, while another part of the data set was generated from a survey of Quantity Surveying firms who participated at the Annual QS Research Conference at the Federal University of Technology, Akure in November 2015.", "In addition, client organizations, comprising government agencies, multinationals (e.g. oil and gas, telecommunication, manufacturing and building construction companies) in Lagos, Abuja and Port Harcourt were also purposively selected to participate in the research.", "In the administration of the questionnaire, the respondents were randomly selected; and to ensure that only one respondent from an organization was included in the survey, the respondents were asked to provide the names and locations of their organizations.", "Although, 500 questionnaires were distributed, 213 valid questionnaires representing around 43% of the total number of questionnaires distributed were retrieved and found useful.", "The data were analysed with the help of Statistical Package of the Social Sciences (SPSS) Version 20." ]
[]
Aerial river management by smart cross-border reforestation
In the face of increasing socio-economic and climatic pressures in growing cities, it is rational for managers to consider multiple approaches for securing water availability. One often disregarded option is the promotion of reforestation in source regions supplying important quantities of atmospheric moisture transported over long distances through aerial rivers, affecting water resources of a city via precipitation and runoff (‘smart reforestation’). Here we present a case demonstrating smart reforestation's potential as a water management option. Using numerical moisture back-tracking models, we identify important upwind regions contributing to the aerial river of Santa Cruz de la Sierra (Bolivia). Simulating the effect of reforestation in the identified regions, annual precipitation and runoff reception in the city was found to increase by 1.25% and 2.30% respectively, while runoff gain during the dry season reached 26.93%. Given the city's population growth scenarios, the increase of the renewable water resource by smart reforestation could cover 22–59% of the additional demand by 2030. Building on the findings, we argue for a more systematic consideration of aerial river connections between regions in reforestation and land planning for future challenges.
[ "AGRI", "ENVI", "SOCI" ]
[ "Bonn Challenge", "Moisture recycling", "Precipitationshed", "Reforestation sites", "Water scarcity" ]
[ "1107", "2308", "2309", "3305" ]
[ "Reforestation has been one of the most active initiatives to mitigate global climate change impact.", "Being potentially a useful tool to sequestrate atmospheric carbon, it also presents co-benefits such as improving status of biodiversity loss and enhancing ecosystem integrity (United Nations Framework Convention on Climate Change (UNFCCC, 2013).", "These co-benefits have been included as objectives of several international agreements addressing those issues, e.g. the Aichi Targets (Convention on Biological Diversity (CBD, 2010) and the Bonn Challenge (Bonn Challenge, 2019).", "However, an undesirable effect of such intervention is the trade-off with downstream water availability (Connor et al., 2016; Cunningham et al., 2015; Farley et al., 2005).", "A dramatic decrease in river runoff is normally observed downstream of the reforestation sites compromising water supply and other ecosystem services from the river (Filoso et al., 2017; Jackson et al., 2005).", "This has become a major factor of low societal acceptance impeding reforestation projects and invoking conflicts (Cao, 2011; Cao and Zhang, 2015) which poses challenges for local implementation of such an intervention despite the top-down forces.", "However, a usually neglected aspect of reforestation is that it can also enhance water availability through invisible aerial river connections (van Noordwijk et al., 2014; Ellison et al., 2017, 2018).", "Similar to surface river networks, aerial rivers (preferential pathways of moisture flows in the atmosphere; Arraut et al., 2012) connect regions, often across administrative borders and topographic watersheds.", "Upwind land activities govern evapotranspiration (Gordon et al., 2005; Silvério et al., 2015), the moisture input to terrestrial aerial rivers, and influence precipitation downwind via atmospheric circulation (D’Almeida et al., 2007; Ellision et al., 2012; Pitman and Lorenz, 2016; Spracklen and Garcia-Carreras, 2015).", "Through the hydrological cycle, this influence propagates to rivers and groundwater, thus impacting water availability (Bagley et al., 2012; Coe et al., 2011; Lima et al., 2014; Swann et al., 2015; Ramírez et al., 2017; Weng et al., 2018).", "Reforestation in general enhances evapotranspiration resulting in more water loss at the catchment scale compared with non-forested land cover (Brown et al., 2005; Dean et al., 2015; Farley et al., 2005).", "However, released from the land surface, these moisture fluxes to the atmosphere are important inputs of continental aerial rivers (Gordon et al., 2005).", "Through this mechanism, reforestation strengthens the delivery of water to downwind regions.", "A prerequisite of integrating this concept in land and water management is an assessment on how relevant it is for downwind water availability.", "Since aerial river connections are not directly observable, utilization of scientific tools are required to recognize connections between upwind and downwind regions (Ellison et al., 2018).", "Via numerical modelling or isotopic tracing it is possible to reveal the aerial river network connecting regions.", "Utilizing numerical modelling as a tool, we quantify the effect of strategic reforestation in the upwind regions on those downwind via aerial rivers, thus exploring the potential of the latter as a water resource management option.", "Expanding on a previous study’s methods of structuring aerial rivers (Weng et al., 2018), we use the results of the WAM-2layers moisture back-tracking algorithm (van der Ent et al., 2014) to identify the most important upwind regions influencing precipitation in our study region, Santa Cruz de la Sierra.", "Based on a Eulerian approach, the model traces the origin of rainfall using the water balance principle and an assumption of well-mixed atmosphere in two vertical layers according to given input data.", "The WAM-2layers was shown to agree well with other moisture tracking approaches in the Amazon region while having lower computation cost (van der Ent et al., 2014; Zemp et al., 2014).", "For our analysis, we use a simulation experiment (MOD experiment, described in Zemp et al., 2014) that utilizes observation-based climatic input data (precipitation, evaporation, humidity and wind field) spanning 2000-2010.", "By averaging annual means of the simulations, we get a network quantifying the contribution of evapotranspiration from each grid cell (1.5° x 1.5° longitude and latitude) to rainfall at the Bolivian economic capital, Santa Cruz de la Sierra.", "This network can therefore be used to determine the precipitationshed (upwind surface area providing evapotranspiration to a specific sink area’s precipitation; Keys et al., 2012, 2018) of the city.", "It is known that for a given location, its upwind regions can have different influences on its water resource through the aerial rivers (Keys et al., 2014; Weng et al., 2018).", "For planning purposes, it is actually possible to identify influential upwind regions which have greatest impact influencing a given region’s water availability.", "We investigated a reforestation intervention in these important upwind regions of Santa Cruz de la Sierra to estimate the optimal potential of such an intervention as a water management option for the city.", "In order to do this for our example location, we rank the regions’ importance in contributing to rainfall of Santa Cruz de la Sierra and outline the Most Influential part of Precipitationshed (MIP) of the city (see the blue region in Fig. 1) by a boundary designating the smallest area which contributes 40% of the total continentally recycled precipitation in the city.", "This threshold was proven applicable in reflecting the most important moisture source regions for assessing land use change impacts on aerial rivers (Weng et al., 2018); also see Appendix A. for more details outlining the MIP.", "Santa Cruz de la Sierra in the Plurinational State of Bolivia, is one of the world’s most rapidly growing cities (annual population growth rate 3.7% between 1992 and 2012; Trohanis et al., 2015) and the home of 1.4 million residents (INE, 2012).", "Migration flows, the main reason of the city’s growth in the past decades, are expected to persist.", "Though the city has the highest coverage of potable water in the country, the current groundwater resources that the city relies heavily on are under stress with a continuous deepening of modern recharge front and deterioration in quality (Morris et al., 2003).", "Water availability for both the growing population and peri-urban agriculture is becoming uncertain (Castelli et al., 2017).", "In addition, more frequently occurring severe droughts in the region also intensify the water challenge faced by the city (Erfanian et al., 2017; Jiménez-Muñoz et al., 2016; Marengo et al., 2011).", "Following the moisture tracking and the identification of the city’s MIP, we tested the potential of managing aerial rivers by smart reforestation as an option to ease the city’s water stress.", "Here “smart” refers to the selective decision of reforestation sites (in the MIP) considering their impacts on aerial rivers and thereby on the water reception of Santa Cruz de la Sierra.", "Our smart reforestation scenario in each MIP component grid cell was built according to the restoration opportunities map of the International Union for Conservation of Nature (IUCN)/World Resources Institute (WRI) for the Bonn challenge (Potapov et al., 2011; Maginnis et al., 2014; see Appendix B.) where reforestation potential was assessed by ecological conditions and local land use culture.", "The additional evapotranspiration input from smart reforestation in each MIP cell was determined using the measured evapotranspiration per area of a neighboring forest reference cell (with forest fraction > 95%).", "We then subtracted the evapotranspiration typical of pasture-land (Sakai et al., 2004), which is the major current land-use type in areas selected for smart reforestation, from the reference forest evapotranspiration per area before multiplying the area reforested.", "The measured evapotranspiration used for forest evapotranspiration reference was derived from the Moderate Resolution Imaging Spectroradiometer (MODIS) evapotranspiration product MOD16ET (Mu et al., 2013).", "Thus the additional evapotranspiration input considered is corresponding to the forest structure and age during the observational period of the data (2000–2010).", "We then calculate the newly added evapotranspiration’s influence on the city’s water availability including annual rainfall and runoff reception.", "Changes in annual rainfall could be directly quantified from our networks assuming that changes in atmospheric moisture flow is proportional to changes in the contribution of this moisture flow to local precipitation (Zemp et al., 2017).", "This assumption is justified by a positive relationship between atmospheric moisture and rainfall in the region (Boers et al., 2014) which also implies sufficient supply of condensation nuclei in the region (van Noordwijk et al., 2015).", "In turn, the new runoff budget of the city’s upstream basin (belonging to the larger Madeira River basin) was evaluated by balancing the changed annual basin precipitation and annual evapotranspiration.", "We also analyzed changes in seasonal rainfall and runoff budgets in the smart reforestation scenario based on dry (June, July, August and September) and wet (December, January, February and March) months’ basin precipitation and evapotranspiration, in line with previous studies showing significant seasonal differences in the influence of aerial rivers on hydrological cycle (Trenberth, 1999; Zemp et al., 2014).", "For an operational scheme of the study case, we refer the reader to Appendix C.", "We found that smart reforestation of 7.1 million ha in the MIP region of Santa Cruz de la Sierra lead to an increase of 1.25% in annual rainfall reception of Santa Cruz de la Sierra (absolute rainfall gain 5.86 × 108 m3).", "This scenario further leads to a rise of 2.23% (absolute runoff gain 2.00 × 109 m3) in the city’s annual runoff enabled by the spatial relationship between its upwind aerial river (more specifically the MIP) and upstream surface river basin.", "As shown in Fig. 1, the MIP region of Santa Cruz de la Sierra is located in Brazil (states of Acre and Rondônia), Peru (departments of Madre de Dios), and Bolivia (departments of Pando and El Beni).", "The increase in runoff in Santa Cruz de la Sierra is due to the fact that smart reforestation in the MIP of the city also enhances rainfall in the city’s upstream surface river basin.", "Even more interestingly, the MIP region of Santa Cruz de la Sierra is largely separated from the upstream surface river basin of the city.", "Thus the expected local runoff decrease resulting from increasing evapotranspiration of reforestation in the MIP is not fully experienced by the city’s upstream surface river basin.", "In fact, the precipitation increase in the basin (through the aerial rivers) results in a marked gain in runoff reception of the city.", "The runoff increase is more prominent in the dry season (June, July, August and September) reaching a 26.93% increase.", "In the wet season (December, January, February and March), gains are moderate (1.85%).", "Slight seasonal variation in rainfall increase following smart reforestation is also observed (dry season 1.27% and wet season 1.24%).", "The rainfall and runoff gain of the study region from smart reforestation are listed in Table 1.", "Uncertainties in our estimates may stem from the moisture tracking model, but moisture recycling ratios in the Amazon region estimated from the MOD experiment agree well with other studies’ estimation in the same region using other datasets and other moisture tracking approaches (see the table 2.", "in Zemp et al., 2014).", "We have also validated our runoff estimation in the Madeira river basin with the historical river observation data (Molinier et al., 1996) and found a slight (8%) overestimation.", "This has only a small impact on our estimation of the runoff gain by smart reforestation (-8.6% of the annual runoff gain).", "Our calculation of the reforestation impact on aerial rivers was based on the condition of the transferability of measured forest evapotranspiration from neighboring sites and minor wind field changes after changing land cover (Bagley et al., 2014).", "Local convection that may be decreased by a lower local land cover heterogeneity (Baidya Roy and Avissar, 2000; D’Almeida et al., 2006) after reforestation is not considered in our study.", "However, this effect is minor compared to changes in aerial river flows after land-use change in the Amazon (Bagley et al., 2014).", "Potential increase of soil infiltration (Bruijnzeel, 2004) after reforestation are not considered in the runoff calculation, but this is unlikely to affect our results since most of the reforestation sites are located remotely (in the downstream areas or out of the upstream catchment) of the city.", "Our results show that smart reforestation is an option to enhance water supply especially during dry seasons.", "An increase of 26.93% in dry season runoff can be beneficial for sustaining ecosystem (Anderson et al., 2013; Brienen et al., 2015) and livelihood (Castelli et al., 2017) given the past (Fu et al., 2013; Vicente-Serrano et al., 2014) and projected (Marengo and Espinoza, 2016; Seiler, 2013) lengthening and intensification of the dry season.", "The amount of water added to the region by smart reforestation can be used to ease the growing water stress brought about by fast urbanization (Castelli et al., 2017).", "Using the population growth projections implied by different Shared Socioeconomic Pathways (SSP) (Jones and O’Neill, 2016), we calculated the additional water resources needed to secure the current water consumption per capita (Instituto Nacional de Estadíticas de Bolivia (INE, 2017a,b) in the city of Santa Cruz by 2030.", "In the case of withdrawal being proportional to available water resources, our results imply that an early implementation (2020) of smart reforestation can gradually increase the renewable water resource of the city and ultimately cover between 22% and 59% of the additional water demand by 2030.", "Though the city’s current water system does not extract directly from runoff, the already confirmed large dam projects (Ministerio de Hidrocarburos y Energía, 2012) will allow to benefit from our proposed management option.", "Such benefits might be particularly relevant given the fact that the glaciers currently sustaining runoff are retreating under climate change (Rabatel et al., 2013; Rangecroft et al., 2016; Vuille et al., 2018).", "In addition, induced increases in both rainfall and runoff reception will have a positive impact on groundwater recharge which the city currently relies heavily on.", "Smart reforestation therefore has the potential to increase water supply being beneficial in mitigating the stress on the existing water system under both population growth and climate change.", "Additional benefits of smart reforestation include sequestering atmospheric carbon at the reforestation sites (Don et al., 2011; Post and Kwon, 2000) and sustaining ecosystem integrity (Coe et al., 2013).", "Our results add new insights into trade-off between carbon sequestration and fresh water supply (Connor et al., 2016; Farley et al., 2005; Gao et al., 2014), while a win-win situation between those is presented in our case.", "Nationally in Bolivia, under the commitment of Ley 1333, reforestation has been also one of the priority land management targets to preserve ecosystems and their services.", "Under the commitment of Decreto Supremo Nº 2912, there is a Bolivian national target of reforesting 4.5 million hectares by 2030.", "The smart reforestation scenario includes reforestation areas of 7.1 million hectares and is therefore in line with the national target while the Bolivian part covers 45% of these areas.", "A full implementation of smart reforestation will require cross-border cooperation.", "Current implementation of reforestation is mainly planned at the upstream catchments (the Piraí river banks) aiming at the improvement of water quality downstream.", "Nevertheless, following this strategy, tension usually arises from a reduction of water quantity in downstream areas.", "In addition, the feasible sites in upstream catchments are usually limited while most of these areas are not easily accessible for development or are protected.", "Thus, a gap for fulfilling the national reforestation target hectares can be expected if prioritizing this traditional strategy.", "In this context, smart reforestation might be a good alternative to fill the gap, even more, it might be worth considered before the traditional strategy when aiming for a more water – resilient city of Santa Cruz in the future.", "In order to implement smart reforestation, it will be necessary to negotiate with other departments out of the Santa Cruz department, which the city belongs to.", "It is true that blue water and sediments losses can be expected from the reforestation sites locally and downstream.", "Nevertheless, the friction stemming from compromise in downstream water supply is likely small while those departments (Beni and Pando) and their downstream regions have relatively rich runoff resources.", "The concept of “right tree at the right place for a clear function” (Creed and van Noordwijk, 2018) can be a national strategy prioritizing those areas facing challenges.", "A full implementation of smart reforestation will require international cooperation because a part of the city’s MIP is located in Peru and Brazil.", "This could be feasible when included in the context of both the Bonn Challenge (http://www.bonnchallenge.org/content/challenge) and the Initiative 20 × 20 (https://www.wri.org/our-work/project/initiative-20×20), where the Latin American countries have a common target of reforesting 20 million hectares by 2020.", "In addition, smart reforestation is in line with the Intended Nationally Determined Contributions (INDC) targeting atmospheric carbon reduction which increases individual country’s will to participate.", "A recent call for the regional joint effort mitigating drought may also impose momentum for the joint management on the aerial rivers (United Nations Convention to Combat Desertification (UNCCD, 2017).", "Our case implies that, similar to integrated surface river basin management requiring collaboration between upstream and downstream entities, joint management between the upwind and downwind regions is necessary for the implementation of aerial river management and smart reforestation.", "This requires improved understanding of spatial connections by the aerial rivers (Dirmeyer et al., 2009; Keys et al., 2017) and the effect of land use practices at upwind regions propagating to downwind regions through hydrological cycle (Ellison et al., 2017, 2018; Weng et al., 2018).", "Nationally, an assessment that outlines critical regions (e.g. the MIPs) to preserve aerial rivers will be a precondition of involving relevant regions for cooperation.", "By the time the present study was written, there is no inter-regional or international agreement explicitly governing aerial river connections.", "The Convention on Long-Range Transboundary Air Pollution (United Nations Economic Commission for Europe (UNECE, 1979; Sliggers and Kakebeeke, 2004) might provide a feasible framework to develop on (Ellison et al., 2017).", "Moreover, establishment of bridging organizations can facilitate co-production of knowledge and collaborative decision making between actors (Cash et al., 2006; Olsson et al., 2007; Crona and Parker, 2012).", "Interestingly, different roles in aerial river regime and surface water regime may also foster positive policy environment for cooperation between aerial river sharing entities.", "Take our case for instance, the upwind regions of Santa Cruz de la Sierra are located downstream of the Madeira River, receiving impacts from the city through the surface rivers, but they can exert influences on the city through the aerial rivers since they are located upwind.", "The reverse roles in this case imply that the shared aerial rivers might challenge current relationships between entities established from surface water regime and the according paradigms of land-water management.", "Nevertheless, the aerial rivers are indeed the key to bring out a more holistic one of those.", "To sum up, by taking advantage of a model recognizing teleconnections through the aerial rivers, we have exposed the potential of smart cross-border reforestation as a water management option mitigating challenges of future population growth and climate change.", "Through transportation by aerial rivers, atmospheric water added by smart reforestation is collected and delivered to the downwind region, presenting potential in increasing both rainfall and runoff (especially in the dry season) in our study region, and certainly beyond.", "Different from traditional upstream catchment approaches, smart reforestation projects attest the possibility of breaking the usual trade-off in reforestation projects between carbon sequestration and fresh water supply by enhancing both for specific target regions.", "Prioritization of smart reforestation projects in the important upwind regions of those experiencing or expecting water stress can present significant benefits but be also in line with national and global efforts in reducing atmospheric carbon.", "For example, the smart reforestation project can provide preferable results watering Bolivia’s fastest growing city and at the same time fulfilling the Bolivian national INDC.", "Beyond the study region, a more systematic consideration of the interconnection between the land and water system while planning reforestation projects should be taken.", "Further studies should focus on exploring smart reforestation sites that optimizes the aerial river impacts downwind for such regions.", "The relevance of other land use types e.g. wetlands in aerial river management should be explored as well.", "However, full reception of aerial river benefits from smart reforestation or other approaches will, in most cases, require cross-border cooperation, which is arguably the key to sustainably managing the interrelated systems that underlie a livable planet." ]
[ "We show a case of reforesting smartly to manage aerial rivers.", "Smart reforestation presents potential in increasing water supply for a city.", "This is relevant to the city’s adaptation to climate change and population growth.", "It breaks the reforestation trade-off between carbon sequestration and water supply.", "The aerial river aspect should be considered by forthcoming reforestation projects." ]
Efficient removal of Pb(II) and Zn(II) ions from aqueous solutions by adsorption onto a native natural bentonite
The new Native Natural Bentonite (NNB) for adsorption of Pb(II) and Zn(II) ions from aqueous environments was investigated at 27 ± 10C by in batch laboratory experiments. Chemical and mineralogical structure of the NNB adsorbent was characterized by XRF and cation exchange capacity (CEC). The effect of pH, metals concentration, adsorbent dose, and agitation time were also studied. Langmuir and Freundlich isotherm and the Giles classification isotherm were used for describing the equilibrium data. The results show that the NNB contains silica (SiO2) and alumina (Al2O3) as a major chemical compound. The maximum adsorption capacity (mg/g), based on Langmuir isotherm were 8.55 and 7.90 for Pb(II) and Zn(II), respectively. Pb(II) and Zn(II) removal efficiency was increasing by increasing the initial pH of solutions, adsorbent dose, and contact time. Therefore, the results of this study show that the equilibrium is reached slowly (180 min), indicating the adsorption sites are not well exposed. By increasing the initial metals ion concentration, the capacity of adsorption decreased and the uptake of Pb(II) and Zn(II) per unit weight of the adsorbent (mg/g) increased. The adsorption efficiency of Pb(II) was higher than Zn(II).
[ "BIOC", "HEAL" ]
[ "Adsorption", "Application of a native natural bentonite adsorbent for removal of Pb(II) and Zn(II) ions from aqueous solutions", "Heavy metals", "Lead", "Natural bentonite", "Zinc" ]
[ "1308", "3607" ]
[ "The material used, as adsorbent for this study was a NNB from Sirjan region in Kerman province, located in the south east of Iran.", "First, the NNB was crushed using jaw crusher (Pulverisette, Fritsch, Germany) then they were sieved.", "Particles of less than 50 mesh were selected for tests.", "In addition, the adsorbent was dried for two weeks in the laboratory temperature and air flow [1–3].", "Chemical and mineralogical composition of the NNB was identified by X-ray Florescent (XRF) (PHILIPS PW1730, Netherlands).", "The Cation Exchange Capacity (CEC) of the NNB was measured by Methylene Blue Index, according to the ASTM C 837 -81 [4].", "Tables 1 and 2 represent chemical composition, mineralogical structure, and CEC of the NNB adsorbent, respectively.", "Chemical composition analysis by XRF showed that the NNB contains silica (SiO2) and alumina (Al2O3) as a major compound and other metal oxides, such as Fe2O3, CaO, MgO, Na2O, and K2O as minor compound.", "The adsorption isotherms were studied by mixing 0.5 g of NNB with 50 ml of meal ions of Pb(II) and Zn(II) solutions at concentrations 10, 30, 50, 75, and 100 mg/L.", "The pH of solutions was adjusted in 3.0 ± 0.1 with NaOH and HNO3 concentrations 1.0 N, then the suspension was placed in rotary shaker and shacked for 300 min with speed of 150 rpm [8].", "Langmuir and Freundlich and the Giles classification adsorption isotherms are shown in Fig. 1 and the adsorption parameters are presented in Table 3.", "Generally, according to the results, the adsorption data of Pb(II) and Zn(II) on the NNB were fitted well by the Freundlich and Langmuir isotherm equations, respectively.", "However, satisfactory correlation coefficients were obtained using the later equations for Zn(II)(R2>0.99) and, Pb(II)(R2>0.98).", "In addition, the maximum adsorption capacity of Q0 (mg/g), based on Langmuir equation were 8.55 and 7.90 for Pb(II) and Zn(II), respectively.", "Fig. 2 shows the effect of Pb(II) and Zn(II) ions initial concentration on adsorption efficiency of the NNB.", "The results show when initial metal ions concentration increased, the percentage of adsorption with gentle slopes decreased.", "Moreover, the results show that about 99% of Pb(II) and 90% of Zn(II) were removed when initial metal ions concentration was 10 mg/g.", "The increasing amount of absorbent increases removal of lead and zinc ions from aqueous solution on the NNB (Fig. 3).", "This trend was expected; since by increasing sorbent dosage, two phenomena may occur.", "The first is increasing the adsorption sites; therefore, the adsorption of surface area increased for metal ions [9].", "The second is, as the results show in Table 1, the bulk chemical composition of the NNB is alkali and alkaline earth metal oxides.", "To determine the effect of pH, the tests were carried out using 0.5 g of NNB added to 50 ml Pb(II) and Zn(II) solutions at concentration 50 mg/L.", "pH was adjusted in the range of 1.75-5.5.", "Increasing initial pH of solutions, increase the adsorption of Pb(II) and Zn(II) (Fig. 4).", "Based on the results, at low pH the adsorption is negligible, which is typical of metal cations adsorption by natural clay minerals.", "At low pH, there is a large number of H+ ions in the solution which competes with Pb(II) and Zn(II) ions for active sites on the adsorbent.", "Consequently, the removal of metals decreases.", "By increasing pH the number of H+ ions reduced and then the competition between H+ ions and metal ions for adsorption sites decreased.", "Therefore, adsorption of lead and zinc ions into NNB increased [2,10,11].", "In pH values 2.5 and 3.5 these adsorption percentages increase sharply, reaching almost constant values for higher pH values.", "The dependence on the sorption effect on pH has almost a S-like course observed by other authors as well in metal cations adsorption with clay minerals [12–14].", "The effect of agitation time on the Pb(II) and Zn(II) ions adsorption by NNB was conducted by varying the agitation time (1–300 min) and using 0.5 g of NNB per 50 ml Pb(II) and Zn(II) solutions at concentration 50 mg/L.", "pH was adjusted in 3.0 ± 0.1.", "Other conditions included particle size of <50 mesh, temperature of 27 ± 1 °C, and shaking rate of 150 rpm.", "As Fig. 5 reveals the time to reach equilibrium is slowly (180 min), indicating the adsorption sites are not well exposed.", "Accordingly, there are other mechanisms which likely occurred for the removal of metal ions, such as ion exchange, precipitation, co-precipitation, complex formation, and coagulation- flocculation [12].", "This can be considered as one of the advantages of NNB, due to its importance in designing a full scale treatment process." ]
[]
Three-dimensional poroelastic effects during hydraulic fracturing in permeable rocks
A fully coupled three-dimensional finite-element model for hydraulic fractures in permeable rocks is presented, and used to investigate the ranges of applicability of the classical analytical solutions that are known to be valid in limiting cases. This model simultaneously accounts for fluid flow within the fracture and rock matrix, poroelastic deformation, propagation of the fractures, and fluid leakage into the rock formation. The model is validated against available asymptotic analytical solutions for penny-shaped fractures, in the viscosity-dominated, toughness-dominated, storage-dominated, and leakoff-dominated regimes. However, for intermediate regimes, these analytical solutions cannot be used to predict the key hydraulic fracturing variables, i.e. injection pressure, fracture aperture, and length. For leakoff-dominated cases in permeable rocks, the asymptotic solutions fail to accurately predict the lower-bound for fracture radius and apertures, and the upper-bound for fracture pressure. This is due to the poroelastic effects in the dilated rock matrix, as well as due to the multi-dimensional flow within matrix, which in many simulation codes is idealised as being one-dimensional, normal to the fracture plane.
[ "ENGI", "MATE", "MATH", "PHYS" ]
[ "Hydraulic fractures", "Permeable rock", "Poroelasticity", "Stress intensity factors" ]
[ "2210", "2211", "2500", "2604", "2611", "3104" ]
[ "Hydraulic fracturing is the process by which one or more fractures are propagated into a rock formation, driven by the internal flow of a pressurised fluid.", "While fluid-driven fracturing can occur naturally, it is most often studied within the context of the engineering process of injecting fracturing fluid into a reservoir rock, with the aim of increasing well productivity (Adachi et al., 2007; Bazant et al., 2014).", "Although the hydraulic fracturing process is currently often thought of in the context of shale gas reservoirs, in current industry practice, almost all oil and gas wells are hydraulically fractured (Economides and Nolte, 2000).", "Hydraulic fracturing is a complex, multi-physics, multi-dimensional problem, which requires robust models that can simultaneously account for matrix and fracture deformation, fluid flow through the matrix and fractures, fluid exchange between fractures and matrix, and fracture propagation and interaction, all in a fully-coupled, three-dimensional setting.", "Hydraulic fracturing protocols are designed to control the fracture's surface area and aperture distribution, and also aim to control injection pressure, and the dependence of these variables on fracturing fluid rheology, injection rate, and the hydro-mechanical properties of the rock (Detournay and Peirce, 2014).", "Analytical and semi-analytical solutions have been developed to quantify hydraulic fracturing variables of interest, such as injection pressure, fracture aperture, and fracture length (cf. Adachi et al., 2007).", "These solutions provide the foundation for hydraulic fracturing design (e.g. Cleary, 1980; Cleary et al., 1988).", "These solutions are constructed by combining the equations for laminar flow through the fracture, with the equations for elastic deformation of the adjacent rock.", "Fluid flow through the fracture is commonly modelled using lubrication theory, which is derived from the general Navier–Stokes equation for flow of a fluid between two parallel plates (Batchelor, 1967; Zimmerman and Bodvarsson, 1996), whereas the fracture aperture is calculated using linear elasticity in conjunction with Linear Elastic Fracture Mechanics (LEFM) to compute the mode I stress intensity factor at the fracture tip (Geertsma and de Klerk, 1969; Spence and Sharp, 1985).", "Based on the energy-dissipation mechanism, fracture propagation regimes can be classified as viscosity-dominated or toughness-dominated (Detournay, 2004).", "In the viscosity-dominated regime, energy dissipation is dominated by the flow of the viscous fluid, whereas in the toughness-dominated regime, energy dissipated is dominated by the creation of new fracture surfaces at the fracture tip.", "Based on the ability of the rock matrix to dissipate fracturing fluid, two other extremes can be defined: storage-dominated, in which the injected fluid remains mainly inside the fracture, and leakoff-dominated, in which most of the injected fluid dissipates into the surrounding medium.", "The four resulting combined asymptotic regimes are therefore storage-viscosity, storage-toughness, leakoff-viscosity, and leakoff-toughness (Garagash et al., 2011).", "Asymptotic solutions that are valid at the end-members of the parameter space provide a fundamental understanding of the hydraulic fracturing process, and provide benchmarking cornerstones for numerical models.", "However, existing analytical solutions are restricted to simplified fracture geometries in homogeneous rock masses, and are typically constrained to a set of fixed boundary conditions.", "Standard geometries include the PKN fracture (Perkins and Kern, 1961; Nordgren, 1972; Mathias and van Reeuwijk, 2009), the KGD fracture (Geertsma and de Klerk, 1969; Spence and Sharp, 1985; Adachi and Detournay, 2008), and radial (penny-shaped) fractures (Savitski and Detournay, 2002; Bunger et al., 2005; Kovalyshen, 2010).", "Moreover, analytical solutions do not exist for cases that are not at the corners of this parameter space.", "Numerical models that attempt to simulate hydraulic fracturing include the boundary integral method (Peirce and Siebrits, 2001), the boundary element method (Simpson and Trevelyan, 2011), the distinct element method (Marina et al., 2014), the finite element method (Carrier and Granet, 2012), discrete fracture network (Fu et al., 2013), the embedded fracture model (Norbeck et al., 2015), the lattice approach (Grassl et al., 2015) and the extended finite element method (Dahi-Taleghani, 2009; Mohammadnejad and Khoei, 2013; Salimzadeh and Khalili, 2015a).", "However, in the majority of available models, flow through the rock matrix, and fluid exchange between fracture and rock matrix, are either ignored by assuming an impermeable rock formation (e.g. Dahi-Taleghani, 2009), or simplified by using a one-dimensional analytical leakoff model (e.g. Zhou et al., 2015).", "Substantial field evidence has proven the impermeable matrix assumption to be an unrealistic assumption (Economides and Nolte, 2000; Adachi et al., 2007).", "In one-dimensional leakoff models (Carter, 1957), fracture-to-matrix flow is represented as a sink term in the mass balance equation for fracture flow.", "This approach has several shortcomings, such as the assumption of one-dimensional flow, time-dependency of flow instead of pressure-dependency, and more importantly, this approach cannot model matrix dilation.", "Although flow from the fracture into the rock matrix is by definition locally one-dimensional at the fracture wall, where the flux vector must be normal to the fracture wall, in a global sense it is three-dimensional, unless the permeability in the direction normal to the fracture plane is significantly higher than in other directions (Hagoort et al., 1980).", "As time elapses, the leakoff rate predicted by Carter's model, at each position along the fracture, decreases proportionally to square-root of time; consequently, a scenario of fracture arrest is not possible (Mathias and van Reeuwijk, 2009).", "Finally, this model does not account for the fact that seepage of the fracturing fluid into the rock formation increases the fluid pressure in the matrix, causing dilation of the rock matrix.", "A dilated matrix applies stresses back onto the fracture, referred to as ‘back-stresses’ in the hydraulic fracturing literature, which tend to close the fracture (Kovalyshen, 2010).", "These factors also affect the available semi-analytical solutions for leakoff-dominated regimes that use a simplified one-dimensional leakoff model in their formulation.", "These solutions therefore fail to accurately predict hydraulic fracturing parameters in leakoff-dominated regimes, as shown by Carrier and Granet (2012), and Salimzadeh and Khalili (2015a) for single-phase flow, and by Salimzadeh and Khalili (2015b) for two-phase flow in two dimensions, as well as in the present study for three dimensions.", "In addition to poroelastic effects due to the aforementioned back stress phenomenon, there is a further environmental consequence of fluid seepage through the rock matrix, as it may promote the possible migration of injected fluid towards drinking water aquifers (Birdsell et al., 2015).", "Therefore, robust modelling of matrix flow is essential for both hydraulic fracture engineering and environmental aspects of subsurface fracturing.", "Only a few attempts have been made to incorporate flow in the rock matrix, coupled to mechanical deformation and flow in fracture.", "Rethore et al. (2008), Mohammadnejad and Khoei (2013) and Khoei et al. (2014), using the extended finite element method, introduced enriched pressures at the fracture to capture the discontinuous flow velocity at the fracture boundary.", "However, the enriched pressure represents the fluid pressure in the rock matrix near the fracture, and does not represent the pressure inside the fracture.", "Therefore, when coupled with mechanical deformation, the enriched pressure will be scaled by the Biot coefficient, whereas the fracture pressure actually does not require such scaling.", "Carrier and Granet (2012) introduced independent flow through the fracture and the rock matrix into their hydraulic fracture model.", "Their model was a combination of zero-thickness elements for the propagating fracture, and conventional bulk finite elements with a cohesive zone model.", "The equality of pressure between fracture and matrix at the fracture walls was enforced in the numerical model using Lagrange multipliers.", "Salimzadeh and Khalili (2015a, b) proposed an XFEM model that included two independent flow models in the fracture and the rock matrix, with a leakoff mass transfer between fracture and rock matrix to link the two.", "The leakoff depends on the pressure gradient in the matrix adjacent to the fracture, as well as on the fluid viscosity and matrix permeability.", "Norbeck et al. (2015), using an embedded fracture model, also considered two flow domains for matrix and fracture in two dimensions, and linked them through a similar mass transfer term.", "A three-dimensional fully coupled finite element model for hydraulic fracturing is presented in the present paper, validated against known analytical solutions, and subsequently applied to study the influence of fluid exchange between fracture and matrix on fracturing.", "In particular, 3D diffusion and its related poroelastic effects on the propagation of fractures are investigated.", "The present model accounts for fluid flow within fracture and matrix, the propagation of the fracture, and fluid leakage into the formation rock.", "Fluid flow through the permeable rock matrix is modelled using Darcy's law, and is coupled with laminar flow within the fracture.", "Fracture growth and the direction of growth are estimated using an energy-based criterion that is based on the modal stress intensity factors along the fracture tip (Paluszny and Zimmerman, 2013).", "This model is validated against available asymptotic solutions for penny-shaped hydraulic fractures.", "Fifteen cases with varying fluid and rock matrix properties are run, to investigate the impact of fluid and rock matrix properties on the leakoff and fracturing.", "Numerical simulations conducted over a range of parameter values delineate the limits of validity of the various available asymptotic solutions.", "Fractures are represented discretely using two-dimensional surfaces embedded in a three-dimensional domain.", "When deriving the governing equations, each fracture is represented by a discontinuity Γc in the domain Ω with boundary Γ, as shown in Fig. 1.", "The fully coupled model is constructed on three separate yet interacting sub-models, including models for mechanical deformation, fracture flow, and matrix flow.", "The solid matrix is assumed to be linear elastic, homogeneous, and isotropic, with flow modelled using Darcy's law.", "An independent fracture flow model is developed based on lubrication theory.", "The mechanical and fracture flow models are coupled through hydraulic loading on the fracture walls, and by ensuring the compatibility of fracture volumetric strains.", "The mechanical model is coupled to matrix flow through the effective stress concept, and finally, the fracture flow and matrix flow models are linked to each other through the leakoff mass transfer term.", "Tension and compression are assumed positive for stresses and pressures, respectively.", "The components of the stiffness matrix depend on the primary unknown variables, i.e. permeability of the fracture depends on the fracture aperture; therefore, an iterative procedure is required to reach the correct solution within acceptable tolerance.", "The discretised coupled equations are implemented as part of the Imperial College Geomechanics toolkit (Paluszny and Zimmerman, 2011), which interacts with an octree volumetric mesher and the Complex Systems Modelling Platform (CSMP++, also known as CSP), an object-oriented application programme interface (API), for the simulation of complex geological processes and their interactions (formerly CSP, cf. Matthäi et al., 2001).", "The set of linear algebraic equations are solved with the algebraic multigrid method for systems, SAMG (Stüben, 2001).", "Two types of discretisation are used: quadratic tetrahedra for volume elements, and quadratic triangles for surface elements (fractures).", "The triangles on two opposite surfaces of a fracture are matched with each other, but they don't share nodes, and duplicate nodes are defined for two sides of a fracture.", "The triangles are matched with faces of the tetrahedra connected to the fractures.", "Therefore, they share the same nodes.", "However, the model presented in this study can also be applied to non-matching elements.", "Fracture flow equation (Eq. 17) is solved only on one-side of fracture (i.e. matrices Hf and Mf are accumulated over triangle elements on one side of the fracture); however, the coupling matrices (Cf and L) are accumulated on both sides of the fracture.", "Mechanical deformation and matrix flow equations are accumulated over the volume elements.", "The propagation angle (θp) is determined using a modified maximum circumferential stress method that takes into account modal stress intensity factors.", "The equivalent stress intensity factors are computed locally at 100 locations along the fracture front, i.e. tips, and are used to determine if the fracture will or will not advance.", "Fractures are extended by deforming their geometry, and the mesh is regenerated and optimised at every growth step (Paluszny and Zimmerman, 2011).", "Fluid is injected through a horizontal well that perforates the centre of a vertical penny-shaped fracture.", "The size of the well is assumed to be negligible with respect to the size of the fracture, and so the wellbore is modelled as a point source boundary condition in the simulations.", "Asymptotic solutions available for this geometric case, under storage regimes (viscosity-storage and toughness-storage), are used to validate the presented numerical model.", "Further simulations, in which leakoff is modelled by considering a permeable matrix, are performed to investigate the limits of validity of asymptotic solutions under leakoff regime, and to highlight the effects of poroelasticity and the three-dimensionality of flow within the matrix.", "A single penny-shaped fracture of initial radius 1 m is located in the centre of a 90 × 90 × 60 m model.", "The model is spatially discretised using 17,441 tetrahedra and triangles.", "Convergence is achieved using an average of four iterations to reach a tolerance of 1%.", "Fracturing fluid is injected at a constant rate of Q = 0.01 m3/s into the centre of the fracture.", "A total of fifteen cases are simulated (seven cases in the viscosity regime, and eight cases in the toughness regime), with varying fluid and matrix properties, including four extreme regimes as well as intermediate cases.", "The properties for these cases (cases 1–15) are defined in Table 1.", "The Young's modulus and Poisson's ratio for all cases are set to 17 GPa and 0.2, respectively.", "The minimum in situ stress acting in the direction normal to the fracture plane for each case is given in Table 1, and the in situ stresses in the other two directions are set to 1 × 107 Pa.", "Cases in which M ≫ 1 are viscosity-dominated, whereas M ≪ 1 represents toughness-dominated cases.", "In this equation, μ′ = 12μf, and K′ = 4(2/π)1/2Kic.", "Note that the dimensionless viscosity is time-dependent, and so the behaviour moves from viscosity-dominated towards toughness-dominated as time increases.", "The fluid viscosity and fracture toughness values for viscosity-dominated cases are set to μf = 0.1 Pa s, and Kic = 1 × 106 Pa m0.5, respectively.", "For storage-dominated cases, the permeability of the matrix is assumed to be negligible (km = 0), whereas for leakoff dominated cases the matrix permeability is taken to be 1 × 10−12 m2.", "The Biot coefficient α is varied between 0 and 1, corresponding to uncoupled and fully coupled poroelasticity scenarios, respectively, although 0 is an unrealistic value, as the Biot coefficient is bounded below by the porosity (Zimmerman, 2000).", "For the given parameters, the dimensionless viscosity is M = 39, which corresponds to a viscosity-dominated case (M ≫ 1).", "The simulation results for injection pressure, fracture aperture at the well, and fracture length (radius) for viscosity-dominated cases 1–7 are shown in Fig. 2.", "Included in these figures are the asymptotic solutions for reference cases.", "Cases 1–3 with zero matrix permeability correspond to the storage-dominated regime, and cases 5–7 with matrix permeability 1 × 10−12 m2, correspond to leakoff-dominated regime.", "Case 4 is an intermediate case.", "The simulation time for leakoff dominated cases 5–7 is increased to 200 s.", "Fig. 2a shows the net injection pressure versus time when injecting a viscous fluid.", "Good agreement is found between the analytical solution for the storage case and the present model with an impermeable matrix (case 1).", "The growth increment in case 1 is 0.5 m, and it has been changed to 1.0 and 0.25 m in cases 2 and 3, respectively.", "Results show a negligible effect due to the growth increment.", "When leakoff is allowed by assuming a permeable rock matrix in cases 4–7, the injection pressure increases, compared with the no leakoff cases 1–3.", "The leakoff is increasing by increasing the permeability, resulting in higher injection pressure, lower fracture aperture, and lower fracture radius.", "In case 5, the permeability in the matrix is assumed to be “one-dimensional”, such that the permeability in direction normal to the fracture is 1000 times higher than the other two directions (one-dimensional leakoff).", "This case is used for validation against the analytical solution.", "A very good match is observed between present model results and the analytical solution for the fracture radius for case 5.", "In case 6, the permeability in all three directions is assumed equal to 10−12 m2 (three-dimensional leakoff).", "Leakoff is also increased by allowing the flow within the matrix to be globally three-dimensional, rather than one-dimensional.", "Increasing leakoff, again, leads to higher injection pressure, lower fracture aperture, and lower fracture radius.", "In case 7, the Biot coefficient is increased to 1.", "Injection pressure increases substantially with increasing Biot coefficient.", "This is due to the poroelastic coupling, which results in a back-stress on the fracture due to the dilated matrix.", "The fracture aperture at the injection well is shown in Fig. 2b as a function of time.", "For the storage cases 1–3, the results of the present model match the analytical solution very well, whereas for the permeable cases 4–7, the fracture aperture has been reduced, due to the increased leakoff from the increase in matrix permeability.", "Furthermore, the fracture aperture reduces when considering poroelastic coupling, through the Biot coefficient in case 7.", "Included in this figure is the simulation of case 5 with a coarser mesh.", "Very good agreement is found between the results for two different meshes, confirming that the leakoff is mesh-independent (38,142 elements for the fine mesh, versus 20,342 elements for the coarse mesh).", "The two discretisations for the fracture are shown in Fig. 3.", "The fracture radii predicted from the present model are compared with available solutions in Fig. 2c. Again, good agreement is found between analytical solutions and the present model, for storage cases 1–3.", "The fracture radius decreases with increasing permeability in cases 4–7.", "A similar trend is observed between the fracture radius and the Biot coefficient.", "Increasing the permeability and the Biot coefficient increases leakoff, and reduces fracture volume.", "The increase in fracture pressure and decrease in fracture aperture and radius are due to leakoff, which causes dilation of the rock matrix.", "Good agreement is also found between the present model results in case 5 and the analytical solution for fracture radius.", "In cases 6 and 7, the present model predicts lower fracture growth than the leakoff-viscosity asymptotic solution; this is due to the combined effects of poroelasticity and multi-dimensional flow through the matrix.", "The analytical solution applies for one-dimensional leakoff without poroelastic effects.", "The fluid viscosity and fracture toughness values for toughness-dominated cases are set to μf = 0.0001 Pa s, and Kic = 2 × 106 Pa m0.5, respectively.", "Again, for storage-dominated cases, the permeability of the matrix is assumed to be negligible (km = 0), whereas for leakoff dominated cases the matrix permeability is taken to be 1 × 10−15 m2.", "The Biot coefficient α is varied between 0 and 1, corresponding to uncoupled and fully coupled poroelasticity scenarios, respectively.", "The dimensionless viscosity for given parameters is M = 0.003, which corresponds to a toughness-dominated case (M ≪ 1).", "In toughness-dominated cases, the effects of matrix permeability, minimum in situ stress normal to the fracture plane, and Biot coefficient on the injection pressure, fracture aperture and fracture radius are illustrated in Figs. 4–6.", "Fig. 4a shows the net injection pressure versus injection time for cases 8–11.", "These cases show the effect of changing the matrix permeability.", "Good agreement is found between the present model and the analytical solution for storage case 8.", "In permeable cases 9−11, the injection pressure increases.", "Bunger et al. (2005) proposed solutions for a penny-shaped hydraulic fracture in the leakoff-toughness dominated regime.", "In their solution, leakoff is modelled using Carter's equation (Carter, 1957).", "The asymptotic solution for the leakoff-dominated regime also shows an increase in the injection pressure.", "In case 10, the flow through the matrix is considered one-dimensional along the direction normal to the fracture.", "Good agreement is found between the present model results and the analytical solution for this case, validating the present model.", "Leakoff increases by increasing the flow dimensions within the matrix, as well as by considering poroelasticity effects through the Biot coefficient.", "Therefore, in case 11, the present model predicts higher leakoff than does the analytical solution.", "This is due to multidimensional flow in the rock matrix, a feature that is not captured in the analytical solution.", "In case 11, the growth increment has been reduced to 0.25 m.", "A smaller growth increment corresponds to more growth steps and more computational time.", "In Fig. 4b, the fracture aperture at the well versus injection time is shown for cases 8–11.", "Again, good agreement is found between current model's results and the analytical solution for both storage case 8 and one-dimensional leakoff case 10.", "As permeability increases in cases 9–11, leakoff increases, and the fracture aperture reduces.", "Similar trends are observed for the graph of fracture radius versus injection time, as shown in Fig. 4c.", "In cases 12 and 13, the in situ normal stress is set to zero, which substantially reduces the leakoff, as shown in Fig. 5.", "Again, the injection pressure increases with increasing permeability, whereas fracture aperture and radius both decrease.", "However, the effect of changing the permeability in the absence of an in situ stress is not as significant as changing the permeability in the presence of an in situ stress (Fig. 4).", "Therefore, the in situ stress has a significant impact on leakoff; meaning that the leakoff could still be important in low permeability reservoirs provided that the minimum in situ stress is high enough.", "As the fracture pressure is not explicitly computed using Carter's model, but is often approximated as being equal to the minimum in situ stress, Carter's model predicts lower leakoff when the in situ stress is reduced.", "The fracture net pressure in the present simulations varies between 1 to 10 MPa (higher values for the viscosity-dominated regime, and lower values for toughness-dominated regime), which could be considerable compared to the actual minimum in situ stress.", "The effect of poroelasticity on leakoff is investigated through cases 14 and 15, in which the Biot coefficient is increased to 0.5 and 1.0, respectively.", "The results for net injection pressure, fracture aperture at the well, and fracture radius versus injection time, are shown in Fig. 6.", "The increase in injection pressure is more dominant when the poroelastic coupling between the matrix fluid and mechanical deformation is considered, as shown in Fig. 6a for cases 14 and 15.", "This is due to the back-stress from the dilated rock matrix.", "Increased fracture pressure increases leakoff, which in turn reduces fracture aperture and radius, as shown in Fig. 6b and c, respectively.", "Included in Fig. 6c is the simulation of case 15 with stress intensity factors computed using displacement correlation (DC) method.", "Good agreement is found between the two results, which validates the computation of stress intensity factors.", "Fig. 7 shows the fracture pressure distribution over the fracture surface, together with matrix pressure contours over a cut plane perpendicular to the fracture plane for cases 7 (Fig. 7a), 11 (Fig. 7b) and 13 (Fig. 7c) in the viscosity, toughness, and intermediate regimes, respectively.", "Higher viscosity of the injected fluid results in a non-uniform fracture pressure distribution.", "The matrix pressure distribution in all cases illustrates the three-dimensional nature of flow through the matrix.", "The time-step shown in Fig. 7a is the immediate step prior to growth-step 7.", "However, the minimum fracture pressure is less than the in situ stress, which means that the fracture growth is controlled by the flow of the viscous fluid; the fracture toughness has negligible effect.", "In the viscosity-dominated regime, the fracture propagates as soon as the fluid reaches the fracture tip.", "The distance between the fluid front and the fracture tip is called the fluid lag.", "The size of the fluid lag depends on the in situ stress, and it reduces by increasing the in situ stress.", "The fracture and matrix pressure distribution for toughness-dominated case 11 is shown in Fig. 7b. Fracture pressure distribution in this case is almost uniform compared to the fracture pressure distribution in the viscosity-dominated case shown in Fig. 7a.", "This is due to the negligible energy required to move the low-viscosity fluid (case 11).", "This result supports the assumption of a uniform pressure distribution in the fracture in the toughness-dominated regimes.", "The minimum fracture pressure is above the in situ normal stress in the time-step shown in Fig. 7b; indicating that the flow has reached the fracture tips, but the fracture is not growing, as its equivalent stress intensity factor is less than the fracture toughness of the rock formation.", "This verifies the fact that the fracture growth is controlled by fracture toughness, as is expected for the toughness-dominated regime.", "For case 13, the matrix pressure distribution is totally different than its distribution in the previous cases 5 and 10.", "Negative matrix pressures are observed in front of the fracture tips, where the tensile stresses develop matrix expansion.", "The Biot coefficient is set to 1 in this case, so matrix expansion develops negative matrix pressures due to poroelastic coupling.", "Zero in situ stress significantly reduces leakoff, such that the leakoff flow cannot compensate the negative pressures due to the matrix expansion.", "Effective normal stress contours in a cut-plane perpendicular to the fracture surface for cases 6 and 7 are shown in Fig. 8.", "Whereas for uncoupled case 6, the effective normal stress contours follow the fracture pressure pattern shown in Fig. 7, in coupled case 7, the increased matrix pressure alters the effective stress, so that the contours in the vicinity of the fracture are parallel to the fracture.", "In the uncoupled case, the maximum effective stress is located at the injection point, and is equal to the maximum fracture pressure, whereas in the coupled case, the maximum effective stress is less than the maximum fracture pressure, and is located away from the fracture.", "The results shown in Figs. 7 and 8 are for one timestep prior to growth occurrence.", "In this step, the fluid pressure at the fracture tips is almost equal in magnitude to the in situ stress (7 MPa), and so the stress singularity is very weak, and does not show up at the scale at which the stresses are plotted.", "In summary, the simulation results show that leakoff increases with increasing the permeability of the rock matrix, in situ stress, the Biot coefficient of poroelasticity, and the dimensionality of the flow within matrix.", "Available asymptotic solutions for the leakoff regime do not account for the effects of poroelasticity and multidimensional flow within the matrix, and consequently they predict lower leakoff.", "Lower leakoff is associated with lower fracture pressure, and higher fracture aperture and radius.", "Actual field cases may not correspond to any of the extreme cases for which asymptotic solutions are available, warranting the need for a robust numerical model that accounts for all the processes that occur during hydraulic fracturing.", "Convergence tests has been also performed to investigate the robustness of the model.", "For this test, the case 2 is considered with three types of mesh: coarse (9540 elements), medium (17,441 elements) and fine (29,080 elements).", "The error between injection pressures calculated from the present simulations and from the analytical solution at a time of 20.85 s is computed and shown for different meshes in Fig. 9.", "The error reduces from 5% for the coarse mesh to close to 1% for the fine mesh.", "A fully coupled three-dimensional finite element model has been presented for the simulation of hydraulically driven fractures in poroelastic rocks.", "The model was validated against available analytical solutions for penny-shaped fractures in different regimes of propagation.", "The simultaneous impact of fluid and rock matrix parameters on the hydraulic fracturing variables such as injection pressure, fracture aperture, and fracture length, has been studied.", "It was shown that leakoff increases with increasing matrix permeability, increasing in situ stresses, increasing Biot coefficient, and increasing flow dimensions.", "The analytical asymptotic solutions for hydraulic fracturing in an impermeable rock matrix provide an upper-bound for fracture radii and apertures, and a lower-bound for fracture pressure.", "For leakoff-dominated cases in a permeable rock matrix, the analytical asymptotic solutions cannot represent a lower bound for fracture radii and apertures, or an upper bound for fracture pressure.", "This is because the analytical solutions do not capture the effect of the fracture being compressed by the dilated poroelastic matrix, and underestimate the leakoff due to their neglect of global flow in directions parallel to the fracture wall.", "For intermediate cases, which are most likely to arise in actual reservoirs, there are currently no analytical solutions available.", "The extent of leakoff is also important when evaluating environmental impact.", "In summary, this work has delineated the range of applicability of the available asymptotic analytical solutions, identified the trends (in fracture aperture, fracture length, and injection pressure) due to deviations of the input parameters from the values assumed in the asymptotic analytical solutions, and demonstrated a new code that can, for example, be used for more complex geometries, such as multiple fractures and/or multiple wells (work in progress)." ]
[]
Experimental demonstration of an ultra-low latency control plane for optical packet switching in data center networks
Optical interconnection networks have the potential to reduce latency and power consumption while increasing the bisection bandwidth of data center networks compared to electrical network architectures. Optical circuit-switched networking has been proposed but it is reconfigurable in milliseconds. Although switches operating on nanosecond timescales have been demonstrated, centrally scheduling such switching architectures is considered to be of high complexity, incurring significant delay penalties on the total switching latency. In this paper we present a high-speed control plane design based on a central switch scheduler for nanosecond optical switching which significantly reduces the end-to-end latency in the network compared to using the best electronic switches. We discuss the implementation of our control plane on field-programmable gate array (FPGA) boards and quantify its delay components. We focus on the output-port allocation circuit design which limits the scheduling delay and the end-to-end latency. Using our FPGA-implemented control plane, for a 32 × 32 switch, we experimentally demonstrate rack-scale optical packet switching with a minimum end-to-end head-to-tail latency of 71.0 ns, outperforming current state-of-the-art electronic switches. The effect of asynchronous control plane operation on the switch performance is evaluated experimentally. Finally, a new parallel allocation circuit design is presented decreasing the scheduling delay by 42.7% and the minimum end-to-end latency to 54.6 ns. More importantly, it enables scaling to a switch double the size (64 × 64) with a minimum end-to-end latency less than 71.0 ns. In a developed cycle-accurate network emulator we demonstrate nanosecond switching up to 60% of port capacity and average end-to-end latency less than 10 μs at full capacity while maintaining zero packet loss across all traffic loads.
[ "COMP", "ENGI" ]
[ "Optical crossbar switch scheduling", "Optical interconnects", "Optical packet switching", "Physical-layer control plane design" ]
[ "1705", "2208" ]
[ "Annual global data center traffic has been increasing by 27% every year since 2015 and it is projected that it will continue to do so reaching 15.3 zettabytes by the end of 2020, out of which 77% will be due to traffic within the data centers [1].", "The traffic exchanged between servers within a data center is known as east-west traffic and it is driven by a number of technological trends.", "For example, the growth of cloud computing and virtualization led to servers running multiple virtual workloads which are commonly migrated from one server to another.", "Furthermore, storage replication and new applications that rely on multiple parallel workloads distributed across many servers, generate significant inter-server traffic [1,2].", "The increasing volume of east-west traffic calls for lower latency interconnection fabrics in future data centers; traditional multi-tiered topologies with a high degree of oversubscription are now replaced by flatter full-bisection bandwidth architectures [3], such as the leaf-spine shown in Fig. 1.", "This topology enables scaling to high port counts while delivering full bisection bandwidth and it features low and predictable inter-rack latency as all paths are equidistant and also the shortest in length [4,5].", "However, further bandwidth scaling is limited by the number of high-speed signal pins on electronic chips [6].", "Optically switched networks exploiting wavelength-division multiplexing (WDM) enable increasing the transmission capacity by orders of magnitude effectively breaking the bisection bandwidth bottleneck.", "Optical circuit switching has been proposed to increase the bisection bandwidth of the data center network and reduce its cost and complexity through prototypes such as c-Through [7], Helios [8], Mordia [9] and Proteus [10].", "However, because the optical switches used are micro-electro-mechanical systems (MEMS), their reconfiguration time ranges from microseconds to tens of milliseconds.", "Hence, they are best used when the traffic exhibits high stability [7–9] and thus are applied at the top layers of the network hierarchy.", "Control plane-wise, complex scheduling algorithms are used to estimate traffic demand to improve circuit utilization, incurring high latency in addition to arbitration delay and the round-trip and processing overheads due to switch path/circuit acknowledgement signaling.", "Optical switches with nanosecond reconfiguration times have been demonstrated for packet switching.", "They can be built using micro-ring resonators [11,12], semiconductor optical amplifiers (SOAs) [13,14], Mach-Zehnder Interferometers (MZIs) [15] or by using a combination of technologies [16–19].", "They can handle rapidly-changing bursty traffic as well and could be widely deployed in the network at different layers.", "Also packets are forwarded to the switch without waiting for acknowledgement signaling, effectively speculating on switch paths.", "The challenge, however, is building a control plane that can operate on packet timescales.", "The optical shared memory supercomputer interconnect system (OSMOSIS) demonstrator [20] and the optical cut-through (OpCut) switch [21] use centralized control and an iterative matching algorithm to offer high maximum throughput, at the cost of high scheduler complexity.", "On the other hand, switching systems such as the Data Vortex [22] and the scalable photonic integrated network (SPINet) [23] distribute control to the switch building blocks to simplify scheduling and improve scalability, at the expense of reduced global fairness and port throughput.", "Yet another approach is to build an optical network architecture which uses wavelength routing to avoid the need for global scheduling, for example [24,25].", "This approach tends to increase optical component counts and, in particular, requires the use of costly wavelength conversion.", "In previous work [26] we experimentally demonstrated an FPGA-implemented centralized control plane for a 32-port SOA-based crossbar switch, enabling optical packet switching with a 75 ns end-to-end latency.", "At this size, the switch is suitable for top-of-rack (ToR) application at the leaf layer, as shown in Fig. 1, however scaling to larger sizes it could be used at the spine layer too.", "The proposed optical switch architecture uses electronic buffers at the input ports to reduce the minimum latency while avoiding packet loss.", "The main focus of this paper is to present our high-speed control plane design.", "We implement it on FPGA boards, identify the end-to-end latency components including control plane contributions and discuss challenges on the minimum scheduling delay.", "We demonstrate experimentally nanosecond switching by means of high speed scheduling and speculative transmission of packets delivered across parallel WDM channels.", "We evaluate the minimum end-to-end latency and discuss the significance of control plane synchronization.", "Furthermore, we present a new highly-parallel output-port allocation circuit design for our switch scheduler and implement it on the same FPGA board as the experimental scheduler to quantify the improvement in scheduler delay and scalability.", "Finally, a cycle-accurate network emulator has been developed to perform an extensive latency analysis and comparison between the two scheduler allocation circuits.", "In this work, control plane demonstrations are for a 32 × 32 optical crossbar switch.", "The system concept is shown in Fig. 2.", "It is based on a nanosecond-reconfigurable N × N optical switch with electronic buffers, implemented as first-in first-out (FIFO) queues, at the input ports.", "An electronic scheduler is co-located with the switch and it is responsible for the switch configuration.", "Implementing buffers at the switch inputs reduces the end-to-end latency and avoids packet loss, as discussed below.", "Every new packet is first stored in a FIFO queue in the network interface at the server.", "When the queue is not empty, the request control reads the destination of the head-of-line (HOL) packet and issues an output port request to the scheduler.", "Next, once the guard time has expired, the packet control releases the packet speculatively; it does not wait for an output port grant from the scheduler.", "This eliminates the transport delay of a grant back to the server and also the overheads for grant synchronization and processing, effectively reducing the control overhead which in turn reduces the total latency [27].", "Therefore, the network interfaces “send and forget” packets as opposed to storing them for possible re-transmission.", "The guard time ensures that packets will reach the switch as soon as it has been configured.", "The transmitted packets are divided into segments each serialized onto a different wavelength using for example a dedicated silicon photonic transceiver [28], integrated on the server chip.", "The segments are multiplexed onto a WDM link and the entire wavelength-parallel structure is transported as one unit, as illustrated in Fig. 2.", "This process, known as wavelength striping, increases the input port capacity (number of wavelengths × wavelength bit rate) and reduces the packet (de)serialization latency (packet size ÷ port capacity), compared to related work [21,29] where packets are transmitted on a single serial channel.", "Also, for a given bit rate, the number of parallel wavelength channels bonded could be dynamically reconfigured to support variable-size packets at low serialization latencies.", "In case of output port contention, the packet is stored at the switch buffer, instead of being dropped, and then recirculated to the output port in a subsequent switch configuration cycle.", "The scheduler always gives priority to packets buffered at the switch over new ones at the server network interfaces to maintain the packet ordering.", "Storing packets at the switch allows for shorter transport overheads of those packets to the switch output and the corresponding control signals between the buffers and the scheduler.", "The switch buffers are electronic and therefore optical to electrical (O/E) and electrical to optical (E/O) conversions are required.", "Although fiber delay line buffers could have been used at the switch inputs for all-optical switching, using fast dense electronic memory and emerging integrated silicon photonic WDM transceiver technology [28] is more compatible with the trend towards electronic and photonic integration.", "There is a one-to-one matching between the server network interfaces and switch recirculation buffers to simplify the scheduling algorithm and also the switch architecture, compared to [21,29] where packets at an input port may be stored in many different buffers.", "A FIFO full signal is asserted when a switch buffer queue is almost full and the corresponding server network interface pauses transmission.", "This is the only control signal from the switch to a server and it is a single bit asserted when there is one free slot in the buffer, to account for a packet possibly in transit.", "This backpressure mechanism avoids buffer overflowing and packet loss.", "In summary, ultra-low latency is achieved through a) high speed switch scheduling, b) speculative packet transmission (enabled by electronic buffering at the switch) and c) wavelength-striped packets.", "The purpose of the experiment is to present the high-speed control plane design which enables ultra-low latency optical switching for the system concept described above.", "We start by identifying the control plane latency components before describing the overall experimental setup.", "The setup used for the control plane demonstration includes two FPGA boards and a 2 × 2 optical crossbar built out of discrete optical components, as shown in Fig. 3.", "FPGA#1 represents the server-side network interfaces and runs at a clock period Ttx while FPGA#2 represents the scheduler located at the switch, running at a clock period Tscheduler.", "The control plane extends across both FPGA boards; it includes the request and packet controllers in the network interfaces and the scheduler circuit.", "Its delay is therefore given by the sum of the individual control processes taking place in both the network interfaces and the scheduler:", "Request generation: The request controller reads the destination of the HOL packet and generates a request.", "This takes one network interface clock cycle, Ttx.", "Cable propagation delay: The request is transported to the scheduler out-of-band taking tcable, given by the cable delay between server and switch.", "The request consists of only the destination output port and a valid bit and hence can be efficiently transmitted using parallel electronic connections, similar to how PCI-e networks are commonly used for control and management.", "Request synchronization: In this demonstration, we assumed that the network interfaces and scheduler are asynchronous as is the case in today's commercial systems.", "The request synchronizer has a worst case delay of two Tscheduler when the request arrives slightly after the scheduler clock edge.", "Synchronization latency is discussed further in the experimental results section.", "Allocation: The optimum switch configuration given the existing switch paths and new requests is determined.", "In this demonstration allocation takes one Tscheduler but forms the critical path in the scheduler design and is discussed in more detail below.", "Switch configuration control: New grants made by the allocation circuit are added and expired grants are removed.", "The new grants are used to generate the switch configuration, including the read-enable signals for the switch buffers.", "Any requests not granted translate into write-enable signals for packet buffering at the switch.", "This takes one Tscheduler plus the switch reconfiguration time, tswitch, which is the time needed to turn on a single SOA.", "Transporting requests out-of-band in digital format eliminates the control overhead for in-band header/label detection.", "The label processor demonstrated in Ref. [30] incurs 100s of nanoseconds delay due to sampling frequency limitations imposed by analog-to-digital conversion, when implemented entirely on an FPGA board.", "Fig. 4 shows a simplified block diagram for the output-port allocation circuit used in the demonstration.", "Allocation is performed based on the well-known round-robin arbitration principle; a currently granted request gets the lowest priority in the next round of arbitration.", "The circuit has 2 pipeline stages, resulting in a 2-clock cycle allocation (including switch configuration).", "In the first pipeline stage two request matrices, one for new server requests and one for switch buffer requests, are read in and registered.", "Requests are initially arranged per input port and each is log2(N)-wide to address the destination output port.", "The size of each request matrix is therefore N log2(N).", "In the second pipeline stage, output-port arbitration and new grants generation takes place, known as allocation.", "First, a request generator rotates each request matrix to arrange requests per output port, so that they can be read by the arbiters, and then multiplexes the two matrices into a single N × N matrix, R.", "It gives priority into the R matrix to requests from the switch buffers to maintain packet ordering and reduce packet delay.", "Also it removes any granted requests, using a feedback from the current grant matrix.", "Next, the R matrix is divided and fed to N N-bit round-robin arbiters to create the new N × N grant matrix G. Finally, G is de-rotated to rearrange grants per input port and then de-multiplexed into two output N-bit vectors, one for the new packets and one for the those buffered at the switch.", "The two grant vectors are registered and will be used next by the switch configuration controller.", "In the allocation circuit, the feedback from the grant register to the request generator logic forms the critical path.", "It extends from the grant register, through one of the arbiters and back to the grant register itself, as marked in Fig. 4.", "Each N-bit arbiter is composed by N bit slices chained together [31].", "It is implemented as a programmable priority encoder using carry-look-ahead to speed up propagation through the slice chain, as proposed by Ref. [32].", "The packet timings are captured on a high-speed oscilloscope at several points along the data paths by placing probes D0-D7 as indicated in Fig. 3 and using optical receivers where necessary.", "As depicted in Fig. 5, the control plane demonstration consists of two scenarios: a) switching packets from a single network interface to either of the two output ports of the switch and b) switching packets from both network interfaces to a single output port.", "The packet generator injects packets periodically, once every 48 ns, with their destination alternating between the two possible output ports of the switch.", "The purpose of this exercise is to verify successfully packet switching and also measure the minimum end-to-end latency.", "Fig. 6 shows the timing of the control processes at the scheduler, captured using ILA probes as illustrated in Fig. 3.", "First, the input requests (probe C0) are successfully detected (only valid bits are shown).", "It then takes two clock cycles to go through the synchronization stage before they are detected by the allocation circuit (probe C2).", "Finally, it takes two more clock cycles to complete allocation and produce the switch configuration signals for the two output SOAs (probes C4 and C6).", "The width of the configuration pulses is two clock cycles.", "As shown in the figure, only one output SOA is active at a time and hence packets are switched to the corresponding output port.", "The packets appear at the same output port on every other scheduling round, as expected.", "Fig. 7 shows the packet timings at different points along the data paths from the server interface to the two output ports, as captured on the oscilloscope.", "The falling edge of a packet's valid bit entering the network interface marks the starting point for the end-to-end latency measurement.", "It takes 36 ns for the packets to leave the FPGA and 12 ns more to arrive at the switch SOA.", "It then takes 28 ns to reach the receivers and 6.4 ns more to deserialize the packet at the receiver.", "This brings the end-to-end latency to 82.4 ns compared with the expected value of 71.0 ns.", "This is due to additional PCB tracks in the control path and fiber pigtails in discrete components in the data path, such as the 2:1 combiners at the switch output ports and off-the-shelf optical receivers, which increase tcable and tfiber to more than 10 ns each.", "The amplification effect of the SOAs is exhibited as a voltage offset every time an SOA is switched ON.", "Although this could have been removed with an optical filter, none was applied in order to show the packet arrival relative to the scheduler SOA configuration pulses.", "In this scenario all packets generated are destined to the same output port but fed to the two network interfaces with a constant time difference.", "Staggering the packets enables investigating the minimum time spacing between packets of different network interfaces, in order to measure the switch throughput.", "Fig. 10 shows the minimum time spacing between packets from different network interfaces.", "That is 32 ns and it is mainly due to the control plane being asynchronous.", "As a result, in this particular demonstration setup, the throughput is (6.4∕32) × 100 = 20%.", "With a synchronous control plane, packets from different sources could be spaced by Tscheduler, which would bring the maximum throughput to (Tscheduler − tswitch)∕Tscheduler × 100 = 69%.", "Nonetheless, the focus here is to demonstrate the latency rather than the throughput of our switch system concept and the implementation of a synchronous control plane is outside the scope of this work.", "The delay characteristics along each data path are the same as in the previous section.", "The packets coming from the two different inputs are all successfully received at the output port.", "Asynchronous control plane communication increases the end-to-end latency because a request synchronization stage needs to be included in the scheduler circuit.", "In our experimental demonstration, this stage contributes 27% of the total latency.", "It also increases the switch configuration duration (to two scheduler clock cycles in the case of this demonstration for 64 B packets) to take into account the uncertainty in the request arrival time at the scheduler.", "This acts to decrease the switch throughput as no new packets may traverse the switch during that time.", "Synchronous operation would significantly reduce latency at the expense of additional wiring to distribute a clock signal.", "In practice, synchronization would also be necessary for data recovery on the receiving end, which could incur a significant latency penalty if implemented using conventional clock recovery circuits on a per packet basis.", "While full frequency and phase synchronization at the bit level has not been demonstrated on the scale of data centers, techniques such as injection clock recovery, either electronic [33,34] or optical [35], and wavelength-striped source-synchronous operation [36,37] are potential low-latency solutions to the receiver synchronization problem.", "These techniques still involve delay due to clock domain crossings.", "However, the proposed leaf-spine network with optical leaf switches, avoids any clock domain crossings at this level of the hierarchy.", "The minimum allocation circuit clock period is crucial on the achievable lower bound of the end-to-end latency in our switch system concept.", "In the scheduler used in the experimental demonstration, although the allocation circuit (Fig. 4) is a two-stage pipeline, it performs the functions of arbitration and new grants generation in a single pipeline stage, thus resulting in a long critical path.", "In Ref. [38] we briefly introduced a faster allocation circuit, shown in Fig. 11.", "Here we discuss in detail the circuit implementation, evaluate reductions in control and end-to-end latencies and investigate scheduling scalability compared to single-stage allocation.", "The new allocation circuit design reduces the critical path in the following ways:", "Arbitration and new grants generation are divided in the two pipeline stages.", "Arbitration is performed separately and in parallel for new server requests and for requests coming from the switch buffers.", "This enables removing the logic for multiplexing and prioritizing requests when generating the requests for the arbiters.", "The priority logic is moved to the 2nd pipeline stage.", "Feedback from the current grant matrix is moved to the 2nd pipeline stage and applies to the logic for multiplexing and prioritizing the new grants.", "This means that instead of dropping already resolved requests, new grants are filtered out.", "As a result, the critical path falls in the arbitration stage, from the input request register through one of the arbiters to the arbiter output register, as illustrated in Fig. 11.", "Each of the two input Nlog2(N) request matrices is fed to a request generator logic for translation into an arbiter readable format; the request matrix is expanded to N × N and then rotated so that requests are grouped per output port.", "The resulting request matrices R and R′ are each divided and fed to N N-bit arbiters.", "Arbitration is performed in parallel for R and R′.", "The two output grant matrices, G and G′, are registered and in the next pipeline stage are multiplexed into a single N × N matrix, giving priority to requests from the switch buffers.", "Feedback from the current grant matrices is used to filtered out any grants already issued.", "Finally parallel logic blocks are used to de-rotate the multiplexed matrix, arranging the grants per input port, and then produce N-bit grant vectors for the server requests and requests from the switch buffers.", "Implementing our work on CMOS application-specific integrated circuit (ASIC) technology would reduce latency even further, for either allocation circuit being used in the scheduler.", "For comparison, an electronic 10 Gb/s 48-port cut-through packet switch has a minimum latency of 170 ns [39].", "Including the same cable and packet serialization delays as in our experimental setup, its minimum end-to-end latency comes to 196.4 ns, without taking into account any control overheads on the transmit side.", "In this section we do a further investigation of the end-to-end latency in our switch system concept, based on a network model developed in SystemVerilog.", "The latency performance is evaluated for both output-port allocation circuits presented above.", "A full-size rack-scale network is emulated for a 32 × 32 optical top-of-rack (leaf) switch, including 32 network interfaces and 32 switch buffers.", "The depth of each buffer is set to 1024 packet entries to avoid packet loss at 100% load.", "We assume a 2 m fiber length from the rack to the switch and another 2 m fiber length from the switch back to the same rack or to the spine switch.", "We also assume wavelength-striped packets with tserial = 6.4 ns.", "This means that for a fixed bit rate, for example 400 Gb/s, the number of wavelengths used may be changed dynamically according to the packet size to preserve tserial.", "The model captures the latency characteristics of our control plane and adds the total fiber transport delay and packet deserialization delay at the receiver to calculate the end-to-end latency in the data plane.", "It includes the buffering latency contributions to both the control plane and data plane.", "The latency performance is measured under a synthetic workload.", "A set of 32 independent packet sources are instantiated each generating packets randomly following a uniform inter-arrival time distribution.", "A universal load parameter is used to set the probability of a packet being generated in a clock cycle, thus controlling the capacity percentage the sources run at.", "A 100% load represents packets generated on every clock cycle, i.e. with no time gaps.", "The packet output port destinations are uniform random.", "Each packet source feeds a dedicated network interface module.", "Table 1 lists the parameter values used in the network emulation.", "Fig. 13, shows how the average end-to-end latency varies as a function of the load for either allocation circuit being used.", "The two-stage allocation design performs better across all port loads.", "At low loads, both curves converge to their expected minimum end-to-end latencies as given by equation (2).", "As the load is increased the probability of contention gets higher and more packets are buffered at the switch, increasing the average end-to-end latency.", "At high loads, beyond 50%, the latency rapidly increases due to a greater number of packets experiencing head-of-line (HOL) blocking.", "With either allocation circuit being used the average latency at 100% load is 10 μs or less, under uniform random traffic.", "Nonetheless, the focus of this work is ultra-low latency switching at low loads which is verified by these results.", "Speculative transmission is one of the main enablers in achieving this at the cost of increased buffering at high loads.", "The switch buffers are assumed to be deep enough (1024 packet entries in this emulation) to avoid packet loss at all loads.", "The average latency at high loads could be reduced by changing the buffer placement at the switch; at every input port one virtual queue per output port may be implemented to store packets per destination, significantly reducing the HOL blocking and thus the buffering time and average latency [40].", "The depth requirement of each queue would also be much smaller.", "Implementing virtual output queues (VOQs) however not only increases the complexity of the switch but also that of the scheduling algorithm, incurring additional delay.", "For instance, for VOQ switches, FPGA-implementations of parallel iterative round-robin-based (iSLIP) or weighted (ipLQF) arbitration have been recently reported [41] with hundreds of nanoseconds delay for scheduling alone and that for only a 16-port switch.", "In Fig. 14 the cumulative distributions of the packet end-to-end latency for the two allocation circuits at 50% load are shown.", "Over the measurement window of 10 μs, more than 10% of the packets were received with the minimum end-to-end latency, for both allocation circuits.", "According to Fig. 13, the average end-to-end latency for the single-stage allocation circuit is 172.5 ns.", "At this value, the two-stage allocation circuit enabled switching 27% more packets.", "Furthermore, 95% of the packets were received with a latency in the range 71.0 ns–378.2 ns for the single-stage allocation circuit design against 54.6 ns–219.6 ns for the two-stage allocation design.", "Thus, for 95% of the packets, the longest possible latency was reduced by 158.6 ns.", "Moreover, two-stage allocation results in a shorter distribution tail; from 95% to 100% of the received packets, the increase in the longest possible end-to-end latency is 85.2% compared to 134.5% for single-stage allocation.", "We have presented the implementation details of our low-latency control plane and demonstrated it experimentally for a 32 × 32 optical crossbar switch.", "For this switch size, in a data center top-of-rack application, the switch scheduling accounts for 70.3% of the minimum control plane delay which in turn accounts for 76.9% of the minimum end-to-end head-to-tail latency, for wavelength-striped 6.4 ns long packets.", "The output-port allocation circuit determines the minimum scheduling delay and therefore we presented here a new circuit design which divides allocation into two stages to reduce the critical path in the design.", "As a result the minimum clock period is reduced from 9.6 ns to 5.5 ns, decreasing the scheduling contribution to the minimum control plane latency to 57.6%.", "Apart from fast scheduling, the control plane delay is also reduced using speculative transmission and storing packets at the switch inputs.", "By speculating on output-port grants the control path is reduced as otherwise grant synchronization and processing would be required at the transmit side.", "Buffering packets at the switch keeps them close to the scheduler and hence reduces the transport overhead of requests and grants between the scheduler and the buffers at the switch.", "Overall, in our FPGA-based control plane demonstration a minimum end-to-end latency of 82.4 ns was achieved, of which 11.4 ns was due to additional fiber/cable delays.", "For 2-meter fiber connections to the switch ports, the minimum latency is 71.0 ns or 54.6 ns when two-stage allocation is used and further reduction is expected when our work is implemented on CMOS ASIC technologies.", "For comparison, equivalent state-of-the-art electronic switch systems have a minimum latency around 200 ns.", "More importantly, the two-stage allocation circuit improves scheduler scalability.", "Indeed, for the same clock period it enables scaling from a 32 × 32 to a 64 × 64 switch size, considerably increasing the number of hosts in the leaf-spine data center network.", "A rack-scale 32 × 32 network emulation was used to measure the packet end-to-end latency in our optical switch system concept, under uniform random traffic.", "The scheduler based on two-stage allocation showed a lower average latency at all port loads.", "Moreover, for the average latency at 50% port capacity, it allowed switching 27% more packets, over a measurement period of 10 μs.", "Synchronous control plane operation would improve both the latency and throughput in our optical switch system, at the cost of extra wiring to distribute the clock signal.", "Practical systems also require receiver synchronization for data recovery, but techniques such as injection clock recovery and wavelength-striped source-synchronous operation could be used to keep the latency penalty small." ]
[]
Numerical investigation of design parameters for optimization of the in-situ ultrasonic fouling removal technique for pipelines
Fouling build-up in engineering assets is a known problem and, as a solution, the application of power ultrasonic for in-situ fouling removal has gained much attention from the industry. Current state-of-the-art fouling removal includes the use of hydraulic, chemical and manual techniques. Much research has been conducted to advance the knowledge on the potential uses of ultrasonics across different fouling applications, primarily in reverse osmosis membranes and heat exchangers. However, the optimization of in-situ ultrasonic fouling removal has not yet been investigated and is still in its infancy. The present study uses a previously experimentally-validated numerical model to conduct a parametric study in order to optimize the technique. Focus was given to the adoption of ultrasonics for large diameter pipes. Therefore, this investigation was conducted on a 6 in. schedule 40-carbon steel pipe. Parameters investigated include: optimum number of transducers to remove fouling in long pipes from a single transducer location; performance at elevated temperature; different fluid domains; optimum voltage; variety of input signals and incremental thickness of fouling. Depending on the particular studied conditions, the possible fouling removal of up to +/−3 m from a single transducer location is demonstrated in a 6 in. schedule 40 carbon steel pipe.
[ "CENG", "CHEM", "ENVI", "MEDI", "PHYS" ]
[ "Acoustic cavitation", "Long-range defouling", "Numerical modelling", "Parametric study", "Ultrasonication", "Wave propagation" ]
[ "1501", "1604", "1605", "2304", "2741", "3102" ]
[ "Accumulation of fouling is a rising problem across many industrial sectors [1], occurring on different structures such as pipelines, offshore structures and heat exchangers.", "The type of fouling which accumulates is dependent on the environmental conditions that surround the structure.", "In pipelines and heat exchangers, a common type of accumulative fouling is crystallization fouling, i.e. calcite formation [2].", "This occurs due to the high operating temperatures causing the high temperature fluid to saturate at the walls of structures, resulting in hard-scale deposition.", "This crystallization fouling is extremely hard to remove and continues to accumulate from carrying the high temperature fluid, potentially leading to blockage of the pipeline.", "The creation of a blockage will immediately affect the efficiency of the structure and result in the operating conditions to drop below acceptable limits.", "The accumulation can also contribute to contamination of the fluid transported in the pipeline [3].", "State-of-the-art fouling removal techniques applied in the industry include hydraulic, chemical and mechanical techniques.", "Each of these methods has disadvantages.", "For example, the use of hazardous chemicals has negative impact on the environment and purity of fluid, leading to production/operation halts for maintenance and, very critically, often posing danger to workers who carry out the procedures [4].", "Another popular method of fouling removal is the use of ultrasound.", "Currently, ultrasonic baths are used for cleaning individual components submerged in a bath where cavitation bubbles are generated within the fluid, implode at the surface of the fouling.", "Research into the use of ultrasonic baths for fouling removal has been carried out for Reverse Osmosis applications [5–7].", "This method demonstrates successful fouling removal but requires halting the production/operation to submerge the components in the ultrasonic bath, and crucially is restricted to small sized components.", "This paper investigates the use of ultrasonic power for in-situ fouling removal and uses a previously experimentally validated finite element model to optimize the process [8].", "The layout of the paper is as follows.", "The ultrasonic fouling removal methodology and state-of-the-art is discussed in Section 2 while Section 3 presents the numerical study.", "Section 4 contains numerical results and analysis which are further discussed in Section 5 with concluding remarks.", "The fouling removal mechanism with High Powered Ultrasonic Transducers (HPUT) is by the generation of acoustic cavitation.", "This is the formation of vapour/gas bubbles which result from sudden decreases in the pressure in a liquid caused by a compressional vibration [9].", "A rarefaction instant is introduced during a decrease in wave pressure which forms a vacuum where a bubble can form.", "During the oscillation of the bubble, the radius of the bubble increases in the rarefaction instants and decreases during the compressional instants.", "In one of these cycles, the compression can burst the bubble to produce a pressure of up to 500 Bar and a temperature of up to 5000 °C.", "In recent studies, it has been found that cavitation bubbles do not nucleate homogenously and instead, heterogeneously from a gas cavity or from existing stabilized micro-bubbles within the liquid [10].", "There are two types of cavitation bubbles that can form within a liquid.", "The first is stable cavitation where bubbles oscillate for long period of time over a number of cycles before its implosion.", "Transient cavitation lasts for less than one cycle and is violent enough to potentially damage any surface in contact with the liquid [11].", "Each type of cavitation results in different fouling removal mechanisms such as microstreaming, shear forces and high temperatures.", "For the present application, transient cavitation is required to generate shear forces and shock waves to dislodge fouling on the surface of the structure.", "Fig. 1 illustrates two types of cavitation bubbles that can be generated with resulting fouling removal mechanisms.", "Although cavitation is a known phenomenon that can damage a structure by the strong forces created in the violent implosions [12], literature has demonstrated the development and implosion of cavitation bubbles to be the primary fouling removal mechanism in ultrasonic fouling removal [13].", "Experimental research supports the notion that these implosions do not damage the structure during fouling removal.", "An example of this is Reverse Osmosis Membrane studies [12].", "Prior work has discussed conventional ultrasonic defouling and the disadvantage of needing to halt operations of the facility for cleaning to commence [8,14].", "The technique proposed in this paper is a non-invasive fouling removal solution using ultrasonic waves [15,16] to vibrate a structure and generate cavitation bubbles within the fluid contacting the fouled surfaces.", "This solution places an array of bespoke HPUTs on the outer wall of the pipe structure which is filled with a cavitating liquid (typically water).", "Fig. 2 shows the modification of the HPUT for attachment to curved structures with maximum surface contact.", "The HPUTs are assembled by bolting the front and back masses to sandwich stacked piezoelectric ceramic rings.", "The materials of these components are dependent on the mechanical and chemical properties desired with the HPUT excitation.", "There are various parametric considerations when configuring an array of transducers for successful cleaning, which have been identified as:", "Bespoke transducers for ultrasonic defouling applications.", "Acoustic impedance mismatch between the structure and the HPUT.", "Power amplifier and input signal generation (power level, frequency, waveform, etc.).", "The transducer is excited at its resonant frequency to generate cavitation bubbles within the liquid.", "The resonance of the HPUT is dependent on the modifications carried out on the selected transducer, which shifts the resonant frequency and also affects the impedance.", "The implosion of bubbles on the surface of the fouling layer results in removal of fouling but the vibration of the structure itself also contributes to the removal of fouling.", "Research into improving the ultrasonic fouling removal technique has focused on optimizing the cleaning parameters such as cleaning time, frequency and cleaning agent.", "Different methods have been used to investigate the parameters describing optimization of cleaning, transducers and applications for the specific problem.", "These include experimental testing and modelling.", "Examples of research for improving the ultrasonic technique include boat cleaning [17] and the system uses ultrasonic as a preventative method of fouling.", "The efficiency of the transducers has been determined through testing on contaminated samples experimentally, which is a costly and time-consuming approach to determining the effectiveness of the selected ultrasonic device and its parameters.", "The effectiveness of ultrasonic defouling based on pH level, ionic strength and particle size has also been studied [18].", "The study found various empirical trends such as both low and high pH solutions being more effective compared to neutral solutions.", "The effects of frequency (28, 45 and 100 kHz) on ultrafiltration and membrane cleaning [19] concluded that 28 kHz was the most effective whereas 100 kHz had little effect.", "The use of low frequencies to enhance permeating flux was studied and found that low power enabled at these frequencies can minimize damage to the membrane surface [20].", "Nguyen et al. [21] focused on a green technology for cleaning turbine engine oil filters on ships with ultrasonics.", "The study used a 25 kHz multi-transducer system.", "Under experimental conditions, various parameters were explored including power (300 W and 600 W), temperature effect, ultrasonic defouling times, and pressure losses through oil filters and solvent for washing.", "The cleaning efficiency was compared for three cases; hand washing, preliminary washing and ultrasonic washing.", "The experimental data were correlated using a statistical analysis method and it was concluded that ultrasonic washing was the most effective and time efficient.", "Experimental work has been carried out to ultrasonically remove contaminants on plastic parts [22].", "The study investigated different cleaning agents and temperature, and also suggested an optimal operation time and frequency for cleaning.", "After selecting the optimal parameters, a field trial was implemented to demonstrate the practical effectiveness and time-efficiency of the method.", "Further work has been carried out on optimising the ultrasonic defouling technique for membrane fouling removal using time-domain reflectometry (TDR) [23].", "TDR was shown capable of detecting membrane fouling in real time and also detected the removal of the fouling during ultrasonication.", "This method can optimise the cleaning runtime.", "Generally, experimental investigation of optimisation of the ultrasonic fouling removal technique is very time-consuming and costly.", "Very few parameters can be investigated as it can be hard to obtain transducers of various frequencies and power.", "Also, creating an experiment that can control different structural variables such as fluid temperature, fouling thickness can also be difficult to achieve.", "In contrast to experimental parameter studies, numerical simulations allow studies with lower cost and time implications.", "Finite Element Analysis has been used in 2D conditions to calculate the sound field distribution within a water tank to optimize the parameters governing effective cleaning [24].", "FEA has also been used to design and test the flow through a separator [25].", "Three models were investigated, separation model (particle fluid mixture), flow model (CFD flow profile) and transducer model (electroacoustic model of the cell).", "Overall, the models contributed towards fine tuning the performance of the cell.", "Another approach that has been investigated combines numerical simulation and automated optimization differentiation to design ultrasonic Langevin transducers [26].", "This work focused only on the modelling aspects without any experimentation to prove the method works for designing ultrasonic horn attachments.", "An in-depth review has been carried out on modelling the acoustic pressure within a sonochemical reactor [27].", "Various models have been discussed, including linear and non-linear modelling, of the two, non-linear modelling was able to account for the attenuation caused by cavitation bubbles within the sonochemical reactor.", "The ultrasonic transducer has also been discussed.", "This is a relatively easy model to create using electromechanical physics.", "Overall, numerical simulations of sonochemical reactors can model the vibration of the walls and the acoustic pressure field.", "The effect of cavitation on wave propagation is significant and can only be taken into account within a nonlinear model.", "Some work has been done to model the generation of cavitation bubbles.", "Numerical modelling has also been used to improve the ultrasonic fouling removal technique by correlating the high intensity sound field with the generation of cavitation [28].", "The modelling investigates the wave propagation into a cavitating liquid model (two-phase continuum).", "The results show qualitative agreement with the pressure antinodes and locations of cavitation generation in the experimental validation.", "In recent work, the use of multiple transducers at moderate power levels has shown to be more effective than using a single transducer at higher power levels [29], which promises improvement of the ultrasonic defouling coverage using a transducer array.", "The use of FEA for advancing the design and upscaling of the ultrasonic defouling technique for reactors is promising, and it was been reported by Jordens et al. [29].", "Effective extensions of linear acoustics have been proposed in Caflish et al. [30].", "The effective equations have been applied [31] where they are most suitable for low amplitude waves or weakly nonlinear conditions [27].", "Similar methods have been applied for reasonable amplitude waves [32,33] but their use is restricted for inertial cavitation due to the amplitude of waves ranging below the Blake threshold.", "Some attempts have been made to extend the Caflisch model with the purpose of simulating larger wave amplitudes; however, this is limited to small spatial regions [34].", "In recent years, COMSOL Multiphysics’ capabilities in coupling a variety of physics phenomena has made it popular for modelling sonochemical reactors and for optimizing design parameters.", "Cavitation activity generated by ultrasonic horns has been investigated using COMSOL [35,36].", "The optimization of reactor dimensions has also been carried out in COMSOL to couple the vibration of the walls and the sonochemical activity within the fluid domain [37,38].", "Several variables have been considered for reactor optimization, including frequency and ultrasonic intensity, however, the reactor geometry has the most significant effect [37–40].", "COMSOL has also been used to generate a model based on the Caflisch model, producing reasonably good estimates of the acoustic pressure field and the approximate cavitation zones [41,42–44]; however, although this work includes a rigorous approach, the tests have some unjustified simplifying assumptions.", "From the various linear and non-linear modelling methods, the prediction of cavitation is a phenomenon which is not close to being solved numerically.", "However, FEA does provide insight into the potential wave propagation and can assist as a useful design tool but this cannot completely avoid experimental investigation and validation.", "An experimental set-up was constructed to first prove that the ultrasonic fouling removal technique can in fact remove fouling from the inner walls of a pipe sample.", "This experimental work was presented in the thesis of de Carellan [45].", "Having produced a sample with successful cleaning patterns, COMSOL Multiphysics 5.2a was used by the present authors to create a model of the experimental configuration to match the cleaning results, specifically the pressure distribution and solid displacement [8,14].", "A reasonable correlation was achieved on the FEA results compared to the experiments conducted using the 3D Laser Scanning Vibrometer.", "Once the COMSOL model was validated with successful cleaning results achieved in previous experiments, this numerical methodology is applied to a 6 in.", "schedule 40 API 5L/A106 carbon steel seamless tube with 168 mm outer diameter, 7.11 mm wall thickness and 6 m length.", "The present paper conducts a parametric study to investigate the optimal HPUT array (see Fig. 3) with water filled within the pipe (static), fouling, signal input and fluid temperature.", "The model focuses on a 40 kHz Langevin bolt-clamped HPUT due to its successful cleaning in prior experiments [8,14,46].", "The pipe is monitored at different locations along its length to compare the various parameters as shown in Fig. 4.", "The points are placed at 1 m distances on the inner and outer walls of the pipe to monitor the acoustic pressure within the liquid and the solid displacement on the outer wall of the pipe.", "The geometry is designed assuming symmetry to reduce the computational time of the model as shown in Figs. 5 and 6.", "The number of planes of symmetry is dependent on the number of transducers under investigation.", "Physics are assigned within the model to account for the transducer, solid pipe and fluid domain within the pipe.", "The fluid surrounding the outer pipe is considered to be atmospheric air, which is not modelled in this work; instead the walls in contact with the air are assumed as ‘free’.", "Pressure Acoustics, Transient – fluid domain", "Electrostatics – piezoelectric ceramic components of the transducer", "Solid Mechanics – elastic components of the transducer and pipe", "Piezoelectric Effect – coupling of the piezoelectric components with elastic components", "Acoustic-Structure Boundary – coupling of the interface between the inner pipe wall and fluid domain", "Multiphysics modules are assigned to couple the pressure acoustics and solid mechanics physics across the acoustic-structure boundary between the fluid and solid domains.", "This allows the radiation of the wall due to transducer excitation to be taken into account and creates high and low pressures to propagate into the fluid domain [47].", "For this reason, COMSOL is used to incorporate the required physics to simulate the experimental configuration for the present study.", "The COMSOL model mimics the transducer attachment by using integration on the boundary between the transducer contact surface and pipe surface.", "This model ignores the transducer holder and instead, a fixed constraint is placed on the top of the transducer bolt to represent the fixed contact between the transducer holder and the transducer itself.", "As this model neglects the presence of cavitation bubbles, the resulting attenuation and acoustic radiation effects are not considered [48].", "However, attenuation has been achieved by altering the bulk viscosity of the fluid domain.", "The dispersion curves for the 6 in.", "diameter schedule 40 carbon steel pipe is shown in Fig. 7 [49].", "The dispersion curve graph illustrates the wave velocity in relation to the frequency which separate curves for each of the existing wave modes in a frequency region.", "Fig. 7 shows the fundamental axisymmetric wave modes (longitudinal and torsional) as well as the corresponding first flexural wave modes produced over frequency range.", "The selected study for this model is Transient, so that the simulation can generate results as the modulated wave propagates from the transducer.", "To ensure that standing waves are achieved, the time of arrival must be calculated based on the signal arriving at the end of the pipe and travelling back to the excitation location, totaling 6 m in travelling distance.", "Extra time must also be taken into account to accommodate multi-modal responses (higher order flexural modes); therefore, the time duration has been selected for a total of 15 m of distance.", "The number of HPUTs within a configuration is investigated, as a single transducer may not develop high enough values of pressure within the fluid to generate cavitation, depending on the diameter of the pipe, wall thickness and pipe length.", "For the pipe sample of interest, the diameter allows multiple transducers to be placed around the circumference to potentially increase the acoustic pressure within the fluid and the outer wall displacement.", "The numbers of transducers being investigated are 1 (90°), 2 (90° and 270°) and 4 (0°, 90°, 180° and 270°).", "The configuration is placed at the mid-length of the 6 m pipe.", "Symmetry is applied to each configuration to reduce computation time.", "The selected optimal transducer array for long-range cleaning is utilized for further investigation of the following removal parameters:", "temperature of fluid (20 °C, 50 °C and 100 °C)", "input signal (sinusoidal and square)", "layer of fouling (calcite)", "Number of cycles (10 and 20)", "The data obtained will primarily focus on the pressure acoustics and solid displacement at the monitored points.", "The results of the FEA parametric study were compiled to compare the solid displacement and acoustic pressure at monitored points along the length of the pipe (0, 1, 2, 3 m).", "To compare the coverage and amplitude achieved from each configuration, the maximum amplitude of solid displacement and acoustic pressure is measured from the monitored points along the length of the pipe.", "Polar plots are created for each configuration at the maximum amplitude at different locations to analyze the coverage over the cross-section of the pipe wall.", "Comparing the amplitude at the monitored points for the different configurations in Fig. 8, the trend shows that the 1-Transducer achieves its maximum displacement and acoustic pressure amplitude at the transducer location; along the length of the pipe, the 1-Transducer case reduces in amplitude, resulting in an average acoustic pressure of 3 KPa.", "Comparing each configuration at the excitation location, the 2-Transducer case achieved the lowest displacement, due to the configuration resulting in superposition of the signals.", "The remainder of the 2-Transducer results follows a similar trend to the displacement amplitude achieved for the 1-Transducer case.", "For the acoustic pressure, with the increase of transducers, the achieved amplitude at the transducer location also increases.", "For the remainder of the pipe length, the 1-Transducer and 2-Transducer case follow a similar trend with an average of 3 KPa.", "The 4-Transducer case has shown to achieve the highest displacement and acoustic pressure at each monitored point, averaging 8 KPa for the remainder of the pipe length.", "The polar plots in Fig. 9 for the 1-Transducer case show high amplitude at the transducer location for both the displacement and acoustic pressure.", "For the remainder of the circumference, the displacement averages below 0.5 × 10−4 mm.", "the acoustic pressure shows some peaks reaching 4 KPa at 0° and 180°.", "The remainder of the circumference has an average of 2 KPa of pressure being achieved.", "This coverage is not ideal for long distance due to the drop in amplitude and the lack of uniform coverage being achieved.", "The 2-Transducer case achieves its highest amplitude at the transducer locations (90° and 270°) as shown in Fig. 10.", "Over the length of the pipe, the amplitude of these peaks drop.", "The remainder of the circumference averages below 0.5 × 10−4 mm and 5 KPa.", "At 0° and 180°, the peak achieved in the acoustic pressure shows a blunt feature, this could be due to superposition related to the transducer spacing.", "The highest amplitude for the displacement and acoustical pressure is achieved in the 4-Transducer configuration at 0°, 90°, 180° and 270° as shown in Fig. 11.", "The configuration achieved a more uniform coverage as well as higher averaging coverage for the displacement (1 × 10−4 mm) doubling the average shown in the 1-Transducer and 2-Transducer cases.", "The acoustic pressure averages approximately 7 KPa.", "The study of the HPUT array has shown that the transducer location should typically achieve the same displacement due to the vibration of the transducer but the 2-Transducer case has a reduction as mentioned previously due to superposition.", "The increase in the number of transducers improves the coverage achieved at the transducer location and has increased the acoustic pressure within the fluid domain.", "The 4-Transducer case has also shown to achieve high amplitudes at further distances compared to the 1-Transducer and 2-Transducer cases showing its promise for achieving long distance cleaning coverage.", "The 4-Transducer configuration was selected for further parametric analysis of the cycle input, fluid temperature, addition of fouling and signal input.", "The number of cycles of the sinusoidal input signal is investigated and displayed in Fig. 12.", "For the displacement, the increase in cycles has in increase in the displacement being achieved on the outer wall of the pipe; however, for the acoustic pressure, this does not have an effect.", "This implies that the number of cycles does not improve the delivery of the signal through the pipe wall thickness, into the fluid domain.", "The effects of temperature of the fluid domain is investigated and displayed in Fig. 13.", "At the transducer location, the displacement increases at 50 °C whereas the 20 °C and 100 °C remain the same.", "Along the length of the pipe, the displacement amplitude increases for 50 °C and 100 °C compared to 20 °C.", "This suggests that the increase in temperature improved the propagation of the vibration from the transducer for longer distances.", "The 50 °C case increases the acoustic pressure of 3 Bar at the transducer location whereas the 100 °C achieves 2 Bar, this could be due to the temperature being at the boiling point of water, the fluid used in this model.", "This can suggest that the increase of temperature can increase the acoustic pressure however, when reaching boiling temperature, this will decrease.", "Overall, acoustic pressure achieved along the length of the pipe does not fluctuate compared to the 20 °C case.", "The addition of a 3 mm thick calcite layer on the inner wall of the pipe is then investigated (see Fig. 14).", "The displacement shows to have a small variation at the excitation location however, along the length of the pipe, the amplitude fluctuates between high and low amplitudes.", "This could suggest that the addition of the calcite has affected the wave propagation across the structure, resulting in a shift in the nodes and antinodes at the monitored points.", "The acoustic pressure shows a reduction due to the addition of calcite at the transducer location, which could be explained by the calcite layer attenuating the vibration through the pipe thickness into the liquid domain.", "At 1 m, there is an increase in acoustic pressure which matches to the high displacement.", "The remainder of the acoustic pressure of the calcite matches similarly to the increase and decrease in displacement.", "This shows that the calcite layer attenuates the wave propagation and this was experimentally studied by the present authors in 2018 [49].", "When investigating an alternative waveform to the sinusoidal input in Fig. 15, it is shown that the square wave input has increased the displacement and the acoustic pressure across all monitored points.", "The acoustic pressure achieves the minimum 1–2 Bar of pressure which is the known threshold to generate cavitation for a 40 kHz transducer [53].", "This provides more evidence that the 4-Transducer case can potentially achieve long-range cleaning by creating pressure amplitudes above the cavitation threshold.", "Fig. 16 shows the displacement on the outer wall of the pipe when exciting the 4-Transducer configuration.", "The outer wall achieves a uniform displacement propagating from the transducer location.", "This work uses a non-invasive method of fouling removal using power ultrasonics.", "The technique itself is carried out by using tailored bespoke HPUTs attached directly to the outer wall of the structure (inner wall of pipe is filled with liquid).", "The excitation of the HPUT occurs at its natural resonant frequency and results in cavitation bubbles within the liquid.", "The implosion of these bubbles occurs in regions of high pressure, favorably on the fouled surface results in forces large enough to remove the fouling.", "FEA methodology has been validated in previous work by the present authors [8,14] and was used in this study to investigate the effects of different parameters and to optimize the transducer configuration and input variables for cleaning a 6 m long 6 in.", "schedule 40 carbon steel pipe.", "The 1-Transducer case showed itself suitable only for localized fouling removal as it does not propagate far, and can be used for specific applications such as fouling removal in pipe elbows or bends.", "The study showed that the 4-Transducer case was the most suitable for long range fouling removal for the studied conditions as it had achieved high pressure and displacement along the length of the pipe.", "This configuration also achieved a uniform distribution at the circumference at different locations along the length of the pipe.", "The parametric study also showed the square waveform input has an increasing effect on the amplitude of both the solid displacement of the pipe and the acoustic pressure of the fluid.", "The sinusoidal input achieves a lower displacement and acoustic pressure across the structure; however, this can be used for fouling prevention of a clean structure.", "Ultrasonic prevention will be investigated in further work and also transducer miniaturization to accommodate more transducers around the circumference to achieve more defouling coverage from one transducer location." ]
[ "A validated numerical model is used to investigate various fouling removal parameters.", "This includes the number of transducers, signal input and fluid temperature.", "This investigation focuses on a 6 m long, 6 in.", "diameter, schedule 40 carbon steel pipe.", "From the investigated transducer array, the 4-Transducer configuration is selected due to its potential to achieve long-distance fouling removal." ]
Glass ionomer cements with milled, dry chlorhexidine hexametaphosphate filler particles to provide long-term antimicrobial properties with recharge capacity
Objective: Glass ionomer cements (GICs) are a versatile material, offering the opportunity for ion exchange with the oral environment. The aim of this study was to develop a GIC that delivers a controlled, rechargeable dose of chlorhexidine (CHX) over an extended period without compromising mechanical properties. Methods: GICs were supplemented with finely milled particles of chlorhexidine hexametaphosphate (CHX-HMP). CHX release into artificial saliva was measured over 660 days, and recharge with CHX and CHX-HMP was investigated. Mechanical properties were investigated, and an agar diffusion test was carried out to assess antimicrobial properties using Streptococcus mutans and Scardovia wiggsiae. Results: Dose-dependent CHX release was observed, and this was ongoing at 660 days. Compared with related studies of GICs containing CHX-HMP, the fine, dry particles resulted in fewer adverse effects on mechanical properties, including tensile, compressive and biaxial flexural strength, with 1% CHX-HMP GICs indistinguishable from control specimens. The GICs could be recharged with CHX using both a conventional CHX digluconate solution comparable to commercial mouthrinses, and a suspension of CHX-HMP of equivalent concentration. Recharging with CHX digluconate increased subsequent CHX release by 50% compared with no recharge, and recharging with CHX-HMP increased subsequent CHX release by 100% compared with no recharge. The GICs inhibited growth of St. mutans and Sc. wiggsiae in a simple agar diffusion model. Significance: These materials, which provide sustained CHX release over clinically relevant timescales, may find application as a restorative material intended to inhibit secondary caries as well as in temporary restorations and fissure sealants.
[ "DENT", "ENGI", "MATE" ]
[ "Antimicrobials", "Caries", "Chlorhexidine", "Glass ionomer cement", "Restorative materials" ]
[ "2211", "2500", "3500" ]
[ "Glass ionomer cements (GICs) are a mainstay of modern dentistry.", "Their uses include temporary and permanent restorations, lining and luting materials, fissure sealants and in atraumatic restorative therapy.", "Although GICs exhibit fluoride release, and antimicrobial effects of this can be demonstrated in vitro, the data in support of an anticariogenic effect in vivo is inconclusive [1–3].", "Nevertheless, a bioactive material that participates in ion exchange with its local environment offers an adaptable vehicle for delivery of other useful molecules to the locale of a restoration.", "Chlorhexidine (CHX) is a broad-spectrum antimicrobial in widespread use as a topical agent.", "It owes its antimicrobial properties to membrane disruption and is efficacious against a wide range of microbes including those implicated in caries.", "CHX in commercial products is typically present as the CHX digluconate salt which is highly soluble and thus has poor substantivity, usually providing only short term antimicrobial function before it is depleted.", "CHX concentrations in saliva fall rapidly after an oral rinse, with 90% of the initially retained CHX cleared from the mouth within 6 h and 98% cleared within 12 h [4].", "One product used for treatment of periodontal disease provides sustained CHX release, but 80% of the CHX is released within the first 2 days, with a much lower release over the following 3–4 weeks [5].", "Owing to the useful antimicrobial functionality of CHX, coupled with its low cost and well-understood pharmacological properties, there have been attempts to incorporate CHX into dental materials, including GICs.", "GICs supplemented with CHX diacetate and CHX digluconate inhibited growth of oral microbes Streptococcus mutans and Lactobacillus acidophilus, but the antimicrobial effects were sustained for only 40–90 days, with no bactericidal effect observed after this time [6].", "In another study where GIC was doped with CHX digluconate and antimicrobial efficacy again assessed using St. mutans and L. acidophilus, there was an exponential decay of the zone of inhibition over the first 30 days [7].", "These short-term antimicrobial effects may be explained by the release profiles of soluble CHX from GICs doped with these conventional salts, which show a high initial release which rapidly diminishes leaving little or no sustained release [8].", "A recent report of core–shell microparticles containing CHX chloride salts reveals potential for graduate release of CHX, although this study only probed CHX release to a maximum of 430 h (18 days) [9].", "Supplementation with these CHX salts has also been found to be associated with a negative impact on mechanical properties [6,10–12].", "More recently, some attempts have been made to incorporate CHX when loaded into particles that may facilitate gradual rather than rapid release, and there has been some success in this, but still the CHX release was heavily front loaded and decayed rapidly after the first 1–3 days [13].", "Although in vivo studies of these experimental materials are rare, there are a few small studies of note.", "Supplementation of a resin-modified GIC with 1.25% CHX digluconate resulted in elimination of St. mutans populations following indirect pulp treatment in vivo [14], and incorporation of CHX digluconate into a GIC reduced microbial counts in cavities in first primary molars in 6–9 year olds, although the CHX digluconate GICs also exhibited a higher failure rate than the controls [15]; similar results were observed in a study of CHX digluconate doped GICs in children receiving atraumatic restorative therapy, where the CHX GICs resulted in reduced St. mutans counts at 7 days but not 3 or 12 months after placement [16].", "These tentative findings highlight the potential benefit of CHX-functionalised restorative materials as well as the challenges, in that the other properties of the GIC are often compromised by the new component.", "We have recently reported a novel salt of CHX: CHX-hexametaphosphate (CHX-HMP) [17].", "CHX-HMP has a much lower solubility than CHX digluconate or CHX diacetate, and when in or on biomaterials, can confer a sustained release of CHX [18].", "We have described a pilot study demonstrating the use of manually ground CHX-HMP as a filler for GICs [19], and another where the CHX-HMP was incorporated into a GIC as a viscous paste formulation [20].", "In the first study [19], large clusters of CHX-HMP particles were used, and this had adverse effects on the mechanical properties; furthermore, another limitation of that study was the short time over which CHX release from the prototype cements was recorded, and the large particle size would have had a detrimental effect on this CHX release.", "In the second report [20], the incorporation of the particles in a wet aqueous paste resulted in deterioration of the mechanical properties when the substitution of CHX-HMP was at 0.34% by mass or greater, although the CHX release was improved on the earlier approach, being sustained for at least 14 months.", "The aim of the study described here was to investigate the use of CHX-HMP particles as GIC fillers but incorporating a mechanical milling process resulting in much smaller CHX-HMP filler particles.", "The hypothesis was that the smaller particle size and thus larger specific surface area would create a more sustained CHX release coupled with fewer adverse effects on mechanical properties.", "Furthermore, given that any antimicrobial incorporated into a biomaterial has a finite duration of release, the capacity of these materials to be “recharged” with CHX, in analogy to the well-known fluoride recharge phenomenon, was investigated.", "A simple microbiological test was incorporated into the study to determine whether CHX release from the prototype GICs had an effect on two cariogenic bacteria in an agar diffusion test.", "The conventional and well-characterised St. mutans was selected for the test, as well as Scardovia wiggsiae, a recently recognised anaerobic pathogen associated with aggressive childhood caries [21,22].", "100 mL 10 mM solutions of each of CHX digluconate and sodium HMP (Sigma Aldrich, Gillingham, UK) were prepared in deionised water and were combined in a glass beaker under ambient laboratory conditions.", "The resulting aqueous suspension of CHX-HMP was stirred vigorously for approximately 1 min, then 30 mL 1 M potassium chloride was added and stirred for a further minute.", "The suspension was stored without disturbing for 24 h, allowing the precipitate to sediment at the base of the container.", "The supernatant was gently discarded leaving a concentrated suspension of the precipitate, which was then centrifuged at 4760 g for 30 min.", "The supernatant was again discarded and the pellet dried at ambient temperature under air extraction for 24 h, then removed from the centrifuge tubes.", "100 g total mass of dried CHX-HMP pellets prepared in this way was placed into a cylindrical ceramic mill of 80 mm diameter and 70 mm height containing ceramic balls of sizes 12.5–20.5 mm (Pascal Engineering Company Ltd., Crawley, UK), and this was placed on a rolling platform for 4 h.", "The mill was opened every hour and particles dislodged from the internal walls.", "The resulting powder was passed through a coarse sieve and examined using scanning electron microscope (SEM).", "The specific surface area was measured by nitrogen adsorption following outgassing for 48 h at 30 °C under vacuum.", "Elemental microanalysis (CHNP) was used to confirm the composition of the precipitate; CHX-HMP was prepared as described above and analysed for C, H, N and P (Microanalytical Laboratory, School of Chemistry, University of Bristol).", "Diamond Carve™ GIC (Kemdent, Purton, UK) was used as the base material to create experimental cements.", "This commercial GIC comprises a powder consisting of alumina-silica based glass filler particles which also contain calcium fluoride and other minor salt components and freeze-dried poly(vinyl)phosphonic acid, and a liquid, which contains polyacrylic and tartaric acids.", "For the prototype GICs, CHX-HMP particles were substituted for the glass component in the GIC powder at 0, 1, 2, 5 and 10% by mass.", "All dopings were tested for each parameter below, unless otherwise specified.", "The CHX-HMP particles were mixed with the GIC powder in the ball mill for 10 min as described above to ensure homogeneous incorporation of the components.", "This powder was then mixed at a 4:1 ratio by mass with the GIC liquid according to the manufacturers’ instructions.", "Mixing was completed in 40–50 s and packing into the moulds took a further 10 s, such that the process of creating the specimens was complete within 60 s.", "GICs were packed, using a stainless steel dental spatula, into stainless steel moulds with dimensions dependent on the purpose of the specific specimen.", "Dimensions were: 6 mm height and 4 mm diameter for compressive strength determination; 4 mm height and 6 mm diameter for measurement of diametral tensile strength, release of soluble CHX, and antimicrobial testing; 2 mm height and 15 mm diameter for determination of flexural strength.", "The moulds were lined with a thin layer of petroleum jelly to aid removal of the set cement.", "Immediately after packing, the moulds were placed between two sheets of acetate and a 2 kg weight placed on top of the specimens on a flat surface to ensure even distribution of the cement.", "After 5 min the specimens, except those for biaxial flexural strength testing, were ground on the two flat surfaces using a P120 grit SiC disc and were then placed into small, sealed plastic vessels containing wet tissue paper packed into the lid to achieve 100% humidity without direct contact with water.", "Specimens were stored at 37 °C for 7 days prior to testing.", "CS was measured by applying a compressive force to the flat surface of the cylindrical specimens (6 mm height, 4 mm diameter) using a universal testing machine (Instron, Buckinghamshire, UK) with cross-head speed of 1 mm/min and recording the load at fracture.", "Specimen dimensions were measured three times using a digital micrometer.", "Load at fracture LF was used with diameter D to calculate CS according to the relationship CS = 4L/πD2.", "Specimens were examined after fracture for evidence of flaws on the internal or external surfaces and flawed specimens were rejected.", "n = 24 specimens per group were used.", "Data were analysed using a one-way ANOVA followed by a Tukey Honestly Significant Difference post-hoc test at α = 0.05.", "DTS was measured by applying a compressive force to the curved sides of the cylindrical specimen (4 mm height, 6 mm diameter) using a universal testing machine (Instron, Buckinghamshire, UK) with cross-head speed of 1 mm/min and recording the load at fracture.", "Specimen dimensions were measured three times using a digital micrometer.", "The load at fracture LF was used in conjunction with the average diameter D and height h of the specimens to calculate DTS according to the relationship DTS = 2L/πDh.", "Specimens were examined after fracture for evidence of flaws on the internal or external surfaces and data from specimens found to be flawed were rejected.", "Data were analysed using a one-way ANOVA followed by a Tukey Honestly Significant Difference post-hoc test at α = 0.05.", "As well as freshly prepared specimens (n = 24 per group), the specimens that were subjected to the long-term CHX elution experiments were also tested for DTS to ascertain whether strength deteriorated after long-term exposure to artificial saliva (n = 15 per group).", "GIC specimens were weighed using a precision balance then placed in individual cuvettes transparent to ultraviolet light (Z637157; Sigma–Aldrich, Gillingham, UK).", "1.5 mL artificial saliva was added to each cuvette.", "The artificial saliva was composed of 0.9 mM CaCl2, 0.2 mM MgCl2, 4.0 mM KH2PO4, 30.0 mM KCl, 20.0 mM HEPES buffer, titrated to pH of 6.8 [23].", "The cuvettes were sealed with tightly-fitting lids (SEMA2533; VWR, Lutterworth, UK) and then placed onto an orbital shaker (SSM1, Stuart, Staffordshire, UK) at 150 rpm and readings taken initially once a day, and less frequently as CHX release decelerated.", "Artificial saliva was refreshed at two-week intervals to avoid any decrease in CHX release that could be attributed to saturation of the artificial saliva with respect to CHX salts.", "Adsorption of light at wavelength 255 nm was measured at regular intervals and calibration standards of 5–50 μM CHX used as references to establish CHX release from the GICs into the artificial saliva [24].", "This was converted to μmoles CHX released per unit surface area for each specimen and normalised by subtracting the mean reading for the 0% substitution, correcting for any systematic error due to background readings.", "n = 15 specimens per group were used.", "Cuvettes were inspected carefully at each reading and, in the rare event that leakage occurred, the specimen was discarded.", "CHX elution was measured for 660 days.", "Specimens were prepared as described above and subjected to CHX elution measurements as described in the previous section, with modification to permit the investigation of the capacity for recharge and subsequent release of the CHX.", "Three sets of specimens (n = 15 each of 0, 1, 2, 5, 10% CHX-HMP for each set) were stored in artificial saliva and CHX release was assessed using UV spectrophotometry as described above.", "Every 4 weeks the specimens were removed from the artificial saliva and were immersed in one of three “recharge” preparations: deionised water (negative control), 2.2 mM CHX digluconate (“CHX”) and a CHX-HMP suspension containing the equivalent of 2.2 mM CHX (“CHX-HMP”).", "CHX release of the three groups was assessed to determine whether the specimens periodically exposed to CHX or CHX-HMP had a different subsequent CHX release than those exposed only to water.", "Readings were converted to μmoles CHX released per unit surface area for each specimen and normalised by subtracting the mean reading for the 0% substitution.", "Cuvettes were inspected carefully at each reading and, in the rare event that leakage occurred, the specimen was discarded.", "CHX elution was measured for 315 days.", "St. mutans GS-5 [25] was maintained in Brain Heart Infusion broth supplemented with 0.5% (w/v) Yeast Extract (BHY) and incubated for 16 h at 37 °C in a candle jar.", "Sc. wiggsiae C1A_55 [26] was maintained in Tryptic Soy broth supplemented with 0.5% (w/v) Yeast Extract, 25 μg/mL haemin and 5 μg/mL menadione (TSBYHM) and incubated for 5 days at 37 °C anaerobically under N2:CO2:H2 (80:10:10).", "Bacterial cells were harvested (5000 g, 7 min), washed and resuspended in PBS at OD600 1.0 (approximately 2 × 108 cells/mL).", "Adjusted cell suspensions (100 μL) were spread evenly over BHY agar plates (St. mutans) or fastidious anaerobe agar (FAA) plates (Sc. wiggsiae), before placing GIC specimens (0 and 2% CHX-HMP) (6 mm diameter, 4 mm height) onto the agar.", "These lawn plates were then incubated as before for 16 h (St. mutans) or 5 days (Sc. wiggsiae), before zones of inhibition around the GIC specimens were measured.", "SEM images of the milled CHX-HMP particles are shown in Fig. 1.", "The milled particles were polydisperse, with the most common sizes in the 1–20 μm range and some aggregates as large as 50–150 μm.", "The specific surface area of the powder as determined using BET was 1.55 m2 g−1 and no micropore volume could be measured indicating a very low porosity.", "Elemental microanalysis indicated that the composition of the precipitate was 20% N, 40% C, 5.3% H and 8.6% P, which corresponds most closely to a composition of 3 CHX to 1 HMP (theoretical values 21% N, 40% C, 4.9% H, 9.3% P).", "Given the relative molecular masses of CHX and HMP this indicates that the CHX-HMP solid is approximately 76% CHX by mass.", "CS data are shown in Fig. 2 with error bars representing standard deviations.", "Rejection of specimens with internal voids, imperfections or non-linear force-distance curves resulted in final n per specimen group of 17–22 (mean n = 18.6; initial n = 24).", "According to the ANOVA and Tukey HSD test, there was no statistically significant difference between CS of control, 1% or 2% CHX-HMP specimens; 5% and 10% CHX-HMP had significantly lower CS; that is, substitutions up to and including 2% had no impact of CS compared with control specimens, whereas 5% or more CHX-HMP had a negative impact on CS.", "DTS data are shown in Fig. 3 with error bars representing standard deviations.", "For specimens tested without aging, rejection of specimens with internal voids, imperfections or non-linear force-distance curves resulted in final n per specimen group of 19–24 (mean n = 21.2; initial n = 24).", "For specimens tested after immersion in artificial saliva for 660 days, rejection of specimens with internal voids, imperfections or non-linear force-distance curves resulted in final n per specimen group of 8–14 (mean n = 11.2; initial n = 15).", "According to the ANOVA and Tukey HSD test, the only statistically significant differences in these data were between unaged 2% and 10% CHX-HMP specimens, where 10% CHX-HMP had lower DTS than 2%, and between unaged 2% and aged 2% CHX-HMP specimens, where the aged material had lower DTS than the unaged material.", "That is, for unaged specimens substitutions up to and including 10% CHX-HMP had no impact on DTS compared with control specimens, and after aging one specimen group exhibited a reduction in DTS but there were no differences among the groups of specimens comparing all aged specimens.", "BFS data are shown in Fig. 4 with error bars representing standard deviations.", "Rejection of specimens with imperfections resulted in final n per specimen group of 22–24 (mean n = 23.0; initial n = 24).", "According to the ANOVA and Tukey HSD test, the 1% CHX-HMP was indistinguishable from the control group, whereas the others (2, 5 and 10% CHX-HMP) had lower BFS than the control group.", "Elution of soluble CHX from CHX-HMP doped GICs is shown in Fig. 5.", "A dose-dependent CHX release was seen, with the total CHX released being related, although not directly proportional, to the total CHX content of the material.", "The CHX release had a smooth profile and did not accelerate and decelerate over the period investigated, indicating that the frequent changing of elution medium ensured that saturation was not a factor in controlling the rate of CHX release.", "Elution of soluble CHX from CHX-HMP doped GICs is shown in Fig. 6.", "Specimens recharged with the 2.2 mM CHX solution released more CHX than negative controls exposed to DIW; on average 50% more CHX was released by the CHX recharged specimens than the controls over the 315 day period.", "Specimens recharged with CHX-HMP suspension at the same equivalent total CHX concentration released more CHX again than the CHX solution recharged specimens; on average over the 315 period specimens recharged with CHX-HMP released 33% more CHX than those recharged with CHX solution and 100% more CHX than negative controls.", "Control GICs without CHX-HMP showed no remote or contact inhibition of microbial growth with either St. mutans or Sc. wiggsiae.", "2% CHX-HMP GICs resulted in both contact and remote microbial inhibition of St. mutans, with an average 11.9 mm zone of inhibition (5.7 mm as measured from the edge of the GIC specimen).", "2% CHX-HMP GICs inhibited growth of Sc. wiggsiae but did not result in remote inhibition (i.e. the inhibition of microbial growth was restricted to the area of agar in direct contact with the specimen).", "Incorporation of dry, milled CHX-HMP particles into a commercial GIC resulted in the release of aqueous CHX that was still ongoing at 660 days for all specimen groups.", "There was a dose response, with higher CHX-HMP dopings releasing more CHX, although this was non-linear, as the total release at 660 days for 2, 5 and 10% CHX-HMP was 1.8x, 4.3x and 6.5x that of the 1% CHX-HMP material respectively.", "The rate of CHX release decelerated after approximately 150 days, and from that time was close to constant until the conclusion of the study.", "The kinetics of CHX release are very different from those observed in other CHX-doped GICs, where there is a rapid decay of CHX release after the first few days of measurement.", "The fundamental difference is that most of the studies described above utilise CHX digluconate and diacetate which, owing to their solubility, dissipate rapidly in contact with aqueous solutions.", "CHX-HMP has a much lower solubility than CHX digluconate or diacetate and the rate of CHX release is governed by kinetic factors (agitation of the surrounding medium) and thermodynamic factors (degree of undersaturation of the surrounding medium, which were effectively sink conditions in this study as evidenced by the smooth profile where the 14 day medium changes were not detectable by an acceleration in CHX release).", "Thus the release profile is not characterised by the “burst” release that has been observed in all studies of CHX digluconate and CHX diacetate GICs.", "Utilising the elemental analysis data it can be concluded that the CHX-HMP solid is ∼72% CHX, and this enables calculation of the total CHX contained within the GIC specimens.", "Given a specimen mass of 202 mg taken from the mean of 152 specimens, this equates to a total content of CHX in the specimens as 1%: 2.9 μmol CHX; 2%: 5.8 μmol CHX; 5%: 14.4 μmol CHX; 10%: 28.7 μmol CHX.", "At the end of the 660 day period the total CHX released by the specimens was 1%: 0.027 μmol CHX; 2%: 0.049 μmol CHX; 5%: 0.115 μmol CHX; 10%: 0.175 μmol CHX.", "This indicates that the total CHX release over the 660 day period for all four specimen groups was of the order of 1% of the total CHX contained within the material.", "It is likely that the CHX that was released was primarily from the outer region of the specimen, where ion exchange with the artificial saliva would most readily take place.", "It does indicate that the CHX was far from depleted at the endpoint of the study and that continued CHX release would have been likely for a substantially longer time even without recharge.", "Furthermore, the volume of saliva and the perpetual undersaturation reflects the conditions at the surface of the GIC that would be exposed in the oral environment, and not that interface which would be of most clinical interest, that is, the interface between GIC and tooth tissue.", "At this location the fluid flow would be very much less and therefore the CHX concentration would likely reach a saturation that would decelerate any further CHX release owing to the common ion effect.", "To the extent that these observations can be used to infer clinical behaviour it is likely that the duration of CHX release in these experiments underestimates the duration that would be observed at the material-tooth interface in vivo.", "The capacity to recharge CHX-containing GICs has not previously been investigated.", "The data indicated that when exposed to either CHX digluconate solution with a concentration equivalent to that in widely used oral rinses, or a CHX-HMP suspension with the same total CHX, subsequent CHX release by the GICs was greater.", "This indicates that the GIC can take up CHX from its environment, and subsequently release it again in a reversible process.", "The aim of this part of the study was to determine whether the longevity of the CHX release could potentially be extended by periodic exposure to an oral rinse containing CHX.", "It appears that this could be effective, and furthermore, a rinse composed of a suspension of CHX-HMP is likely to be more effective, leading to greater CHX release than the CHX digluconate solution.", "However, the data must be interpreted with caution, since while GICs have long been accepted to take up fluoride from their environment, this ability is now recognised to be short-lived, and is lost after the first month of maturation [27].", "Therefore, further investigations regarding the recharge of GICs with chlorhexidine will need to be carried out with more mature specimens to determine whether the same limitations apply.", "A primitive assessment of antimicrobial efficacy, the zone of inhibition or agar diffusion method was adopted in this study.", "The method has many limitations but is commonly used as a first attempt to assess antimicrobial materials, including dental restoratives.", "It is commonly used in other fields to assess antimicrobial susceptibility of particular microbes.", "It is important to bear in mind, if comparing different antimicrobials, that the remote effect (the “zone of inhibition”) is affected not only by the interaction between the microbe and the antimicrobial, but also by the ease by which the antimicrobial can diffuse through the agar.", "CHX, being a comparatively large molecule, diffuses through agar less readily than some antiseptics.", "The aim of the disc diffusion study was to take the first steps in assessing whether the CHX release from the prototype GICs was sufficient to have an adverse effect on growth of oral cariogenic microbes, whilst it is acknowledged that more sophisticated, multi-species models would be beneficial in future in ascertaining the optimal dose of CHX-HMP for a given clinical indication.", "In this simple study, it was illustrated that the 2% CHX-HMP doped specimens inhibited the growth of both St. mutans and Sc. wiggsiae, with a greater effect on the former microbe.", "When adding materials such as CHX salts to GICs it is important to consider the effect that the dissolution of the salts may have on mechanical properties, and the higher the concentration of material the greater the likelihood of an adverse effect on properties such as strength.", "When adding conventional CHX salts digluconate and diacetate, a dose response of an adverse affect on strength is indeed observed, although the threshold for a statistically significant effect varies from study to study and is likely to be affected by a number of factors including sample size and means of incorporating the CHX.", "Turkun et al. [6] observed that 0.5% CHX diacetate and 0.5, 1.25 and 2.5% CHX CHX had no adverse effect on CS but higher concentrations of CHX diacetate did reduce CS.", "There was a numerical reduction in DTS for 2.5% CHX diacetate and digluconate of ∼30% but this was not statistically significant; 0.5 or 1.25% had no effect on DTS either.", "Ahluwalia observed similarly that 1% CHX diacetate had no adverse effect on the CS or DTS of a GIC, but did not investigate higher or lower concentrations [28]; whereas Mittal who utilised concentrations of 1.5 and 3% CHX diacetate observed a ∼30% reduction in CS compared to unmodified GIC [29], while Duque observed no decrease in CS when adding 1.25 and 2.5% CHX digluconate to a GIC [30].", "While it is therefore not possible to draw a definitive conclusion as to what concentration of CHX salts has a significant (whether statistical or clinical) effect on strength, a likely explanation of the reduction in CS and DTS observed by some authors with higher concentrations may be explained Marti’s observation that increased concentration of CHX diacetate or digluconate is accompanied by an increase in porosity [10] which is likely to reduce strength.", "The higher the substitution of CHX-HMP, the more likely a deleterious effect on the mechanical properties, with substitutions of 5% or greater adversely affecting CS, and substitutions of 2% or above adversely affecting BFS, although none significantly affected DTS.", "At the higher concentrations of 5 and 10%, the CS was 100 MPa which is the minimum CS stipulated by the relevant ISO standard for restorative cements [31]; lower concentrations brought the strength more comfortably within the acceptable range at 130–150 MPa.", "The threshold for adverse effects on the mechanical properties of these materials appears therefore to be broadly comparable, or perhaps a little higher, than that for more soluble CHX salts, although it would be interesting to explore how these mechanical properties vary over time as the CHX is released from the various GIC materials.", "It is considered encouraging that for the lower dopings of CHX-HMP the effects on the mechanical properties are moderately small; should such materials become commercially viable, it is plausible that other aspects of the formulation could be modified to compensate for this loss of strength.", "On the other hand, these GIC materials could be considered as prototypes for GIC-based fissure sealants, where absolute strength is of lesser importance and the local delivery of antimicrobial to the fissure areas could be considered beneficial, and as temporary materials designed to stabilise the region before the definitive restoration is placed.", "However, given the calculations presented above regarding the proportion of CHX released from the specimens, and the reflections on relative fluid flow rate and therefore CHX saturation at the tooth-tissue interface, it is plausible that even the lower concentrations of CHX-HMP used here may still offer sufficient CHX release to operate as long-term restoratives without compromise to mechanical properties.", "GICs have been developed which are supplemented with fine, milled, CHX-HMP particles.", "CHX release was sustained for considerably longer and at a more consistent rate than with any previous published studies, and with fewer adverse effects on mechanical properties than many formulations with short lived and front-loaded CHX release.", "Earlier studies with CHX-HMP prepared in different ways have achieved long term CHX release, for example ongoing release after over 400 days in artificial saliva, but this was at the expense of mechanical properties even using much smaller CHX-HMP dopings.", "The materials presented in this manuscript may prove beneficial as technologies with the aim of inhibiting secondary caries in mainstream restorative materials, as well as related materials such as temporary restorations and GIC-based fissure sealants.", "The study was funded by the Medical Research Council as part of a CASE PhD studentship awarded to Candice Bellis.", "Kemdent were the CASE partner and provided in-kind support but did not take an active role in the data acquisition, interpretation or analysis of data, or the decision as to whether and how to publish the data.", "PFD was supported by EPSRC under its ACCIS Centre for Doctoral Training grant, EP/G036772/1." ]
[ "Milled, dry chlorhexidine hexametaphosphate (CHX-HMP) was incorporated into a GIC.", "CHX release was dose-dependent and sustained for at least 660 days.", "GICs could be recharged with CHX and CHX-HMP.", "1% CHX-HMP did not affect compressive, tensile or biaxial flexural strength.", "CHX-HMP GICs inhibited growth of cariogenic microorganisms in an agar diffusion model." ]
Contaminant loading and competitive access of Pb, Zn and Mn(III) to vacancy sites in biogenic MnO2
The scavenging properties of MnO2 are largely attributed to sorption on layer vacancy sites. However, co-occurrence of Mn(II, III) with MnO2 can change mineral reactivity by modifying its Mn(III) content or number of free vacancy sites. These processes are critical in biogenic MnO2 because nascent precipitates are in contact with aqueous Mn(II) and redox processes involving the biomass can modify the Mn(III) content of the oxide. Here we studied the mechanism of Zn(II) and Pb(II) sorption by the biogenic MnO2 precipitated by Pseudomonas putida GB-1. Sorption isotherms on biogenic MnO2 at pH 5.2 ± 0.3 showed considerably higher loadings for Pb(II) (0.49 mol Pb mol−1 Mn) than for Zn(II) (0.12 mol Zn mol−1 Mn). For loadings above 0.1 mol mol−1 Mn, Zn(II) and Pb(II) sorption was concomitant, albeit to different extents, with Mn(II) accumulation in solution. Wet chemical measurements and analysis of Zn K–edge and Pb L3-edge EXAFS spectra showed that the difference in metal loadings on the oxide and extent of Mn(II) release to solution originates from the ability of the cations to displace interlayer Mn(III), which then undergoes disproportionation and accumulates as Mn(II) in solution. In addition, the formation of nanoscale precipitates rich in Pb-P-Cl in the biomass matrix, as shown by energy dispersive X-ray analysis, leads to greater accumulation of Pb than Zn on the biomass. Our findings show how the reactivity of biogenic MnO2 towards co-occurring trace or contaminant metals in natural systems depends directly on the mineral Mn(III) content and competitive sorption processes.
[ "EART" ]
[ "Biogenic minerals", "Biomass-mineral assemblage", "Birnessite", "Competitive sorption", "Contaminant metals", "EDX", "EXAFS spectroscopy", "Lead", "Pseudomonas putida GB-1", "STEM", "Trivalent manganese", "Zinc" ]
[ "1906", "1907" ]
[ "The distribution and bioavailability of inorganic species in environmental systems is regulated by their sorption to solid phases (Brown, 2001; Bradl, 2004; Stumm and Morgan, 2012; Xyla et al., 1992).", "Layer-type manganese oxides (MnO2) are ubiquitous nanoparticulate minerals that are characterised by an unusually high affinity for metal cations.", "Most naturally occurring Mn oxides precipitate upon the biologically-mediated oxidation of Mn(II) and Mn(III) to Mn(IV) (Tebo et al., 2004; Tebo et al., 2005; Spiro et al., 2009; Santelli et al., 2011).", "Biogenic Mn oxides typically are phyllomanganates with hexagonal sheet symmetry, have up to one out of every six Mn(IV) absent from MnO6 octahedral layer sites, and average Mn oxidation numbers ranging from 3.7–3.9 (Tebo et al., 2004; Spiro et al., 2009; Santelli et al., 2011; Webb et al., 2005; Villalobos et al., 2003; Droz et al., 2015).", "The presence of vacancy sites in these oxides leads to local undersaturation of surface oxygen atoms, which can be charge-compensated by cation adsorption upon their diffusion to the interlayer (Spiro et al., 2009; Manceau et al., 2002).", "Oxygen undersaturation at the lateral edges of MnO2 layers creates additional sorption sites that become increasingly abundant as particle size decreases (Manceau et al., 2002; Peña et al., 2015; van Genuchten and Peña, 2016; Takahashi et al., 2007).", "These scavenging properties ensure layer-type Mn oxides an important role in the mobility and bioavailability of contaminants and trace elements in natural systems (Post, 1999; Villalobos et al., 2005a) and motivates their use for the remediation of metal-polluted environments (Haack and Warren, 2003; Gardner et al., 2013; Fuller and Bargar, 2014; Han et al., 2006).", "In regions of active redox cycling, Mn(II, III) often co-occurs with MnO2(s).", "For biogenic MnO2, the active biogeochemical cycling of Mn and heterogeneity in concentration gradients that the biofilm can maintain (Haack and Warren, 2003; Flemming and Wingender, 2010; Stewart, 2003) suggests that local Mn(II) concentrations may be significantly higher near the cells than in the pore water and that nascent precipitates will be in contact with aqueous Mn(II) (Learman et al., 2011).", "The sorption of Mn(II) on the mineral, which increases with increasing pH (Lefkowitz et al., 2013; Zhu et al., 2010a), can be a major pathway for Mn(III)-enrichment in MnO2.", "Specifically, comproportionation between Mn(II) and Mn(IV) (Learman et al., 2011; Mandernack et al., 1995; Bargar et al., 2005; Elzinga, 2011; Q. Wang et al., 2018), results in both layer and interlayer Mn(III).", "Under low Mn(II)/Mn(IV) ratios relevant to natural systems, comproportionation does not cause phase transformations (Zhao et al., 2016; Hinkle et al., 2016; Hinkle et al., 2017).", "However interlayer Mn(III) may migrate into a layer position and the Jahn-Teller distortion of the newly incorporated Mn(III) may modify the stacking and symmetry of the layers from hexagonal to orthogonal (Zhao et al., 2016; Hinkle et al., 2016; Hinkle et al., 2017).", "High pH values (Zhu et al., 2010a; Zhao et al., 2016; Miyata et al., 2007; Kwon et al., 2013) and reaction of Mn(II) at the edges of the particles may facilitate the enrichment of structural Mn(III).", "For lower pH regimes and in particular for biogenic MnO2 (Simanova et al., 2016), where sorption of microbial biomass may slow the reaction at the particle edges relative to abiotic MnO2, structural Mn(III)-enrichment may be less important than its sorption at vacancies sites.", "For biomineral assemblages, Mn(III)-enrichment of the mineral may also result from partial reduction of the oxide by organic residues (Julia, 2018).", "The capping of vacancies by Mn(II) or Mn(III) can decrease the extent of metal sorption as shown for Ni(II) and Zn(II) in abiotic systems (Hinkle et al., 2017; Simanova et al., 2015).", "However, based on the position of the adsorbing metal in the affinity sequence for MnO2 relative to Mn(II) and Mn(III), incoming metals may be able to displace adsorbed Mn(II, III).", "Selectivity sequences have been established through macroscopic measurements for a number of metals (Murray, 1975; McKenzie, 1980; Tamura and Furuichi, 1997; Tani et al., 2004) The selectivity sequence Ni < Zn ≤ Co ≤ Mn < Cu < Pb has been determined for MnO2 through sorption experiments (Murray, 1975; McKenzie, 1980; Tamura and Furuichi, 1997) and can be explained by the stability of the surface complex formed, which in turn depends on factors such as the ionic potential, electron configuration and acidity of the adsorbing cation (Sposito, 2008).", "This sequence is consistent with the selectivity sequence obtained through competitive sorption experiments of transition metal cations on biogenic MnO2 at pH 7 (Tani et al., 2004).", "While Mn is included in these selectivity sequences and studies have shown the ability of Mn(III) to limit metal sorption at vacancy sites, sorption data have not been interpreted from the viewpoint that metal cations may potentially displace Mn(III).", "The hypothesis guiding this work is that the position of cations on the affinity serie relative to Mn(II) and Mn(III) governs the cation exchange mechanism and thus their sorption by MnO2.", "Furthermore, depending on the accessibility of metals to vacancy sites, metal cations may partition to other surface sites, including those on the biomass (Droz et al., 2015; Haack and Warren, 2003; Toner et al., 2005; Toner et al., 2006; Peña et al., 2011; Zhu et al., 2010b; Nelson et al., 1999a; Peña et al., 2010) or at the mineral particle edges (Manceau et al., 2002; Peña et al., 2015; van Genuchten and Peña, 2016; Takahashi et al., 2007), given their availability of the latter in biogenic MnO2.", "To test these hypothesis, we compared the relative sorption behaviour of Zn(II) and Pb(II) on biogenic MnO2.", "These common contaminants are found on both sides of Mn on the affinity sequence for MnO2 surfaces (Murray, 1975; McKenzie, 1980) and therefore are ideal probes of mineral reactivity.", "Furthermore, spectroscopic studies have identified the coordination geometries of Zn(II) and Pb(II) sorbed by MnO2.", "Specifically, Zn(II) adsorbs as triple-corner-sharing (TCS) complexes at vacancy sites, adopting a tetrahedral geometry (ZnIV) at low loading and an octahedral coordination (ZnVI) at high loading (Manceau et al., 2002; Takahashi et al., 2007; Hinkle et al., 2017; Toner et al., 2006; Kwon et al., 2009).", "For Pb(II), in addition to TCS complexes, stable complexes on the edges of MnO2 have been described (van Genuchten and Peña, 2016; Takahashi et al., 2007; Villalobos et al., 2005b; Kwon et al., 2010).", "In the current study, sorption isotherms of Zn(II) and Pb(II) were measured on the biogenic MnO2 precipitated by the bacterium Pseudomonas putida GB-1 at pH 5.2 ± 0.3.", "Since neither Zn(II) nor Pb(II) undergoes redox changes upon reaction with biogenic MnO2 (Manceau et al., 2002; Toner et al., 2006; Villalobos et al., 2005b), aqueous Mn(II) was used to measure the displacement of Mn(II, III) by Zn(II) and Pb(II).", "Sorption isotherms were also measured on biomass-only sorbents to estimate the highest possible surface loadings of the metals to the organic fraction on the bio-mineral sorbent.", "Analysis of Zn K-edge and Pb L3-edge EXAFS spectra was used to identify the sorption mechanism as a function of the affinity of the metal cations for MnO2 and availability of reactive surface sites.", "Finally, scanning transmission electron microscopy combined with energy dispersive X-ray analysis of selected samples was used to determine whether polymerization or precipitation occurred in the samples.", "These findings improve our understanding of competitive sorption processes on biogenic MnO2 and the fate of contaminant metals in systems containing Mn(III)-rich MnO2.", "All solutions were prepared using ultrapure water (>18 mΩ cm) and ACS-grade reagents.", "The Mn(II)-oxidizing bacterium Pseudomonas putida GB-1 (P. putida) was used to produce biomass-only and biogenic MnO2 suspensions.", "All cultures were propagated under sterile conditions in a nutrient-rich growth medium (Toner et al., 2005; Peña et al., 2010; Tebo et al., 2007).", "Leptothrix medium was prepared by dissolving glucose (1.0 g L−1, 5.5 mM), casamino acids (0.50 g L−1), yeast extract (0.50 g L−1) and HEPES acid (2.38 g L−1, 10 mM) in ultrapure water and autoclaved at 120 °C for 20 min.", "After cooling, the medium was amended with filter-sterilised CaCl2 (0.5 mM), MgSO4 (0.83 mM), FeCl3 (3.7 μM), CuSO4 (40 nM), ZnSO4 (152 nM), CoCl2 (84 nM), Na2MoO4 (54 nM), and either 0 or 1 mM MnCl2, which are denoted as (−)Mn or (+)Mn cultures, respectively.", "Biomass-only and biogenic MnO2 suspensions were prepared by inoculating (−)Mn or (+)Mn growth medium (125 mL) in Erlenmeyer flasks (250 mL) capped with foam stoppers and incubating on a table-top shaking incubator in the dark (27 °C, 150 RPM).", "The innocula for biomass-only and biogenic MnO2 suspensions were transferred from P. putida suspensions grown in (−)Mn or (+)Mn medium, respectively, for 30 ± 12 h.", "The resulting sorbents were harvested after 120 ± 24 h of incubation and washed from their spent growth media by centrifuging the suspensions (3700 RCF, 10 °C, 30 min) and re-suspending the pellets in a NaCl solution (10 mM), with 30 min equilibration between centrifuge cycles on an end-to-end rotator.", "The washing procedure was repeated twice, and the washed sorbents were suspended in 10 mM NaCl.", "Given the ability of HEPES buffer to reduce Mn(IV) in suspensions of abiotic Mn oxides (Simanova et al., 2015; Ling et al., 2015), HEPES buffer was excluded from the electrolyte.", "Sorbent concentrations (csorbent, kg L−1) were determined gravimetrically.", "A 10 mL aliquot of the biomass-only or biogenic MnO2 suspensions was sampled and the sorbent was collected in Epi-Tubes 1 mL at a time by centrifuging (14,000 RCF, 5 min) and discarding the supernatant.", "The pellets were oven-dried for 48 h at 45 °C.", "Reported csorbent values are the average of triplicate measurements.", "The concentration of MnO2 in biogenic MnO2 was measured by ICP-OES (Perkin-Elmer Optima 8300) as described below.", "Zinc(II) and Pb(II) isotherms were measured at pH 5.2 ± 0.3.", "Biomass-only and biogenic MnO2 suspensions (15 ± 0.1 mL) were transferred to opaque high-density polyethylene (HDPE) bottles (30 mL) from the sorbent suspension under vigorous stirring (Peña et al., 2010).", "Zn(II) and Pb(II) additions were made from stock solutions of ZnCl2 (10 mM and 100 mM) and Pb(NO3)2 (10 mM and 100 mM) to yield metal concentrations ranging from 0 μM to 600 μM.", "Sorption experiments were done in the absence of HEPES buffer and no pH adjustments were made during the experiments; suspension pH was regulated by the buffering capacity of the sorbents themselves.", "All experiments were performed in triplicate.", "Sorption samples were equilibrated for 24 h on an end-to-end rotator before sampling for chemical analysis.", "Concentrations of Mn, Zn(II) and Pb(II) were measured by ICP-OES (Perkin-Elmer Optima 8300) using 2 or 3 emission lines for each element.", "At the end of the reaction, aqueous metal concentrations (cPb, cZn, and cMn) were measured from 5 mL aliquots obtained by filtration (0.20 μm pore size, polyethersulfone PES) and acidification with 50 μL of HNO3 65%.", "To determine the total metal concentrations in biomass and biogenic MnO2 suspensions (cPb_TOT, cZn_TOT, cMn_TOT), 1 mL aliquots of the suspensions were acid-digested for 10 min with 50 μL of 1 M oxalic acid and 50 μL of HNO3 65% to achieve complete dissolution of the mineral phase and/or release of all metal ions to solution.", "After centrifugation of the digested suspensions (14,000 RCF, 5 min), the supernatants were diluted and analysed for Zn(II), Pb(II) and Mn(II).", "All solutions were undersaturated with respect to common oxide, hydroxide and carbonate minerals [ZnO, Zn(OH)2, ZnCO3(s) Pb2(OH)3Cl(s), PbCl(OH)(s), Pb2O(OH)2(s), PbO(s), Pb(OH)2(s), PbCO3(s)] as determined by speciation calculations in MINEQL+ (C. W. J., 1976).", "Saturation indices for all solids are shown in Table A1.", "Because Pb can easily precipitate, additional controls were run in triplicate to verify the absence of Pb precipitates when adding 10, 50, 100, 250, 450 and 650 μM PbNO3 to 10 mM NaCl (pH 5.5 maintained by NaOH addition).", "Control samples were equilibrated on an end-over-end shaker for 24 h before sampling (0.2 μm PES filter) and were not diluted prior to chemical analysis.", "The difference between the Pb concentration in the filtrates and in acid digests (50 μL of 1 M oxalic acid and 50 μL of HNO3 65% added to 5 mL sample) was within instrument uncertainty (<2.6%) (Table A2), confirming the absence of homogeneous precipitation of Pb.", "Surface excess qi (with i = Zn or Pb), which is defined as the number of moles of i partitioned to the sorbent relative to its concentrations in the supernatant, normalised by the quantity of sorbent (Sposito, 2008), was expressed in both units of mol i kg−1 sorbent and mol i mol−1 Mn.", "Values of qi were calculated according to (ci_TOT − ci_aq)/csorbent or (ci_TOT − ci_aq)/(cMn_TOT − cMn_aq).", "All wet chemical data is provided in Tables A3–6.", "Separate samples for X-ray absorption spectroscopy were prepared as described above and are named according to metal_% loading (mol i mol−1 Mn) (Table A7).", "Metals were added dropwise from 10 mM ZnCl2 and Pb(NO3)2 solutions to the sorbent suspensions and equilibrated for 26 h on an end-to-end rotator before chemical analysis.", "Finally, pellets were collected through successive centrifugation cycles.", "Excess moisture was removed with a Kim-wipe before freezing the samples in a 1.5 mL EpiTube at −4 °C.", "At the beamline, wet pastes were thawed, homogenized and loaded on aluminum sample holders sealed with Kapton tape.", "X-ray absorption spectra were collected at beamline 4–1 of the Stanford Synchrotron Radiation Lighsource (SSRL, Stanford, US) using a Si(220) ϕ = 90° crystal monochromator.", "The monochromator energy was calibrated using Zn(0) and Pb(0) foils with binding energies of 9659 eV and 13,035 eV respectively, in a double-transmission configuration.", "Data were acquired under liquid‑nitrogen conditions (77 K) using a Lytle ion chamber detector in fluorescence mode.", "Scattering of the incident beam was attenuated by Cu-3 and Ga-3 filters for Zn(II) and Pb(II) analysis respectively, and by soller slits.", "Three to four replicate spectra were collected for each individual sample.", "The EXAFS spectra were analysed using the SixPack program (Webb, 2005), which is built on the IFEFFIT engine (Newville, 2001).", "Replicate spectra were aligned to a common energy scale and averaged.", "The averaged spectra were then background subtracted with the following parameters for Zn K-edge and Pb L3-edge respectively: E0 = 9659 (Zn) and 13,035 (Pb) eV, Rbkg = 1, no clamps, splined between 1.0 and 11 Å−1 (Zn) and 1.0 and 10.8 Å−1 (Pb) with a linear pre-edge function and a quadratic post-edge function, a Kaiser-Bessel Fourier-transform window and k-weight = 3.", "Zinc K-edge EXAFS spectra of the Zn-biogenic MnO2 samples were analysed by linear combination fitting (LCF) of reference spectra using the least-square fitting module in SixPack to determine the partitioning of Zn(II) between the biomass and mineral fractions of the sorbent, as well as the distribution of Zn(II) between tetrahedral (ZnIV) and octahedral (ZnVI) coordination geometries.", "The sum of components was allowed to vary during the fitting of the experimental spectra and the reduced chi-squared (χ2red) values were used to evaluate the quality of the fits.", "Reference spectra collected for Zn(II)-sorbed on P. putida biomass (Ref. Brown (2001): q ~ 0.1 mol Zn kg−1 biomass, pH 6.0, 10 mM NaCl and HEPES) and on biogenic MnO2 (Ref. Bradl (2004): q = 0.09 Zn mol−1 Mn, pH 6.5, 10 mM NaCl and HEPES, 100% ZnIV and Ref. Stumm and Morgan (2012): q = 0.12 mol Zn mol−1 Mn, pH 7.0, 10 mM NaCl and HEPES, 59% ZnIV/41% ZnVI).", "The fraction of ZnIV in Ref. Bradl (2004) was inferred from shell-by-shell fitting, which showed characteristic structural parameters for tetrahedral Zn [R(Zn-O) = 1.96 ± 0.01 Å, CN(Zn-O) = 3.72 ± 0.66; σ2 = 0.005 ± 0.002 Å2; R(Zn-Mn) = 3.34 ± 0.01 Å, CN(Zn-Mn) = 7.12 ± 1.61, σ2 = 0.008 ± 0.002 Å2] (Pena et al., 2010).", "For Ref. Stumm and Morgan (2012), the average coordination of Zn(II) was determined by LCF using Ref. Bradl (2004) and chalcophanite (ZnMn3O7⋅3H2O, 100% ZnVI) as reference spectra; the fit returned 41% and 59% ZnIV and ZnVI, respectively.", "Due to the lack of reference spectra acquired under the same conditions as the experimental samples, the Pb L3-edge EXAFS spectra of Pb-sorbed biogenic MnO2 were analysed by shell-by-shell fitting using the FEFF EXAFS fitting interface in SixPack.", "The CrystalMaker® software (CrystalMaker®, 2013) was used to visualise the coordination environments of Pb(II) on MnO2 and to generate a list of atomic coordinates needed to calculate phase and amplitude scattering paths for the Pb-MnO2 cluster in FEFF6 (Newville, 2001; Rehr et al., 1991).", "An amplitude reduction factor (S02) of 0.84 was used in all fits, which was determined previously for Pb(II) sorbed on δ-MnO2 (Villalobos et al., 2005b).", "All EXAFS spectra were fitted in R-space over the k-range of 3–10.8 Å−1 and R-range 1–4 Å, and plotted alongside reference EXAFS spectra for Pb(II) sorbed on abiotic δ-MnO2 [0.2 mol Pb mol−1 MnO2, pH 5.8, 7 days equilibration, collected at room temperature (Villalobos et al., 2005b)] and Pb(II) sorbed on Penicillium chrysogenum cell wall powder [0.01 mol Pb kg−1, pH 6.0, 24 h of equilibration, collected at room temperature on dried samples (Sarret et al., 1998)].", "Separate samples were prepared as described above using both the biomass-only and biomineral sorbents for microscopy analysis.", "Two samples were generated for each metal and each sorbent using low (200 μM) and high (600 μM) metal concentrations; data are presented for all samples except for the high Zn concentration added to the biomass-only sorbent.", "The loadings of the biomass-only sorbents were approximately 0.07 mol Zn kg−1 biomass and 0.2 mol Pb kg−1 biomass.", "For biogenic MnO2 samples, the loadings were approximately 0.2 mol Zn kg−1 biooxide (0.1 mol Zn mol−1 Mn), 0.3 mol Pb kg−1 biooxide (0.2 mol Pb mol−1 Mn) and 0.7 mol Pb kg−1 biooxide (0.5 mol Zn mol−1 Mn).", "At the end of the sorption reaction, an aliquot of the sample was transferred to a 2 mL epi tube and washed twice using a 10 mM NaCl solution to remove any aqueous Zn or Pb from the slurry.", "After resuspension, a 5 μL aliquot was added by pipette onto a formvar carbon-coated copper grid that was previously glow discharged.", "The sample was allowed to settle for 15 min.", "Subsequently, the excess liquid was wicked with filter paper and replaced with a 5 μL aliquot of ultrapure water.", "The ultrapure water was then wicked with filter paper and the grid was transferred to the sample holder and into the instrument.", "Imaging was conducted in a Zeiss GeminiSEM 500 using a STEM detector with annular diode detection in high-angle annular dark-field mode at 30 kV, a back-scattered electron detector at 30 kV or an in-lens detector at 5 kV.", "Due to strong scattering of biogenic MnO2, images were taken at different gain values in HAADF mode, or with both back-scattered and in-lens detectors and the layers were blended in Adobe Photoshop for visualization of the full information in the image.", "Original images are available at DOI:https://doi.org/10.5281/zenodo.1322364.", "EDX mapping was performed at 30 kV, high current mode and 120 μm aperture using an Oxford AZTec microanalysis system with an X-MAX 150 mm2 detector.", "The EDX system is set up for SEM, not STEM.", "Therefore, it can perform qualitative but not quantitative maps.", "Hence, the intensity of each element in their corresponding map does not correspond to relative amounts between elements (i.e., each element has its own relative scale) and the maps could thus be digitally enhanced using Photoshop for better visualization without altering information.", "The sorption isotherms of Zn(II) and Pb(II) on the biomass-only sorbent are presented in Fig. 1A. Both isotherms are L-type and show moderate sorption of the metals by organic functional groups on the biomass.", "The maximal surface excesses reached by Zn(II) and Pb(II) were 0.07 ± 0.04 and 0.20 ± 0.02 mol kg−1 respectively.", "The greater sorption capacity of the biomass for Pb(II) as compared to Zn(II) is consistent with the soft character of Pb(II) and reported literature values.", "At high metal concentrations, moderate and large scatter was observed in the surface excess values measured for replicate experiments.", "This variability in Zn(II) loadings was especially pronounced at cZn > 200 μM and may arise from a biological response of the bacteria to high Zn(II) concentrations.", "On the contrary, the high toxicity of Pb(II) or difference in its sorption mechanism may have inhibited any biological activity during the sorption experiments.", "Similar scattering observed in analogous sorption isotherms studies conducted using Ni (Droz et al., 2015; Peña et al., 2010) has been ascribed to a non-linear biological response to high concentrations of toxic contaminants.", "Sorption isotherms for Zn(II) and Pb(II) on biogenic MnO2 are presented in Fig. 1B. Both isotherms are H-type, which demonstrates the extremely high affinity of MnO2 for these metal cations.", "The sorption isotherms showed a significantly higher sorption affinity and capacity for Pb(II) compared to Zn(II), which is consistent with previous studies (van Genuchten and Peña, 2016; Villalobos et al., 2005a; Toner et al., 2006; Nelson et al., 1999a; Villalobos et al., 2005b; Matocha et al., 2001).", "While the mass of sorbent only increased by only 16–24% in biogenic MnO2 compared to biomass-only suspensions, the sorption capacity of the bio-mineral assemblages was 2–3 times greater than in the biomass-only sorbent, with surface excesses reaching 0.15 ± 0.04 mol Zn kg−1 and 0.67 ± 0.03 mol Pb kg−1.", "The large increase in loadings attained in the presence of MnO2 (up to 0.08 mol Zn kg−1 and 0.47 mol Pb kg−1) is consistent with significant partitioning of both metals to the inorganic phase.", "The Zn and Pb sorption isotherms on biogenic MnO2 also showed less scatter in replicate experiments at high loadings relative to the biomass-only sorbent.", "This difference may reflect the reduced exposure of the bacterial cells to the contaminants in the biogenic MnO2, either due to differences in cell viability between the two sorbents or physico-chemical protection of the cells by the mineral.", "However, any impact of metal toxicity on metal sorption (adsorption or absorption) by the biomass is not accounted for in the calculation of surface excess, which considers only the total mass of sorbent per unit volume.", "Because of the significant sorption of Zn(II) and Pb(II) to the inorganic phase inferred from Fig. 1A–B, surface excess is expressed in units of mol i mol−1 Mn in Fig. 1C.", "The lack of a clear x-intercept in plots of Kd (qi/ci) versus qi did not support the calculations of qmax values.", "Thus, we defined qmax for Zn(II) and Pb(II) as the highest loading measured (± standard deviation of the 7 and 12 highest loadings).", "For Zn, this value was calculated for samples where cZn < 200 μM due to the scatter in the data at greater concentrations.", "The sorption isotherms showed qmax of 0.12 ± 0.01 mol Zn mol−1 Mn and 0.49 ± 0.02 mol Pb mol−1 Mn and indicated the presence of 0.06 and 0.35 mol of high affinity surface sites per mole of MnO2 for Zn(II) and Pb(II) (0.09 and 0.50 mol kg−1respectively) as indicated by the negligible concentration of aqueous Zn(II) or Pb(II) in equilibrium with the sorbent (ci_aq < 3.0 μM).", "Fig. 2 shows that the sorption of Zn(II) and Pb(II) was accompanied by a loading-dependent accumulation of aqueous Mn(II).", "This figure includes the data from Zn (cZn < 300 μM) and Pb (all samples) but excludes the data from experiments where cZn > 300 μM given the large scatter between replicates and generally decreasing surface loading.", "The amount of Mn(II) released for both metals followed the same S-curve but was significantly higher in the Pb(II) experiments than in Zn(II) experiments.", "For both metals, <3 μM Mn(II) was detected at loadings below 0.10 mol mol−1.", "For Zn(II), up to 0.04 mol Mn(II) mol−1 Mn was measured at loadings >0.10 mol Zn mol−1 Mn.", "For Pb(II), the data showed three distinct trends (R1, R2, R3): no release of Mn(II) for 0.0–0.10 mol Pb mol−1 Mn (R1), linear Mn(II) release with increasing loading up to qPb = 0.49 ± 0.02 mol Pb mol−1 Mn, with a slope of 0.60 mol Mn(II)released mol−1 Pbsorbed (R2 = 0.97)(R2), and, finally, no additional Mn(II) release once the maximal surface excess was reached (R3).", "For qi values >0.1 mol i mol−1 Mn, when sorption of i is associated to the displacement of sorbed Mn, a plot of qi/(ci/cMn) versus qi should yield a straight line with a slope of - Kapp.", "Because of the large variability in q values at high Zn(II) concentrations, qZn/(cZn/cMn) versus qZn were plotted only for cZn < 200 μM (Fig. A1).", "The selectivity coefficients Kapp for Zn(II) and Pb(II) were 0.11 ± 0.12 (95% confidence interval, R2 = 0.41) and 108.19 ± 16.65 (95% confidence interval, R2 = 0.93) respectively, indicating no selectivity for Zn(II) over Mn and a very large selectivity of Pb(II) relative to Mn at pH 5 (Sposito, 2008).", "For Zn(II), the low selectivity coefficient and poor R2 value did not allow us to constrain the total number of reactive surface sites (qTOT).", "For Pb(II), 0.49 ± 0.02 mol reactive sites mol−1 MnO2 was estimated.", "Given structural constraints (i.e., maximum number of vacancy sites of 0.2 mol mol−1 Mn), this value for the number of total surface sites is likely too large and thus indicate additional uptake mechanisms for Pb as discussed below.", "The Zn K-edge EXAFS spectra and corresponding Fourier transforms (FT) for Zn(II) sorbed on biogenic MnO2 are reported in Fig. 3A–B, alongside reference spectra for Zn(II) sorbed on bacterial biomass and two biogenic MnO2 samples showing Zn in tetrahedral and a mixture of tetrahedral and octahedral coordination on MnO2, respectively.", "The loadings of Zn(II) on the organic and inorganic fractions of the sorbent were determined by scaling the measured surface loading by the fractions obtained from linear combination fitting (LCF, Table 1).", "We found between 0.06 and 0.09 mol Zn kg−1 sorbent partitioned to the biomass.", "The significant contribution of the biomass to Zn(II) sorption in the lowest loading sample suggests the presence of surface sites with a sufficiently high affinity for Zn(II) to compete with MnO2.", "These EXAFS-derived loadings of Zn(II) on the organic fraction of biogenic MnO2 are consistent with the data for the biomass-only system, which show a qmax of 0.07 ± 0.04 mol Zn kg−1 biomass.", "In the biogenic MnO2 samples, the EXAFS-derived loading of Zn(II) on the mineral fraction increased with increasing loading and ranged from 0.07 to 0.21 mol Zn kg−1 sorbent (0.03 to 0.10 mol Zn mol−1 Mn) This trend is consistent with studies that place Zn(II) towards the low-affinity end of the selectivity sequence for MnO2.", "In addition, all samples showed a dominant contribution from Zn in tetrahedral coordination in all samples (0.03–0.07 mol ZnIV mol−1Mn) and a moderate but consistent increases of Zn in octahedral coordination with increasing loading (0.0–0.03 mol ZnVI mol−1 Mn).", "The above interpretation assumes that Zn(II) does not partition to the particle edges.", "Because the Zn-Mn distances in the Zn-TCS complex on vacancies and DCS complexes on particles edges are presumably too similar to be distinguished by EXAFS (Hinkle et al., 2017; Simanova et al., 2015), the LCF analysis cannot distinguish sorption on edges to that on biomass.", "Linear combination of the spectra for Zn-TCS complex on vacancies (CNZn-Mn 6) and of Zn on biomass (CNZn-Mn 0) would yield a spectrum very similar to that expected for Zn(II) sorbed on edges (CNZn-Mn 2) (Droz et al., 2015; Peña et al., 2011).", "However, Zn partitioning to the particle edges was not observed in biogenic MnO2 in the reference spectrum (CN ~ 6, obtained from shell-by-shell fitting) at loadings comparable to those measured in this study.", "Thus, Zn partitions preferentially to vacancy sites, if available, and not particle edges.", "Furthermore, the loadings of Zn(II) on biomass in biogenic MnO2 as predicted by LCF are consistent with those measured for the biomass-only sorbent and thus argues against significant partitioning of Zn(II) to edge sites.", "The Pb L3-edge EXAFS spectra and corresponding Fourier transforms (FT) for Pb(II) sorbed on biogenic MnO2 are reported in Fig. 3C–D, alongside reference spectra for Pb(II) sorbed on bacterial biomass and δ-MnO2.", "The EXAFS spectrum of Pb(II) sorbed on biomass showed one main oscillation and no significant second-shell structure in the FT. The EXAFS spectra from biogenic MnO2 samples showed a strong splitting of the second oscillation around k = 5.5–6.0 Å−1 and large amplitude in the second-shell (Pb-Mn) peak in the FT. This splitting was much more pronounced in the EXAFS spectra from biogenic MnO2 than δ-MnO2, where the latter was reported as having 75% of the Pb(II) adsorbed at the particle edges and 25% adsorbed at vacancy sites.", "The shape of this oscillation, which correlates with the second-shell amplitude, thus indicates that Pb(II)-TCS complexes at vacancy sites represent the dominant binding mode in biogenic MnO2.", "The EXAFS spectra for the experimental samples and thus average coordination environment were similar to each other irrespective of the Pb(II) loading, which ranged from 0.04 to 0.44 mol Pb mol−1 Mn.", "The EXAFS spectrum from the lowest loading sample, however, appeared slightly shifted to lower k values relative to the higher loading samples.", "The superposition of the rising edge across all samples, as seen by plotting the first derivative of the mu spectra in Fig. A2, confirmed that the differences did not originate from energy misalignment, but indicate a slightly different coordination environment for Pb(II) at low loading that is not captured well by the EXAFS spectra.", "The Fourier transforms of Pb(II) sorbed biogenic MnO2 samples consist of two main peaks at R + ∆R = 1.7 Å and at R + ∆R = 3.3 Å, which correspond to Pb-O and Pb-Mn single scattering interactions, respectively.", "Shell-by-shell fitting using two single scattering paths was used to fit the EXAFS spectra from biogenic MnO2 and the δ-MnO2 reference.", "Fits (Figs. 3C–D and A3D) and fitting parameters are reported in Table 2.", "The quality of the fits was not improved by adding a second shell for a Pb-Mn scattering interaction near 3.5 Å (Fig. A3A–C and Table A8) and therefore did not justify the addition of a second Pb-Mn shell.", "Overall, fit parameters were largely independent of loading, which indicates similar average coordination geometry of Pb(II) at all loadings.", "The first shell Pb-O atomic distance determined was 2.27 ± 0.02 Å and the Pb-Mn interatomic distance was 3.71 ± 0.03 Å.", "These interatomic distances are consistent with those reported for Pb(II) binding to MnO2 vacancies as TCS complexes (Manceau et al., 2002; Villalobos et al., 2005b; Matocha et al., 2001).", "Similarly, the σ2 values (0.008 ± 0.003 Å2 and 0.009 ± 0.002 Å2) were consistent with those reported in the literature (0.008–0.011 Å2) (Manceau et al., 2002; Villalobos et al., 2005b; Matocha et al., 2001).", "The coordination numbers (CN) for both Pb-O (2.49 ± 0.71) and Pb-Mn (3.76 ± 1.38) pairs were consistently low compared to the ideal CN of 6 expected for TCS coordination and were the same for all samples when considering the fitting uncertainty.", "Low fitted CNs, are typical for Pb-MnO2 samples (Manceau et al., 2002; Villalobos et al., 2005b; Matocha et al., 2001) due to the large degree of disorder inherent to Pb(II) and the variety of first-shell coordination geometries that Pb(II) can adopt (Manceau et al., 2002).", "The low coordination numbers obtained for the Pb-Mn shell may also indicate that Pb sorbs in part to the biomass or in other configuration(s) where the second-shell neighbour has a low coordination number and is a low-scattering atom (e.g., C, P or O) that contributes little amplitude to the Pb-Mn shell.", "This point is underlined by the highest measured surface excess value (0.49 mol Pb mol−1 Mn), which is greater than twice the vacancy content (and hence the upper limit for Pb sorption at vacancy sites assuming double-capping of the vacancies).", "Thus, Pb must partition to the biomass, the particle edges or polymerize/precipitate as observed for Cu (Peña et al., 2015).", "Below we discuss plausible scenarios as constrained by the sorption isotherms, measurements of Mn(II) in solution as a function of Pb(II) loading, the modelling of the EXAFS spectra and EDX analysis.", "Figs. A4–5 and 4 show the STEM-EDX images for Zn(II) and Pb(II) sorbed on biomass-only and biogenic MnO2 sorbents, with elemental maps showing the distribution of C, Ca, P, Cl, and if present Mn, Zn and Pb.", "Carbon, and P were selected to locate cells and/or EPS components and Mn to locate MnO2 deposits.", "For Zn(II) sorbed on biomass (Fig. A4A), Zn appears to be homogenously distributed over the sample and co-located with C and P.", "In the presence of the oxide however, for both the low and high Zn(II) samples (Fig. 4A–B), the images show a strong colocation of Zn and Mn.", "While Zn appears to be strongly enriched in areas containing Mn, vis MnO2, Zn is more weakly distributed in areas rich in C but not Mn, suggesting a preferential partitioning of Zn to the oxide or to EPS components associated with the oxide.", "The latter scenario, that Zn partitions preferentially to EPS components associated to MnO2 is supported by the analysis of the Zn K-edge EXAFS spectra.", "Furthermore, while EXAFS analysis shows 26–44% Zn partitions to the biomass, the organics contribute >80% of the sorbent on a mass basis, which further corroborates the weak to moderate distribution of Zn on the cells and/or EPS in Mn-poor areas within the EDX maps.", "For Pb(II) spiked to biomass samples, both at low and high loadings (see Fig. A4B–C), the EDX maps show strongly scattering Pb(II)-rich areas that collocate with both P and Cl, thus suggesting the formation of Pb-P-Cl-containing pyromorphite-like precipitates (Rhee et al., 2012).", "At low Pb(II) concentration and in the presence of the oxide (see Fig. 4C), no precipitates were observed, with Pb(II) appearing to be homogeneously distributed with a distinct colocation with Mn, and no distinct colocation with either C or P.", "At high Pb(II) concentration (Figs. 4D, A5B–C), Pb was always strongly collocated with Mn, but two out of three maps showed a grainy texture in some portions of the sample, which correlated with the distribution of P and Cl.", "These observations may indicate some precipitation and/or a change in aggregation state of both MnO2 and organics as surface charge changes with very high Pb loadings.", "While the thickness of the sample limits our ability to determine whether the precipitates are formed on the organic or the mineral fractions, several lines of evidence favour the interpretation that the precipitates are associated with the biomass: i) no precipitation was observed in control solutions containing Pb-NO3-Cl (Table A2), strong evidence for Pb-P-Cl co-precipitation was observed in the biomass-only samples (Fig. A4), and no precipitates were observed in the low loading biooxide samples but some evidence for precipitation was observed in the high loading biooxide sample (Figs. 4 and A5A–B).", "Combined these results are consistent with the preferential sorption of Pb(II) on MnO2 as identified from EXAFS spectroscopy and moderate partitioning on the biomass/EPS components, such that Pb precipitation may be induced at high Pb concentrations upon sorption and subsequent saturation of phosphate-rich functional groups present on cell surfaces, EPS components or secreted by the cells from internal stores (Tobin et al., 2007).", "Biogenic MnO2 consists of microbial biomass and mineral particles in close physico-chemical association and therefore forms a dynamic system with potential for active redox recycling of Mn and multiple types of surface sites available for contaminant sorption.", "In this study, we investigated the sorption of Zn(II) and Pb(II) on biogenic MnO2, two metals found on both sides of Mn(II) and Mn(III) on the affinity serie for MnO2 vacancies.", "Sorption isotherms showed a significantly larger loading of Pb(II) compared to Zn(II), while TCS complexes at vacancy sites were prevalent for both Zn and Pb(II) as determined from EXAFS spectroscopy.", "The 3–4 fold higher qmax reached upon Pb(II) sorption compared to Zn(II) indicates that Pb(II) had access to 0.52 mol binding sites kg−1(0.37 mol binding sites mol−1 Mn) inaccessible to Zn(II).", "The absence of Mn release during the sorption of the initial 0.10 mol i mol−1 Mn indicates that up to 0.10 mol mol−1 Mn of reactive sites are equally available to both metals, even if the sorption sites accessible to both metals are not necessarily the same.", "The accumulation of up to 150 μM Mn(II) in solution that accompanied the sorption of Pb(II) for qPb > 0.10 mol Pb mol−1 Mn and the invariant average coordination environment for Pb inferred from EXAFS spectroscopy indicate that Pb(II) can displace any Mn(II, III) occupying vacancy sites to achieve remarkably high surface loadings on MnO2.", "Instead, increasing the Zn loading on MnO2 from 0.03 to 0.10 mol Zn mol−1 Mn was accompanied the release of only up to 0.02 mol Mn mol−1 to solution.", "Similarly, Grangeon et al. (2012), observed a 0–2% loss in interlayer Mn(III) as the Zn(II) loading was increased from 0.003 to 0.06 mol Zn mol−1 Mn at pH 5 in vernadite suspensions.", "These results are consistent with the affinity sequence for MnO2 with Zn(II) < Mn(II), Mn(III) < Pb(II) and a competitive access to sorption sites (Murray, 1975; McKenzie, 1980; Matocha et al., 2001).", "Biogenic MnO2 are enriched in Mn(III), with average manganese oxidation number (AMON) reported to be close to 3.7 for the biogenic MnO2 produced by P. putida cultured with 1 mM Mn as done in this study (Tebo et al., 2004; Santelli et al., 2011; Webb et al., 2005; Villalobos et al., 2003; Droz et al., 2015).", "More specifically, agreement between pyrophosphate extractable Mn(III) (~30% (Peña et al., 2017)) and potentiometric titrations indicate little Mn(II) associated with the oxides.", "Therefore, aqueous Mn may derive primarily from reductive dissolution of MnIVO2 or disproportionation of two Mn(III) metal centres into Mn(II) and Mn(IV).", "The reductive dissolution of MnO2 is unlikely both because of the plateau reached in the amount of Mn(II) released upon reaching qmax and because Manceau et al. (Manceau et al., 2002) showed no oxidation of Pb(II) during the sorption by MnO2.", "The release of Mn therefore most likely originates from the disproportionation of interlayer Mn(III), which is destabilised by Pb(II), a soft metal that can form bonds with surface oxygen atoms of greater covalent character than Zn(II), as it approaches from the other side of the vacancies.", "The displaced Mn(III) can then disproportionate, either with a layer-Mn(III) or with interlayer-Mn(III) located on neighbouring vacancies as illustrated in Fig. 5, and generate aqueous Mn(II) and solid-phase Mn(IV).", "This mechanism implies a qPb of about 20 to 40% (for single or double occupancy of vacancies) that derives from vacancy sites initially occupied by Mn(III).", "The slope of Mn(II) accumulated in solution as a function of Pb(II) sorbed (Fig. 2), which shifted from 0 to 0.6 (R2 = 0.97), can be used to constrain possible sorption mechanisms.", "The sorption of Pb(II) on available free vacancies does not require the displacement of any Mn (slope of 0).", "Sorption on biomass, as either a surface complex or precipitate as identified through EDX analysis, or MnO2 particle edges would also yield a slope of 0.", "Any of these sorption mechanisms (or a combination thereof) can explain the R1 phase of sorption (qi ≤ 0.10 mol i mol−1 Mn).", "The analysis of the EXAFS spectra as well as strong metal-Mn correlations in the EDX maps for both Zn and Pb confirm that adsorption on mineral vacancy sites accounts for at least 60% of the sorption observed in the R1 phase.", "At loadings above 0.10 mol i mol−1 Mn, the displacement of Mn(III) by Pb(II) at vacancy sites explains the uptake of Pb from solution and accumulation of aqueous Mn.", "The observed accumulation of 150 μM of Mn(II) is consistent with the disproportionation of the initial ~30% Mn(III) content reported for this biogenic MnO2.", "Because aqueous Mn(III) is unstable, any displaced Mn(III) will disproportionate with a neighbouring Mn(III) atom [2× Mn(III) ➔ 1× Mn(II) and 1× Mn(IV)].", "Disproportionation of interlayer Mn(III) with layer Mn(III) (Fig. 5A) or interlayer Mn(III) (Fig. 5B) would result in the sorption of one (slope of 1) or two (slope of 0.5) moles of Pb(II) for every mol of Mn(II) that accumulates in solution, depending on the ability of Pb(II) to occupy both sides of the vacancy.", "The binding of Pb(II) on both sides of vacancies has been suggested from dPDF analysis of Pb(II) sorption on δ-MnO2 at pH 5.5 (van Genuchten and Peña, 2016).", "In the R2 phase, the measured ratio of 0.6 mol Mn displaced per Pb(II) sorbed (Fig. 2) is consistent with the double-occupancy of vacancy sites by Pb(II).", "However, any of the mechanisms described in the R1 sorption phase (slope of 0) could combine with single occupancy of vacancies (slope of 1) to lower the slope to a value of 0.6.", "Furthermore, aqueous Mn(II) concentrations reflect the net Mn released in solution and therefore do not account any oxidation or reduction of Mn that could result from microbial activity (e.g., enzymatic Mn oxidation and formation of de novo MnO2).", "Therefore additional mechanistic data would be required to quantify the contributions of the different pathways discussed here.", "The measured XAS spectra and surface excesses on biomass only and biogenic MnO2 sorbents, as well as the knowledge of surface site densities (e.g., up to 0.2 mol vacancy mol−1 Mn) (Villalobos et al., 2006) and EDX maps show that in addition to vacancies, other sorption sites are involved in the sorption of Zn and Pb.", "The biomass (EPS and bacterial cells) contains metal binding organic functional groups, in particular carboxyl and phosphoryl functional groups that can contribute to the sorption of Zn(II) and Pb(II) (Droz et al., 2015; Toner et al., 2005; Fein et al., 1997; Fein et al., 2001; Fein et al., 2005; Guiné et al., 2006).", "Through potentiometric titrations, Droz et al. (2015) reported 0.46 mol kg−1 proton exchangeable sites for P. putida GB-1 biomass, of which 0.37 and 0.035 mol kg−1 were modelled as COO and PO groups, respectively.", "Any absorption of metals or secretion of biomolecules (e.g., phosphate (Tobin et al., 2007)) could provide additional organic functional groups for metal sorption.", "In the present study (pH 5.2 ± 0.3), loadings of 0.07 ± 0.04 mol Zn kg−1 biomass and 0.20 ± 0.02 mol Pb kg−1 biomass were measured for Zn and Pb respectively, in the biomass-only sorbents.", "For Zn(II), LCF analysis demonstrated partitioning to the biomass.", "Toner et al. (2005) showed that sorption of Zn(II) by P. putida at pH 6.9 was dominated by phosphoryl (85 ± 10 mol%) and carboxyl (23 ± 10 mol%) functional groups.", "At the acidic pH values used in this study, the relative contribution from carboxyl groups (pKa ~4–5) to Zn sorption may increase as phosphoryl group become protonated (pKa ~7.0).", "The sorption of Zn by the biomass reflects the limited accessibility of Zn(II) to interlayer vacancies, which is supported by limited Mn release, and thus favours partitioning to alternative binding sites.", "While sorption to the particle edges cannot be fully excluded from EXAFS analysis alone as explained above, FTIR measurements combined with EXAFS analysis were used by Simanova et al. (2016) to show that significant organo-mineral interactions passivate the particle edges with respect to metal sorption (Ni(II), pH 6.5) in biomass-δ-MnO2 composites.", "Thus, under the assumption that Zn(II) sorption by particle edges is minimal in our samples, 29% of the total Zn(II) sorbed by the bio-mineral sorbent would thus be partitioned on the biomass (0.09 mol Zn kg−1 sorbent) in the highest loading sample.", "This loading is consistent with the qmax reached for Zn(II) sorption in the biomass-only isotherm (0.07 ± 0.04 mol Zn kg−1 sorbent; see Fig. 1).", "The good agreement between the wet-chemistry and the EXAFS spectroscopy suggests similar reactivity of the biomass in both the biomass-only and the biooxide sorbents.", "Based on previous work (Toner et al., 2006; Peña et al., 2010), we expected saturation of vacancy sites before partitioning to the organics.", "The LCF analysis of the lowest loading sample in this study indicated that Zn(II) partitioned both to sorption sites on the biomass and on the mineral.", "Toner et al. (2006) however used a higher pH (7.0 versus 5.5 in our experiments) and longer reaction times (48 h, versus 24 h in our experiments) compared to the conditions used in this study.", "At higher pH values, the mineral fraction have significantly greater sorption reactivity relative to the biomass and thus outcompete the biomass for Zn sorption; at lower pH values the affinity of the oxide and the biomass for Zn may become comparable.", "Slow diffusion of Zn(II) into the interlayer to reach vacancy sites at low Zn(II) concentrations may also explain the greater partitioning of Zn(II) to the biomass observed in our study (Y.H. Wang et al., 2018).", "In addition, differences in the initial Mn(III) content of the biogenic MnO2, which can vary with the biomineralization conditions (Droz et al., 2015) or cleaning procedure (Zhu et al., 2010b) could lead to differences in the partitioning of metals to organic versus inorganic surface sites.", "Further insight into the partitioning of metals across these different sorption sites might be gained by developing surface complexation models for composite sorbents, which often show different reactivity relative to biomass-only or mineral-only systems.", "For Pb(II) sorption, we estimated that 0.17–0.23 mol Pb kg−1 sorbent must be sorbed on the biomass or particle edges since the highest surface excess attained (0.49 ± 0.02 mol Pb mol−1 Mn or 0.67 ± 0.03 mol Pb kg−1 sorbent) exceeded the amount that could be reached by occupying vacancy sites alone.", "Based on the maximal vacancy content for biogenic MnO2 of 0.20 mol mol−1 Mn (Villalobos et al., 2006), only 40 to 81% of the surface excess can be explained by sorption on vacancy, assuming single or double occupancy (van Genuchten and Peña, 2016) respectively.", "Lead has a high affinity for organic functional groups and thus to bind to biomass components (Nelson et al., 1999a; Sarret et al., 1998; Nelson et al., 1999b; Pardo et al., 2003; Sheng et al., 2004).", "Sorption of Pb on phosphate functional groups may induce the formation of stable Pb-phosphate precipitates (Rhee et al., 2012; Xie and Giammar, 2007) as observed in the EDX maps for Pb-biomass samples.", "Lead may also displace weakly bound organic molecules to access particle edges given the reported high affinity of Pb(II) complex at particle edges (Takahashi et al., 2007).", "In an EXAFS analysis of Pb(II) sorbed on δ-MnO2, Takahashi et al. (2007) resolved two Pb-Mn distances as a doublet in the Pb-Mn shell of the Fourier transform, which they assigned to an edge complex (CN = 2, Pb-Mn 3.2 Å) and interlayer complex (TCS, CN = 6, Pb-Mn 3.7 Å).", "Their data showed the prevalence of the edge complex at low loading and a shift to the TCS complex at higher loading, highlighting the high affinity of the former complex.", "In our samples, we were unable to detect a second Pb-Mn distance that could be attributed to the Pb(II) edge complex.", "In another study based on pair distribution function analysis, sorption of Pb(II) on edges of δ-MnO2 was found to increase with increasing loadings and to show same interactomic distances as the TCS complex (van Genuchten and Peña, 2016).", "Therefore, we cannot exclude that some contribution of edge sites that is concomitant with sorption on vacancies, throughout the sorption isotherm.", "Displacement of contaminants sorbed on MnO2 by incoming ones with greater affinity for the mineral surface sites has important implications for the effective reactivity of MnO2 towards different, often co-occurring contaminants.", "This study shows that the competition for reactive sites between incoming contaminants and Mn(III) will dictate contaminant uptake by MnO2, The incorporation of metals inside the vacancies, which is observed to varying extents for cations such as Ni(II) and Co(II), can lead to largely irreversible sorption (Miyata et al., 2007; Kwon et al., 2013; Simanova and Peña, 2015; Peacock, 2009).", "However, for cations such as Zn(II) (Kwon et al., 2013; Tani et al., 2004) or Pb(II) (Zhao et al., 2009), whose ionic radii and electronic configurations prevent them from entering the layer structure, adsorption is a reversible process.", "Our findings also suggest that the removal of layer and interlayer Mn(III) during sorption of cations may increase the average oxidation state of Mn (see Fig. 5A–B) and therefore will modify the oxidative reactivity of the mineral towards organic and inorganic species (Nico and Zasoski, 2000; Nico and Zasoski, 2001; Ukrainczyk and McBride, 1992; Y. Wang et al., 2018).", "These dynamic interactions between Mn(III) and contaminant metals are especially relevant for biogenic MnO2 where oxidation and reduction of Mn can be mediated by biomolecules and metabolically active cells within the biomineral assemblage.", "Therefore future work should characterize the extent to which lower valent Mn and vacancy content evolve not only during biomineral formation but also during reaction with contaminants.", "Finally, metal sorption in the biofilm can lead to additional sequestration pathways as observed for Pb via the formation of phosphate-rich precipitates." ]
[ "Metal sorption by biogenic MnO2 was studied using EXAFS spectroscopy and STEM-EDX imaging.", "Zn partitions to biomass and MnO2 at all loadings at pH 5.", "Pb can access four times as many reactive sites compared to Zn.", "Pb but not Zn can dislodge Mn(III) adsorbed on vacancy sites.", "Initial Mn(III) content and competitive sorption control contaminant uptake by biogenic MnO2." ]
Pneumatosis cystoides intestinalis revealed after a hand-to-hand aggression: A case report
Introduction: Pneumatosis cystoides intestinalis (PCI) is a condition defined by the presence of multiple gas-filled cysts within the intestinal wall. We demonstrated a case of PCI presenting as pneumoperitoneum following a hand-to-hand aggression. Consent was obtained from the patient for publication of this paper. Presentation of the case: This article describes a case of an 28 year-old man with medical history of gastroduodenal ulcer admitted in the emergency room with an acute abdominal pain secondary to a hand-to-hand aggression. Computed tomography (CT-scan) revealed signs of PCI, the presence of pneumoperitoneum and a small amount of fluid in the Douglas pouch. The patient underwent an urgent laparotomy in front of a high suspicion of a bowel perforation. Per operative findings revealed multiple small cysts of the terminal ileum and there were no bowel perforation. Discussion: Pneumatosis cystoides intestinalis may be related to a wide spectrum of gastrointestinal conditions. The diagnosis of PCI can be established by endoscopic ultrasound or CT-scan imaging. Management of PCI is conditioned by the clinical and radiological presentation which is essentially related to the primary cause. Conservative approach is allowed in a stable patient with no signs of complications. In the presence of predictive factors of pathologic PCI, namely transmural ischemia and bowel perforation, surgical operation is required. Conclusion: The management of PCI may be challenging particularly in the presence of pneumoperitoneum. Complications must be excluded before considering a conservative therapy. Therefore, PCI should be interpreted with relevance to the entire clinical context.
[ "MEDI" ]
[ "CT imaging", "Case report", "Pneumatosis cystoides intestinalis", "Pneumoperitoneum", "Therapy" ]
[ "2746" ]
[ "Pneumatosis cystoides intestinalis (PCI) is not a disease but a condition characterized by the presence of multiple gas-filled cysts within the submucosa or subserosa layer of the intestinal wall.", "Its clinical significance may vary from ischemic origin to a benign finding.", "Since the broad indications of computed tomography (CT-scan), the incidence of pneumatosis intestinalis is becoming more common than previously reported.", "At the present time, there are no consensus to guide surgical intervention.", "Here, we present a case of a PCI revealed by pneumoperitoneum following a hand-to-hand aggression, in a 28 year-old man.", "Consent was obtained from the patient in question for publication of this case report and accompanying images.", "A copy of the written consent is available upon request.", "Our work has been reported in ligne with SCARE criteria [1].", "A 28 year-old man with medical history of gastroduodenal ulcer admitted to the emergency room with an acute abdominal pain secondary to a hand-to-hand aggression.", "Vital signs were normal.", "Physical examination showed a diffuse abdominal tenderness with normal bowel sounds.", "No abdominal distension or contracture were noticed.", "The patient was apyretic.", "Laboratory investigations were normal.", "Initially, the patient was explored by abdominal sonography which revealed a small amount of anechoic fluid in the Douglas pouch.", "However, the exploration of the solid organs was no conclusive because of the interposition of a gas screen.", "Thus, an abdominopelvic CT-scan was performed revealing multiple small gas-filled cysts within the wall of the terminal ileum, the presence of pneumoperitoneum and a small amount of hypodense fluid in the Douglas pouch (Fig. 1).", "No other abnormalities were seen.", "The patient underwent an urgent laparotomy, in front of a high suspicion of a bowel perforation.", "Per operative findings revealed multiple small cysts of the terminal ileum and there were no bowel perforation (Fig. 2).", "The postoperative period was uneventful.", "Pneumatosis cystoides intestinalis tends to occur more frequently in the colon followed by the small intestine.", "Combined involvement was noticed in few cases [2].", "Location confined to small intestine was correlated with the presence of an underlying bowel disease (such as bowel perforation, bowel infarct, enteritis, bowel obstruction) (3).", "In the present case, the terminal ileum was affected.", "The rest of the bowel tract was normal.", "Although the pathophysiology is incompletely understood, two main theories exist [3,4].", "The mechanical theory suggests that intestinal mucosa injury allows normal gas to dissect into the bowel wall, especially under increased intraluminal pressure.", "The bacterial theory postulates that gas-forming bacilli enter the submucosa through mucosal breachs or increased mucosal permeability and subsequently form gas within the bowel wall [4].", "PCI may be related to a wide spectrum of gastrointestinal conditions including peptic ulcer, inflammatory bowel disease, intestinal obstruction, and intestinal necrosis, as well as, endoscopic procedures and post abdominal surgery.", "Connective tissue anomalies and pulmonary disease were seen too [2].", "It may be also associated with chemotherapy and steroid therapy [3].", "Most PCI are asymptomatic and incidentally detected on CT or during surgery.", "Symptoms vary depending on the underlying cause and may consist of abdominal pain, diarrhea, distention, nausea and vomiting, bloody stool, mucous stool or constipation [2].", "CT-scan is a sensitive method for detecting PCI.", "The use of a lung window setting is the key of the diagnosis.", "Gas cysts in the bowel wall may not be apparent at the soft tissue window setting.", "Furthermore, CT-scan allows the detection of combined pathological conditions participating in the decision-making process [3,5,6].", "Lee et al demonstrated that mesenteric stranding, bowel wall thickening and ascites were CT-scan features of clinically worrisome PCI heralding underlying bowel disease.", "Pneumoperitoneum was found in both idiopathic or secondary PCI and did not necessarily imply the presence of an associated underlying bowel disease [3].", "In our case, the pneumoperitoneum was probably related to the gas-filled cyst rupture secondary to the abdominal trauma.", "Ultrasonography can be used to detect PCI.", "This technique is more commonly applied to the pediatric patient in whom avoidance of ionizing radiation is preferred but in most cases it is hampered by the presence of tympanites [7].", "The diagnosis of PCI can also be established by endoscopic ultrasound which demonstrates multiple hyperechoic air pockets with shadowing in the submucosal layer [8].", "This examination can differentiate PCI from others lesions avoiding harmful endoscopic treatment.", "Management of PCI is conditioned by the clinical and radiological presentation which is essentially related to the primary cause.", "Conservative approach is allowed in a stable patient with no signs of complications [2].", "In the presence of predictive factors of pathologic PCI, namely transmural ischemia and bowel perforation, surgical procedure is required [9].", "Management of PCI may be challenging particularly with the presence of pneumoperitoneum.", "Complications must be excluded before considering a conservative therapy.", "Therefore, PCI should be interpreted with relevance to the entire clinical context.", "The authors declare no source of funding for the study.", "This study is exempt from ethical approval in the authors’ institution.", "Consent was obtained from the patient in question for publication of this case report and accompanying images.", "A copy of the written consent is available upon request.", "Belkhir Asma: Writing of the paper and scan interpretating.", "Jrad Mariem: Assistant to the manuscript writing and scan interpretating.", "Soudani Mariem: Assistant to the manuscript wrinting and scan interpretating.", "Sebei Amine: Main surgeon involved in patient care.", "Haddad Anis: Main surgeon involved in patient care.", "The authors don’t need to register this work.", "Dr. Belkhir Asma and Dr. Myriam Jrad, the corresponding authors of this manuscript accept full responsibility for the work and the conduct of the study, access to the data and controlled the decision to publish.", "Not commissioned, externally peer-reviewed.", "The authors declare no conflict of interest." ]
[ "Pneumatosis cystoides intestinalis should be interpreted with relevance to the entire clinical context.", "Positive diagnosis can be established by computed tomography imaging.", "Management of PCI is conditioned by the clinical and radiological presentation which is essentially related to the primary cause.", "Surgical procedure is required when suspecting transmural ischemia or bowel perforation." ]
Pollen and spores from molar folds reflect food choice of late Pleistocene and Early Holocene herbivores in The Netherlands and the adjacent North Sea area
Molars of eight large herbivore species (Megaloceros giganteus, Cervus elaphus, Rangifer tarandus, Alces alces, Bison priscus, Ovibos moschatus, Coelodonta antiquitatis and Stephanorhinus kirchbergensis) were collected ex situ from Pleistocene and Holocene sands dredged in the North Sea, and from Dutch inland sites. Folds in many molars contained compacted masticated plant remains, and also microfossils. We identified pollen, spores, and non-pollen palynomorphs and discuss and interpret food preferences, represented flowering seasons, or parts of flowering seasons, and we discuss effects of changing vegetation composition in relation to climate and age of the molars, based on the pollen spectra. Various confounding factors have contributed to the recorded pollen composition, but nevertheless the pollen spectra show valuable aspects of vegetation composition, food choice, age, and landscapes, from subarctic open areas to interglacial forest. Ecological and statistical analysis of the results shows dietary differences between the mammal species analyzed.
[ "AGRI", "ARTS", "EART", "ENVI", "SOCI" ]
[ "Dentition", "Diet", "Herbivores", "Holocene", "Infundibulum", "North sea area", "Palaeoecology", "Palynology", "Pleistocene", "Pollen" ]
[ "1105", "1204", "1907", "2306", "3302" ]
[ "The mammoth steppe ecosystem contained a combination of plant and animal species that does not occur anywhere today.", "It was characterized by diverse megafauna of mostly herbivorous species (Guthrie, 1990).", "Food choice is an interesting and crucial part of a species’ ecology, and understanding the diet of animals within an ecosystem helps to better understand the system (Owen-Smith, 2002).", "Hofmann (1989) classified extant ruminants, based on extensive dietary work on fresh specimens into concentrate selectors (selecting high quality food, rich in plant cell contents), grass/roughage eaters (selecting low quality food, rich in plant cell wall, cellulose) and intermediate, or mixed feeder types.", "There is a link with body size: the bigger the ruminant, the more suited is its digestive tract to process large volumes of low quality diet.", "Most large ruminants (e.g. bison) are classified as grass/roughage eaters, while most medium sized species of deer (including reindeer and red deer) are intermediate types.", "The moose is an exception, as it can be clearly classified as a concentrate selector, despite its large body size (Hofmann, 1989).", "Herbivores actively select certain plants from a given area based on their physiological needs and past experiences.", "Even large grazing animals, classified as roughage eaters, do not indiscriminately graze all plants (e.g. Villalba and Provenza, 2009; Kleynhans et al., 2011).", "Here, we use observations on fossil molar contents of ruminants and non-ruminants to assess fossil mammal diets in the mammoth steppe ecosystem and during the Early Holocene.", "In 2011 an artificial peninsula called ‘Zandmotor’ was created along the shoreline of The Netherlands south of The Hague.", "For that purpose late Pleistocene and Early Holocene sediments were dredged from the relatively shallow North Sea bed.", "The new land functions as a coastal reinforcement.", "‘Maasvlakte 2’ is a westward extension of the port of Rotterdam and it was also built from dredged sandy North Sea deposits.", "Three sediment units were dredged: from marine Holocene/recent sandbars, from Early Holocene deposits, and from underlying (predominantly) Late Pleistocene sandy sediments.", "The dredged sediments were originally deposited in ancient Rhine and Meuse deltas (Laban and Rijsdijk, 2002; van der Valk et al., 2011; Langeveld, 2013; Kuitems et al., 2015) when, worldwide, sea levels were much lower than today and therefore the southern part of the North Sea area was dry land.", "Maasvlakte 2 and Zandmotor became localities for collecting fossils by citizen scientists, including mammal bones from Pleistocene (Weichselian) and Early Holocene sediments (Mol et al., 2006, 2008; van der Valk et al., 2011).", "The fauna was dominated by large herbivores, such as woolly mammoth, woolly rhinoceros, steppe bison, giant deer and wild horse.", "Radiocarbon dated material shows that representatives of this fauna lived between c. 50,000 and 25,000 BP when North West Europe was part of the mammoth steppe ecosystem (Mol et al., 2006, 2008).", "A study of the botanical infill of folds (infundibula) in a molar of a mid-Weichselian giant deer from Zandmotor yielded well-preserved pollen and shed light on the diet of giant deer (van Geel et al., 2018).", "For the present study we selected a number of species to study the pollen contents of molar infundibula.", "The selected species were the ruminants Megaloceros giganteus (giant deer), Cervus elaphus (red deer), Rangifer tarandus (reindeer), Alces alces (moose), Bison priscus (steppe bison) and Ovibos moschatus (muskox), and the non-ruminants Coelodonta antiquitatis (woolly rhinoceros) and Stephanorhinus kirchbergensis (forest rhinoceros; the fossil record of this species ended shortly after the Eemian interglacial).", "We borrowed molars of these species from various private collections and museums in The Netherlands.", "Most molars were from North Sea deposits but some of the molars were recovered from dredged inland river deposits (Table 1).", "The molars were identified to species level through comparison with molars of identified skeletons in the reference collection of the Natural History Museum Rotterdam (NMR).", "Botanical material present in infundibula was sampled for the analysis of pollen and non-pollen palynomorphs (NPP).", "Quantities of organic material were not sufficient for macrofossil analysis and radiocarbon dating.", "Pollen and spore records from cored lake sediments and peat deposits show regional trends of vegetation changes.", "The vegetation composition in The Netherlands during mid-Weichselian interstadials is recorded in detailed pollen records (Brinkkemper et al., 1987; Ran, 1990).", "For the vegetation history of the Lateglacial period in The Netherlands we refer to Hoek (1997) and for the Holocene to van Geel et al. (1981).", "Palynologists are accustomed to the fact that wind-pollinated taxa produce high amounts of pollen, and that such taxa therefore are over-represented compared to the relative abundance of the source plants in the vegetation.", "In contrast, insect-pollinated taxa produce relatively low amounts of pollen and are thus underrepresented.", "A sediment sample or a peat sample normally represents a multi-annual mixture of pollen deposition.", "The interpretation of the presence and frequencies of pollen and spore taxa from molar infundibula in terms of past vegetation cover is different, and the pollen records will be biased for several reasons.", "For the interpretation of our results we have to keep in mind that there remain some basal, still unsolved questions about the indicative value of such samples (chronology, vegetation composition, phenology, food choice, age of the material).", "The extinct M. giganteus could attain a maximum body weight of 600–700 kg (Moen et al., 1999); this matches modern moose.", "The fossil record can be traced back to at least the early Late Pleistocene in Europe (Kahlke, 1999).", "The North Sea has yielded numerous specimens, some of which were radiocarbon dated.", "The youngest is 36,300 ± 1100 BP and the oldest is > 45,000 BP.", "The species is known from both glacial and interglacial (or interstadial) deposits (Lister and Stuart, 2019).", "Chritz et al. (2009) studied stable isotopes in dentition and concluded a grass- and forb-based diet, supplemented by browsing.", "Rivals and Lister (2016) and Saarinen et al. (2016) studied meso- and microwear and concluded that M. giganteus had a wide range of dietary behaviour including grazing, leaf browsing and mixed feeding, but generally tending towards mixed feeding and grazing.", "The diet of one specimen, based on pollen in molar folds, was reviewed by van Geel et al. (2018): it was completely dominated by Artemisia, indicating that the animal foraged in a steppe environment and may have preferred to eat Artemisia, which contain a high level of mineral nutrients such as calcium and phosphorous (Klein, 1965; Ashraf et al., 2010; van Geel et al., 2018).", "C. elaphus is very flexible in its choice of habitat and diet.", "Hofmann (1989) classifies it as a mixed feeder.", "This large deer (75–340 kg; Nowak, 1991) is known from both glacial and interglacial fossil deposits since at least the Middle Pleistocene (Rivals et al., 2009).", "The Dutch and North Sea fossil record includes many Early Holocene occurrences, especially worked antlers from the Mesolithic (e.g. Louwe Kooijmans, 1970; Mol et al., 2008).", "There are no Late Pleistocene radiocarbon dates, but the state of fossilisation of some of the red deer material suggests that the species was part of the Late Pleistocene fauna as well (Mol et al., 2008).", "C. elaphus currently has a very extensive range, including much of Europe, Asia and North America.", "It occurs in various habitats, ranging from coniferous and deciduous forests to open grasslands, heathlands, moorlands and even semi-arid environments.", "Its diet reflects its broad habitat range and varies from grass to sedges, ferns, mosses, lichens, fungi, twigs, nuts, shoots and bark of trees.", "It usually varies from season to season and whenever possible includes both herbaceous and browsed material (Straus, 1981; Nowak, 1991; Chen et al., 1998; Clutton-Brock and McIntyre, 1999; Gebert and Verheyden-Tixier, 2001; Suter et al., 2004; Augustyniak, 2010; Ligi and Randveer, 2012; Berlioz et al., 2017).", "A similar flexibility in the diet of C. elaphus was found through isotopic and dental wear analyses for Pleistocene populations (Drucker et al., 2003; Rivals et al., 2009, 2010; Saarinen et al., 2016).", "R. tarandus is one of the large mammals that lived on the mammoth steppe and it survived into the Holocene.", "Hofmann (1989) classifies it as a mixed feeder.", "This moderately sized deer (body weight 60–318 kg; Nowak, 1991) has a fossil record that starts at least in the Early Pleistocene.", "It was common in the European Weichselian, especially during the Magdalenian period.", "Its range extended from northern Spain into North America (Kahlke, 1999).", "Radiocarbon dates from The Netherlands and the North Sea range from >45,000 BP till 39,000 + 700, −600 BP and an isolated date at 29,460 ± 250 BP (Glimmerveen et al., 2006).", "Today, reindeer populations may migrate for up to 1000 km between summer grounds in the tundra and winter grounds in forested areas.", "They feed on a variety of plants, including lichens and leaves of shrubs and fine twigs (Nowak, 1991; Augustyniak, 2010).", "Using dental mesowear analysis, Saarinen et al. (2016) found that Pleistocene reindeer browsed for soft forage even in more open vegetation of the mammoth steppe.", "Using carbon and nitrogen isotopes, Fox-Dobbs et al. (2008) reconstructed Pleistocene reindeer diet in eastern Beringia as consisting mostly of lichens, mosses and fungi.", "Utilizing both meso- and microwear, Rivals et al. (2010) and Rivals and Semprebon (2017) concluded that reindeer from the Brown Bank in the North Sea were mixed feeders, possibly with a more abrasive diet than modern populations.", "A. alces is the largest extant deer species with a body weight of 200–825 kg.", "Hofmann (1989) classifies it as a concentrate selector.", "It occurs in the northern parts of Eurasia and North America (Nowak, 1991).", "Fossil moose are known from The Netherlands and the North Sea deposits.", "They date from the Early Holocene until late medieval time (Walch, 2000; van Geel and van Wijngaarden-Bakker, 2002; Mol et al., 2006; Spinney, 2018), but there is one date of 44,560 + 1840/-1490 BP (Mol et al., 2008).", "Moose are rather specialized feeders that live in forested areas.", "They mainly feed on willow (Salix spp.), but they also forage on water plants and can swallow with their head submerged (Dungan and Wright, 2005; Shipley, 2010).", "Rivals et al. (2010) studied meso- and microwear of A. alces from the North Sea (Brown Bank) and reconstructed a strictly non-abrasive browsing diet, comparable to the modern diet.", "Saarinen et al. (2016) reached the same conclusion.", "B. priscus was a characteristic component of the Mammuthus-Coelodonta Faunal Complex (Kahlke, 1999).", "Recent genetic evidence showed that there were two distinct types of Bison in the Late Pleistocene of Europe, including the North Sea plain (Soubrier et al., 2016; Grange et al., 2018), but they appeared to be morphologically indistinguishable and therefore we here use B. priscus.", "They were large animals, with a body weight of around 1000 kg (Boeskorov et al., 2014).", "A radiocarbon date of 45,350 + 2400/-1850 BP (Mol et al., 2008) places B. priscus in the Late Pleistocene of the North Sea area.", "Bison has a very rich fossil record across all of its geographical range.", "It appears to be the most common taxon from the North Sea, but paleoecological data on the diet are scarce.", "Guthrie (1990) provided data on North American B. priscus.", "Using cuticle analysis of plant material obtained from molar infills and mesowear analysis, he concluded that these animals were grazers, with grasses making up 80–90% of their diet (followed by other herbs).", "Rivals et al. (2010) studied microwear of B. priscus from the North Sea (Brown Bank) and concluded that they were primarily grazers, or mixed feeders with a grass-dominated diet.", "Saarinen et al. (2016) studied mesowear of fossils from the United Kingdom and Germany and reconstructed mixed feeding diets with a significant component of grass.", "O. moschatus weigh 200–410 kg.", "Hofmann (1989) classifies it as a mixed feeder.", "Their fossil record in Europe starts in the late Middle Pleistocene (Kahlke, 1999).", "Together with reindeer, muskox is one of the few large mammals of the mammoth steppe that survived the end of the Pleistocene in Eurasia with the youngest radiocarbon date from the Taimyr Peninsula at 2900 ± 60 BP (MacPhee et al., 2002).", "Today they occur only on arctic tundra, with a preference for moist habitats in summer and windy higher areas (thus with little snow cover) in winter.", "Currently they occur in parts of the far north of Eurasia, where they were reintroduced in the 20th century, e.g. in Norway, the Taimyr Peninsula, Yakutia, North America and Greenland (Nowak, 1991).", "Remains of O. moschatus are rare in The Netherlands and the North Sea area; they have been dated to the Late Pleistocene (Mol et al., 2006).", "Modern O. moschatus feed primarily on grasses, sedges and dwarf willows.", "They are not very selective feeders and in winter they do not travel extensively to suitable foraging grounds (Klein, 1991; Oakes et al., 1992; Klein and Bay, 1994; Kristensen et al., 2011).", "Carbon and nitrogen isotopic analysis of fossil O. moschatus from the Holarctic suggests a diet flexibility that followed changes in precipitation (Raghavan et al., 2014).", "C. antiquitatis was a characteristic component of the Mammuthus-Coelodonta Faunal Complex (Kahlke, 1999).", "It was a large animal (over 2000 kg) and it occurred over a large range, stretching from Great Britain and Spain across Europe to northeast Siberia, northern China and Mongolia; it never reached North America (Stuart and Lister, 2012).", "It is a common species in Late Pleistocene fossil assemblages, though never as common as woolly mammoth, steppe bison and wild horse.", "Its fossil record extends at least back to the late Middle Pleistocene (Kahlke, 1999).", "Its range contracted eastwards from 35 ka and it went extinct ca. 14 ka (Stuart and Lister, 2012).", "A radiocarbon date from the North Sea places the species in the Late Pleistocene at 39,910 + 1070/-950 BP (Mol et al., 2006).", "Much of its ecology has been studied from permafrost and natural paraffin wax preserved carcasses, including stomach contents and molar infills.", "Both skeletal and soft tissue morphology (Stuart and Lister, 2012) as well as these food remains (Boeskorov et al., 2011) characterize C. antiquitatis as a true grazer, well adapted to the tough and dry mammoth steppe vegetation.", "While mesowear analysis from the North Sea (Brown Bank) and adjacent areas supports this, microwear analysis of North Sea material suggests periodical inclusion of some woody components in its diet (Rivals et al., 2010; Saarinen et al., 2016).", "S. kirchbergensis was a large animal with relatively long legs (Fortelius et al., 1993).", "It is considered an interglacial species with a fossil record in Europe starting in the Middle Pleistocene at 0.6 Ma and ending shortly after the Eemian interglacial (van der Made, 2010; Mol and van der Plicht, 2012).", "Analysis of plant macro-remains trapped in molar folds and mesowear from specimens recovered from Germany and Russia indicates that the species was a mixed feeder that tended to browse on foliage of trees or shrubs, but also included grasses and herbs in its diet (van der Made and Grube, 2010; van Asperen and Kahlke, 2015; Kirillova et al., 2017).", "Based on mesowear analysis, Saarinen et al. (2016) reconstructed a browsing diet for this species.", "The molar folds of about half of the screened specimens appeared to be empty or too poor in pollen to get sufficient microfossil spectra, while some other molar samples contained badly preserved microfossils.", "Such samples had to be discarded.", "Molar folds of Megaloceros giganteus (Mg), Cervus elaphus (Ce), Rangifer tarandus (Rt), Alces alces (Aa), Bison priscus (Bp), Ovibos moschatus (Om), Coelodonta antiquitatis (Ca) and Stephanorhinus kirchbergensis (Sk) contained plant remains and microfossils were successfully retrieved.", "Fifty-two samples were analyzed for pollen, spores and non-pollen palynomorphs (NPP; van Geel, 2001); the microscope slides are stored in the collection of the Natural History Museum Rotterdam, accession number 19–112.", "For practical reasons we abbreviate the zoological species names in Table 1, in the plotted DCA, and in the text below.", "Table 1 shows the numbering of samples, the collection codes, the location of the sites, and the molar positions in the jaws.", "Sampling of the botanical material occurred by using clean preparation needles in order to separate the plant remains from the molar folds.", "The obtained palynological ‘mini-samples’ were collected in small beakers and a standard pollen preparation method was applied (Faegri and Iversen, 1989).", "Microscopic analysis was applied using Beug (2004) and Punt et al. (1976–2009) for difficult pollen identifications.", "Detrended Correspondence Analysis (DCA; Hill and Gauch, 1980) was applied in PAST 3.16 (Hammer et al., 2001) as an addition to a conventional ecological interpretation.", "In most cases more than three hundred pollen grains and spores were counted per sample, but for some molars that sum could not be reached.", "Calculation of percentages, as shown in Fig. 1A–D, was based on sums of pollen grains and spores of vascular cryptogams.", "NPP frequencies were also calculated as percentages of those sums.", "The diagrams show the percentages of pollen grains, spores and NPP.", "Observations of monospecific aggregates of pollen grains (each aggregate was recorded as 1 pollen grain) are shown in the diagram with a black square.", "Such aggregates (Fig. 2A–D) may indicate that flowering plants with unripe pollen grains were consumed, thus providing extra information about the herbivore diets.", "Mg03, Mg08, Mg10 and Mg11 show relatively high Betula percentages.", "In Mg08 and Mg11 aggregates of Betula pollen show that flowering birch was consumed.", "Alnus and Corylus with some Tilia and Ulmus pollen in Mg11 suggest that the sample may represent post-depositional Holocene contamination, but an upper Eemian or an early Weichselian interstadial age cannot be excluded.", "Artemisia and Asteraceae sf.", "Asteroideae are common in most of the Mg-samples.", "Other light-demanding taxa (Poaceae and a variety of herbaceous taxa, like Caryophyllaceae, Chenopodiaceae, Filipendula, Linum, Plantago, Thalictrum and Helianthemum) are of regular occurrence.", "Humulus was also consumed, considering the presence of aggregated Humulus pollen in Mg04.", "Equisetum shows high values in Mg07 and Osmunda was very common in Mg03, but the absence in other Megaloceros samples shows that they were not regular components of the giant deer food choice.", "Pollen of Poaceae is present in all samples analyzed, but always in low percentages; only in Mg05 a slightly higher percentage and an aggregate of unripe grass pollen was observed.", "This matches work based on isotopes and tooth wear that suggests mixed feeding for M. giganteus.", "The dominance of Artemisia, that was observed by van Geel et al. (2018) for one giant deer molar, was found here in other molars as well, also with aggregates of unripe Artemisia pollen.", "This provides further evidence of M. giganteus selecting Artemisia as a food plant.", "Saarinen et al. (2016) found that M. giganteus was commonly associated with rather open vegetation, but also occurred in localities relatively rich in arboreal pollen.", "Based on the relatively high numbers of Corylus and Alnus and ferns, samples Ce07, Ce11, Ce 12, Ce13, and Ce14 may point to a Holocene (but pre-inundation) age.", "The other Ce-samples are probably of Pleistocene age.", "Remarkable are Ce04 and Ce10 with high Betula values.", "A Betula aggregate in Ce04 points to consumption of flowering birch.", "Ce05 contained a very large number of Helianthemum and aggregates show that Helianthemum was consumed.", "The quantity of Humulus and Impatiens in Ce06 probably points to consumption, while the pollen record of Ce06, Ce15 and Ce16 may point to Urtica as a food plant.", "We suppose that Ce08 and Ce12 point to Filipendula as a food plant, while Ce11 may indicate that Equisetum was eaten.", "Ce08 and Ce14 point to consumption of Symphytum.", "Asteroideae, Apiaceae, Poaceae and Cyperaceae were common in many samples.", "Saarinen et al. (2016) found that Cervus elaphus was clearly associated with both open and closed forest environments.", "Most samples show relatively low tree pollen values, pointing to a sub-arctic open landscape.", "In Rt08 only few individual Betula pollen grains were recorded, but an aggregate of B. nana shows consumption of dwarf birch.", "Rt05 and Rt07 show rather high Salix pollen values and, considering the composition of the pollen spectra of those two samples this Salix pollen may well have been produced by dwarf willows.", "Artemisia is common in most samples; Rt03 and Rt08 yielded aggregates of Artemisia.", "Aggregates in the two samples with high values of Cyperaceae (Rt02 and Rt06) point to a bias (consumption and over-representation in the pollen spectra) of flowering Cyperaceae.", "Several samples show rather high records of Helianthemum, and three even show aggregates, indicating a preference for Helianthemum as a food source.", "According to Milotic et al. (2017) herbivores in the landscape even stimulate the growth and expansion of Helianthemum.", "The frequencies of Pedicularis palustris-type in the Rt-samples (absent in almost all other samples) point to Pedicularis (P. lapponica?)", "as a preferred species for consumption.", "Some other taxa worth to be mentioned are Centaurium, Papaver rhoeas-type and Polemonium in Rt09, Triglochin in Rt11 and Silene dioica-type in Rt07.", "Poaceae pollen generally is slightly more common in R. tarandus than in M. giganteus, but lower than in C. elaphus, suggesting that grasses were part of the reindeer diet, but not to a very large extent.", "The pollen data match studies on meso- and microwear of reindeer specimens from the North Sea.", "Based on pollen records Saarinen et al. (2016) found that Rangifer tarandus was evidently associated with open environments.", "Considering the amount of tree and shrub pollen (Betula, Pinus, Corylus, Alnus, Taxus, Tilia, Ulmus, Frangula alnus) and fern spores, and taking into account the geological information of the dredged deposits, Aa01 and Aa02 are of Early to mid-Holocene age.", "An aggregate of Alnus pollen in Aa02 probably points to consumption of Alnus.", "Aa03 is older than the other two samples (maybe Allerød interstadial, or a Preboreal age), because Alnus and Corylus are almost absent, while it shows high Pinus pollen percentages and even a Pinus tracheid pit.", "Aa03 shows indicators of open water (Nymphaea, including trichosclereid, Myriophyllum, Potamogeton, Sparganium, Typha, and various algae (Botryococcus, Pediastrum, Spirogyra, Tetraedron, Zygnema-type, Mougeotia, HdV-128A and HdV-128B)).", "The Equisetum spores may well have been produced by the wet growing species E. fluviatile.", "Aa01 and Aa02 represent relatively ‘dry’ environments with, among others, Calluna, Lonicera and ferns.", "Aa02 contains Salix epidermis: a clear indication of consumption of Salix.", "Willows are very important food plants for modern moose.", "In concert with our results, Saarinen et al. (2016) found that Alces alces - with low mesowear values - was linked with low non-arboreal pollen percentages, which supports the status as a forest-adapted browser.", "The only pollen spectrum that could be obtained is completely dominated by pollen of Artemisia, Asteroideae and Poaceae.", "Tree pollen is absent, pointing to Weichselian subarctic steppe conditions and a grazing diet.", "This is in accordance with the scarce previous work done on the palaeobiology of steppe bison.", "Saarinen et al. (2016) found that Bison priscus was commonly associated with rather open vegetation, but also occurred in environments relatively rich in arboreal pollen.", "Aggregates of Betula pollen, present in the only available molar point to browsing of flowering birches, but the landscape was rather open, considering the representation of Asteraceae, Ericaceae and Poaceae.", "Polygonum viviparum is an arctic-alpine species and its presence fits in the open landscape under subarctic conditions.", "Tree pollen values are low, and the vegetation was dominated by Poaceae, Cyperaceae, Plantago, Galium-type, Gentianella campestris, Scabiosa, Helianthemum, Artemisia and other Asteraceae.", "Aggregates of pollen of Poaceae in three samples point to consumption of flowering grasses and therefore Poaceae are probably over-represented in the pollen spectra.", "The data clearly point to a grazing diet, which matches previous work done on molar wear and stomach contents.", "Ca05 shows a spectrum pointing to a moist habitat: the aquatic HdV-128A is very common and also some other algal spores point to wet conditions.", "Fungal spores HdV-1400 (unequally 2-celled, longitudinally ribbed; Fig. 2F) that were only known from a Weichselian interstadial deposit (van Geel et al. 2010) now appear to be linked to the habitat of C. antiquitatis (cold, dry climatic conditions).", "The presence of spores of the coprophilous fungi Podospora, Sordaria and Sporormiella may well point to HdV-1400 as another coprophilous fungus.", "The single molar spectrum shows a dominance of tree pollen (mainly Alnus and Corylus, with some Betula, Quercus, Tilia, Ulmus).", "In combination with Viscum album, Hedera helix, ferns and Kretzschmaria deusta (a fungal parasite on a variety of tree species; van Geel and Aptroot, 2006; van Geel et al., 2013) the sample represents a predominantly forested environment and supposedly it has an Eemian interglacial, or an early Weichselian interstadial age.", "We can exclude a Holocene age as S. kirchbergensis became extinct shortly after the Eemian interglacial (van der Made, 2010; Mol et al., 2012).", "Fig. 3 shows the DCA plot of the plant taxa.", "There are three poorly defined clusters.", "The cluster on the right contains mostly thermophilous interglacial taxa like Alnus, Betula, Corylus, ferns, Frangula, Hedera, Lonicera, Myrica, Quercus, Rhamnus, Tilia, Typha, Ulmus and Viscum and most of the wetland taxa.", "Most taxa on the left represent species of tree-less landscapes.", "Fig. 4 shows the DCA of the zoological taxa, as based on the pollen spectra recorded from the molars.", "There is clustering of the various species and variation between those species.", "The samples from C. antiquitatis cluster strongly together (except sample Ca01).", "This is in sharp contrast to the C. elaphus samples that are spread through most of the plot.", "This may be explained by the extreme dietary flexibility of C. elaphus compared to the strictly grazing diet of C. antiquitatis.", "R. tarandus samples cluster close to both M. giganteus and B. priscus and some of the R. tarandus samples are also similar to C. antiquitatis.", "M. giganteus samples show a rather large variation along Axis 1, but not so much along Axis 2.", "This suggests a more specialized diet than that of C. elaphus.", "A. alces and S. kirchbergensis cluster close together; the abundance of arboreal (interglacial) pollen in those samples explains this.", "The dominance of Betula pollen in the O. moschatus sample explains its clustering close to these temperate taxa, but Betula may be strongly over-represented (pollen aggregate).", "The three outliers have unique microfossil compositions.", "Mg03 is the only sample rich in Osmunda regalis.", "Rt11 has abundant Triglochin and Pedicularis palustris-type, which may be caused by grazing of flowers.", "Ce05 contains a remarkably high abundance of Helianthemum, including an aggregate.", "R. tarandus and C. elaphus are both large mammals that survived into the Holocene.", "It is worth noting that both species show a larger variation in the DCA plot than the extinct M. giganteus and C. antiquitatis.", "Species with low diet diversity are most vulnerable for extinction.", "After abrupt climate change, sudden vegetation changes could cause a population crash and possibly extinction (Augustyniak, 2010; van Geel et al., 2018).", "How much time (days, weeks, months, years?)", "does a pollen sample from an infundibulum represent?", "In other words: how much time did it take to fill an infundibulum?", "If different very short periods are represented in the pollen records from infundibula then different parts of the flowering season (and therefore different plant taxa) may be represented in the pollen and spore samples even if the same area was foraged.", "Infundibula that were filled with compacted plant fragments when less or no flowering took place (late autumn and winter) will show low amounts, or may even be devoid of pollen.", "Probably the infill of infundibula directly followed the eruption of molars into the oral cavity.", "This eruption has a set order and a rather narrow timing in the ontogeny of many mammals (Hillson, 2005).", "However, variation in timing within a species of only a couple of weeks may lead to significantly different parts of the flowering season being represented in the infundibulum.", "Can we expect further compression and therefore additional, later infill of an infundibulum when decomposition of the plant remains, and related additional compaction in the infundibulum takes place?", "In other words: may such samples represent large time intervals, or even complete flowering seasons?", "Our questions remain unanswered by our data and require experimental investigations.", "What were random effects of individual food choices of herbivores on our pollen records from molars, and can we anyhow expect that we can derive preferences in the food choice based on pollen and spores?", "The differences between pollen records from the same species do show that there are factors that must cause these differences.", "The abundance of Poaceae pollen in Coelodonta, a known grazer, is a striking example of a link between our pollen records and the animal’s diet.", "Furthermore, the clustering of our samples in the DCA does point to some structure in the dataset that may well be explained by food preferences and selectivity of the species.", "Flowers often are the most sun-exposed (highest) parts of plants and therefore flowers with not yet dispersed pollen may have been consumed.", "We observed that not yet released, unripe pollen grains appeared as pollen aggregates in the microscope slides.", "Consumption of flowers could lead to a strong overrepresentation of some plant species, but an advantage of the presence of pollen aggregates is that direct information on consumption of certain plant species becomes available.", "Individual herbivore specimens lived during a relatively short period, while climatic conditions were not at all stable over longer time intervals (interglacials, glacial stadials, interstadials and periods of transitional climatic conditions).", "Migration of plant species will have taken place after climate change and different successive climatic conditions were linked to the existence of different vegetation types.", "Therefore differences between pollen spectra may occur that are not only influenced by the season and the food choice of the herbivores but also by the age of the molar in relation to changing climatic conditions and related changes in the vegetation composition.", "We also should be aware that the pollen rain - especially pollen of wind-pollinated taxa - will have landed on plants that were consumed by the herbivores and thus various pollen taxa will be represented, independent from the individual food choice of the animal.", "Taking into account the limitations in the interpretations of our pollen and spore record, we nevertheless obtained new information about the food choice of giant deer, red deer, reindeer, moose, steppe bison, muskox, woolly rhino, and forest rhino.", "Based on molar-embedded pollen grains and spores we gained insight into the diet of eight herbivore species that lived during the Eemian interglacial, the Weichselian, and the Early to mid-Holocene (until inundation because of sea level rise), respectively.", "Food plants were represented as relatively high percentages of their pollen or spores, and monospecific pollen aggregates in the microfossil slides gave valuable additional information about food choice.", "Compared to pollen records from stratigraphic sequences, our pollen spectra were strongly biased.", "Some of our questions about the pollen sources (pollen rain versus active food selection) remained unanswered by our data and require experimental investigation.", "By combining ecological data of the recovered plant taxa with statistical analysis, we showed variations in diet between the various sympatric large herbivores of the mammoth steppe ecosystem.", "Although resource partitioning through competition over food is still hotly debated in large herbivores (Owen-Smith, 2002), we conclude that the dietary differences we found may have allowed different herbivore species to coexist.", "B.v.G. took the botanical samples, analyzed the microfossils, led the discussion and wrote part of the manuscript.", "B.W.L. and D.M. identified the molars, wrote the general text about the mammal species and contributed to the discussion and the interpretation of the data.", "B.W.L. executed the DCA of the zoological and botanical data.", "J.F.N.v.L. and P.W.O.v.d.K. identified problematic pollen taxa, made the microfossil diagram and were involved in discussions and interpretation of both zoological and botanical data." ]
[ "Folds in molars of Pleistocene and Holocene herbivores contain pollen and spores.", "Pollen from molars shows vegetation types from subarctic to interglacial settings.", "Pollen composition indicates diet variability within and between herbivore species.", "Monospecific pollen aggregates provide valuable information about food choice." ]
The effects of shape-setting on transformation temperatures of pseudoelastic shape memory alloy springs
Since shape memory alloy (SMA) wires can hardly ever be reliably employed under compressive loadings, SMA springs are developed as axial actuators with the ability of withstanding both tension and compression. In this paper, shape memory alloy helical springs are produced by shape-setting two types of wires: one with shape memory effect (SME) and the other with pseudoelasticity (PE) at the ambient temperature. Phase transformation temperatures of the produced springs are measured by differential scanning calorimetry (DSC), and the influences of effective parameters including cold work, heat treatment temperature and duration, and cooling rate are investigated on transformation temperatures of the products. The results show that phase transition temperatures of the fabricated springs can be tuned by performing cold work and by adjusting temperature and duration of the conducted heat treatment as well as the subsequent cooling rate. It is found that transformation temperatures of the springs fabricated using the SME wire increase as the heat treatment temperature increases. However, for samples manufactured using PE wire, transformation temperatures first increase and then decrease with the increase in the heat treatment temperature. An increase in the cooling rate leads to a decrease in the austenite final temperature (Af), and an increase in the extent of cold work leads to the increase in transformation temperatures especially Af.
[ "MATE" ]
[ "Heat treatment", "SMA", "Shape memory alloy", "Shape-setting", "Transformation temperature" ]
[ "2501", "2502", "2503", "2504" ]
[ "Shape memory alloys (SMAs) are a class of smart materials which exhibit two extraordinary behaviors of shape memory effect (SME) and pseudoelasticity (PE) (or superelasticity) owing to thermoelastic martensitic phase transformation between the two phases of martensite and austenite.", "The so-called shape memory effect is the thermally-driven recovery of the initial configuration of an SMA when residual strains appear after an inelastic loading/unloading cycle.", "In PE, recovery occurs spontaneously upon unloading once a large deformation is induced during inelastic loadings [1].", "Shape memory alloys with several shapes have been widely employed in various fields, such as robotics, biomechanics and microelectromechanical systems [2].", "In particular, beside SMA wires, spring actuators are vastly used owing to their simplicity of fabrication [3] compared to other shapes such as tubes.", "Moreover, in contrary to wires, helical springs can be subjected to both tension and compression.", "Many studies have been so far accomplished to investigate the behaviors of SMA springs both theatrically and experimentally.", "In theoretical modeling of SMA springs, the main goal is to predict force-displacement response of a helical spring.", "Toi et al. [4] presented a finite element formulation for analysis of superelasticity in SMA springs using linear Timoshenko beam elements.", "Aguiar et al. [5] proposed a numerical method based on the operator split technique in this regard.", "Mirzaeifar et al. [6,7] studied pure torsion of SMA bars with circular cross sections to investigate the pseudoelastic response of SMA helical springs under an axial force.", "Heidari et al. [8] proposed an enhanced one-dimensional constitutive model to describe the shear stress–strain response within the coils of an SMA spring.", "Their model was based on the von-Mises effective stress and strain, and it was further extended to take large deformations into account [9].", "To fabricate SMA elements with a desirable shape, specific thermomechanical treatments called “shape-setting” are required [10].", "Shape-setting mainly includes annealing but may be accompanied by cold work and quenching on the products.", "Although several microstructural analyses have been carried out [11–16] to investigate the detailed mechanisms of the various stages in shape-setting, macroscopic studies have gained a great attention in order to directly observe the influences of each stage on the phase transition and other features of an SMA specimen after shape-setting.", "Wang et al. [17] investigated the effect of annealing on the transformation characteristics of TiNi shape memory alloys by differential scanning calorimetry (DSC).", "Their results showed that the R-phase transformation appeared at low annealing temperatures.", "The R-phase disappeared and austenite directly transformed to martensite as the annealing temperature exceeded 550 °C.", "When the R-phase appends to austenite and martensite, several thermomechanical behaviors of an SMA may be affected [18–21,22,23].", "Yeung et al. [24] found that phase transition temperatures can be manipulated by adjusting the heat treatment parameters including duration, temperature and cooling rate.", "Liu et al. [25] showed that the austenite final temperature (Af) mostly increases after ageing.", "Eggeler et al. [26] realized that, in general, the transformation temperatures increase as the aging time increases.", "Sadiq et al. [27] investigated the effect of annealing temperature on the transformation temperatures.", "They found that, after annealing the NiTi alloy above the recrystallization temperature, the R-phase disappears so that a direct transformation from austenite to martensite takes place.", "The transformation temperatures increase as the annealing temperature increases; however, the R-phase transforms at lower temperatures.", "Wang et al. [28] investigated the microstructure, martensitic transformation, shape memory effect and superelastic property of the Ti49.6Ni45.1Cu5Cr0.3 alloy.", "They concluded that, in the course of elevating the annealing temperature, the transformation temperatures increased for annealing temperatures in the range of 623 K–873 K. Then, they decreased for the temperature range of 873 K–1023 K and did not considerably vary after 1023 K until 1273 K. Heidari et al. [8] fabricated SMA helical springs by shape-setting the NiTi wires and evaluated the thermomechanical characteristics of their products.", "They found that transformation temperatures of the fabricated springs increase as the annealing temperature increases; however, the start and final temperatures of the R-phase transition decrease so that the R-phase eventually disappears at the annealing temperature of 600 °C.", "Motemani et al. [29] studied the effect of cooling rate on the phase transformation and mechanical properties of Ni-rich NiTi shape memory alloy.", "They realized that the austenite final temperature Af increases as the cooling rate decreases while the martensite final temperature Mf is reduced.", "Consequently, a furnace-cooled sample has the highest phase transformation hysteresis (Af–Mf) compared to samples with lower cooling rates.", "Mitwally and Farag [30] studied the effect of cold work on the superelasticity, the shape memory effect and the hardness of NiTi alloy.", "They found that the shape memory recovery is diminished by cold rolling as well as bending.", "Lin and Wu [31] observed that, by augmenting the extent of applied cold work, the products would show lower residual strains upon unloading.", "Grossmann et al. [32] proposed a procedure to fabricate an SMA spring and to characterize its microstructural evolution during the production.", "Costanza et al. [33] investigated the shape memory effect in Ni–Ti springs and presented a technique to design a linear actuator made of SMAs.", "Follador et al. [3] fabricated an SMA spring actuator.", "They further described the mechanical characteristics of the SMA spring by a simple linear-elastic model whose parameters depend on the crystalline characteristics.", "In most of the available works on fabrication of SMA springs, the products are martensitic at the ambient temperature.", "Moreover, the influences of all shape-setting parameters on transformation temperatures of the fabricated springs are not thoroughly investigated.", "Since pseudoelastic SMA springs at the ambient temperatures are vastly required, in this paper, the main purpose is to fabricate austenitic springs using shape memory alloy wires.", "Two types of NiTi (Ti-55.87 at % Ni) wires (one of which is martensitic at ambient temperature and the other is austenitic) are utilized.", "The wires are wound and locked on a screw and are then heat treated at temperatures within the range of 300–1000 °C for 5–1260 min.", "The effects of various production parameters such as cold work, heat treatment temperature and duration, and the subsequent cooling rate on transformation temperatures of the fabricated springs are further investigated.", "The present investigation is carried out on commercial SMA wires provided by Memry Co. with the nominal composition of Ti-55.87% Ni and the diameter of 1.5 mm.", "Two types of wires, one with shape memory effect (SME) and the other with pseudoelasticity (PE) at the ambient temperature, were utilized.", "Fig. 1 and Fig. 2 illustrate results of differential scanning calorimetry (DSC), and Table 1 shows the transformation temperatures of these wires.", "To fabricate springs using SME and PE wires, according to Fig. 3, each wire was first wound and locked on a screw.", "Once the temperature of the furnace has reached a desired number, the specimen was placed in the furnace for the required time interval.", "Then, the specimen was cooled down.", "To prevent oxidation of the specimens, the whole wound wire together with the fastening components were covered by a stainless steel foil.", "Heat treatments were performed on the fabricated springs at different conditions so that various combinations of the influential parameters were examined.", "For instance, the springs S-11, S-12 and S-13 were made using SME wire by heat treating at 750 °C for 10 min; the samples S-11 and S-13 were quenched in a water-ice bath, but S-12 was quenched in a dry-ice bath.", "Cold work was performed on S-13, but specimens S-11 and S-12 were fabricated with no cold work.", "Sample S-14 indicates the SME wire which was heat treated at 1000 °C for 10 min and finally was quenched in the water-ice bath.", "For fabricating the spring S-15, sample S-14 was first wound and locked on the screw; then, it was heat treated at 1000 °C for 10 min.", "Eventually, quenching in the water-ice bath was performed.", "This procedure of heat treatment followed by quenching was repeated to fabricate spring S-16 from S-15.", "A summary of heat treatment temperatures, duration, and cooling method is given in Appendix 1.", "DSC tests were used to determine the transformation temperatures of the products.", "DSC samples were cut into 30–50 mg in mass, and the tests were performed at the heating/cooling rate of 5 °C/min between −50 °C and +150 °C.", "To induce a thermal shock in order to reduce the transformation temperatures of the samples S-13 and S-16, heat treatment without using stainless steel foil was carried out in three stages as follows:", "Heat treatment for the initial wire at the required temperature followed by quenching in the water-ice bath;", "Wounding the heat-treated wire on the screw followed by quenching in the water-ice bath;", "Heat treatment of the fabricated spring followed by quenching in the water-ice bath.", "Additionally, cold work was carried out on some of the samples in order to obtain desirable transformation temperatures.", "To this end, cold rolling was used to reduce the thickness by the amounts of 3.5% and 32.1%.", "A wide range of temperatures from 300 °C to 1000 °C was chosen for the shape-setting; however, in order to prevent oxidation of the samples, heat treatment at lower temperatures is more preferred.", "In this section, the effects of temperature and duration of the heat treatments, cooling rate, and cold work on the transformation temperatures of the products are investigated.", "Transformation temperatures of the products are given in Appendix 2.", "Since the utilized instrument was limited to the minimum temperature of −50 °C, determination of Mf and Ms for some specimens was not possible.", "Fig. 4 shows one of these cases for the sample S-09.", "R-phase transition occurs for most of the specimens (for instance, in Fig. 5 where the DSC curve of sample S-13 is shown) while it is not seen for some specimens (for instance, in sample S-24 whose DSC curve is shown in Fig. 6).", "The samples S-18, S-19, S-20, S-21, S-22 and 23 were pseudoelastic springs at the room temperature while the other ones showed shape memory effect.", "Fig. 7 illustrates one of the fabricated pseudoelastic springs from two different views.", "As shown in Appendix 2, the austenitic transformation temperatures of the specimens S-18, S-19, S-20 and S-22 are lower than the ambient temperature.", "It should be noted that sample S-21 is similar to S-22 except the difference that sample S-21 was placed into a quartz capsule under vacuum to prevent from oxidation as much as possible.", "The specimen S-23 was heat treated to find the lowest applicable temperature for achieving a pseudoelastic spring.", "Accordingly, 630 °C was the minimum temperature to optimize the process of fabricating a pseudoelastic spring with the PE wire.", "Samples S-21 and S-23 were mechanically shown to be pseudoelastic: they were examined under inelastic stretch followed by inelastic compression and could return to their original configuration after unloading.", "The samples S-24, S-25 and S-26 are 32.1% cold-worked SME wires.", "As formerly indicated, in some specimens, the reverse transformation (during the heating cycle) occurs in two stages: from martensite to an intermediate R-phase and then to austenite.", "Similarly, for some samples, the forward transformation (during the cooling cycle) occurs in two stages: from austenite (parent phase) to R-phase and then to martensite.", "Table 2 shows transformation temperatures of R-phase during the heating cycle for such specimens.", "Since pseudoelastic springs are planned to be manufactured in the present work, Af of the products should be lower than the ambient temperature.", "Therefore, the effects of the various parameters on the austenite transformation temperatures are investigated in the following subsections.", "Heat treatment parameters including temperature, cooling rate, and the duration affect transformation temperatures of the fabricated springs.", "Here, the findings of this work are presented.", "Fig. 8 shows the influence of the heat treatment temperature on the austenite transformation temperatures of the springs fabricated by using the SME wire.", "It is seen that transformation temperatures increase with the increment in the heat treatment temperature.", "Moreover, for the manufactured spring at the minimum temperature of 750 °C, As and Af are higher than the ambient temperature.", "The rapid growth of Af with the increase in the heat treatment temperature indicates that the fabrication of a pseudoelastic spring with an SME wire is impossible at these conditions.", "Variations of the transformation temperatures for the springs made from PE wire with the heat treatment temperature are shown in Fig. 9.", "It is observed that the transformation temperatures strongly depend on the heat treatment temperature.", "According to this figure, there are three trends for the variations in the transformation temperatures:", "Transformation temperatures are enhanced by elevating the heat treatment temperature until 500 °C.", "Transformation temperatures, except Ms, are reduced by increasing the heat treatment temperature between 550 °C to 650 °C.", "Transformation temperatures except Rs and Rf are nearly constant with the increase in the heat treatment temperature beyond 650 °C until 750 °C.", "In fact, with further increasing the heat treatment temperature, transformation temperatures remain fairly constant.", "This is why heat treatment at temperatures above 650 °C results in pseudoelastic products.", "According to the data provided in Appendix 2, the comparison between the transformation temperatures of specimens S-19 and S-22 and between those of S-10 to S-12 indicates that transformation temperatures of the products mostly decrease when quenching in the water-ice bath is done instead of cooling in the furnace for both series of springs made using PE and SME wires.", "However, by comparing the samples S-11 and S-12, it is seen that Mf increases by increasing the cooling rate.", "This trend coincides with the findings of investigations carried out by Motemani et al. [29] on Ni-rich Ni–Ti shape memory alloys.", "In other words, the furnace-cooled specimen has the highest phase transformation hysteresis (Af–Mf) compared to the other samples.", "The increase in transformation temperatures for the samples quenched in dry ice, compared to the samples quenched in water-ice, is due to the application of the steel foil to prevent oxidation of the parts.", "When the sample is cooled in the water-ice bath, since the steel foil is not completely sealed, water may leak during the cooling time.", "Consequently, cooling using dry ice is performed more gradually.", "Variations of the austenite transformation temperatures with the duration of the heat treatment for springs made from the SME wire are shown in Fig. 10.", "As it is seen, As and Af first increase slightly with the increase in the heat treatment duration.", "Then, by further prolonging the heat treatment, their increase is suppressed so that As is reduced a little.", "Fig. 11 shows the effect of the heat treatment time on the transformation temperatures for springs made from the PE wire.", "It is seen that the increase in duration of the heat treatment leads to a very negligible rise in As and Af compared to the observed increase for the springs made of the SME wire.", "Moreover, the effect of the heat treatment duration on transformation temperatures is less than that of the other production parameters.", "Using a flat rolling machine, the SME wire with the initial diameter of 1.5 mm was cold-rolled to achieve a 0.75 mm-thick strip.", "The cold-rolled specimen was then further wound and locked on a screw.", "Then, the prepared spring was heat treated at 750 °C for 10 min followed by quenching in the water-ice bath.", "Specimen S-13 was manufactured with the use of a 3.5% cold-worked SME wire.", "According to Appendix 2, comparison of transformation temperatures for samples S-11 and S-13 indicates that the SME wire becomes austenitic at the room temperature when the cold work percentage increases to 32.1%.", "Moreover, transformation temperatures of 32.1% cold-worked wires S-24 to S-26 are generally higher than those of the as-received wire.", "This is in agreement with the findings reported by Mitwally and Farag [30].", "According to the findings of this work, the effects of the various shape-setting stages on the transformation temperatures for the fabricated samples using PE wire are schematically illustrated in Fig. 12.", "By elevating the heat treatment temperature, the transformation temperatures increase and then decrease.", "Consequently, in early stages of annealing when the temperature is not high enough, the possibility of achieving pseudoelastic parts may be lessen even with the use of initially austenitic wires.", "In other words, if one needs to reduce the transformation temperatures after annealing, the set temperature is recommended to be chosen as high as possible.", "A more pronounced cooling rate gives rise to the decrease in transformation temperatures; thus, severe cooling such as quenching is beneficial when pseudoelastic products are desirable.", "Cold work on the specimens leads to the rise in transformation temperatures; therefore, such processes may be useful in achieving martensitic parts and are not recommended when austenitic products are desired.", "In this paper, pseudoelastic helical springs were manufactured by shape-setting two types of NiTi wires: one was martensitic at the ambient temperature and the other was austenitic.", "The effects of the various stages such as cold work, heat treatment temperature and duration, and the subsequent cooling rate on the transformation temperatures of the products were investigated.", "The main results obtained in this research can be summarized as follows: Heat treatment duration has the less effect on the transformation temperatures than the other adjustments of the shape-setting process.", "Transformation temperatures of the springs fabricated using the SME wire increase as the heat treatment temperature increases.", "However, for samples manufactured using PE wire, transformation temperatures first increase and then decrease with the increase in the heat treatment temperature.", "The increase in cooling rate results in a decrease in Af which is beneficial to fabricate PE springs at the ambient temperature.", "Cold work leads to an increase in transformation temperatures, especially Af.", "Depending on the desired type of products with either the pseudoelasticity or the shape memory effect, the presented findings regarding the effects of the various shape-setting stages on transformation temperatures of an SMA spring can be considered as guidelines for fabricating SMA springs with a predefined phase (austenite or martensite) at the ambient temperature.", "Metallurgical investigations will improve the present understanding, and such studies are under way.", "In addition to the procedures used in the current study, the various thermomechanical treatments cause a well-developed dislocation sub-structure or nanocrystalline structure and may be used for improving the properties of SMAs in future works.", "The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper." ]
[]
Assessing the performance durability of elastomeric moorings: Assembly investigations enhanced by sub-component tests
The growing marine renewable energy sector has led to a demand for increasingly compliant mooring systems. In response, several innovative mooring tethers have been proposed demonstrating potential customisation to the stiffness profile and reduced peak mooring loads. Many of these novel systems utilise materials in a unique application within the challenging marine environment and their long term durability remains to be proven. This paper presents a multifaceted investigation into the durability of a novel polyester mooring tether with an elastomeric core. Laboratory based functionality tests are repeated on tether assemblies following a 6 month sea deployment. Results show a 45% average increase in dynamic axial stiffness. This is supported by high tension laboratory based fatigue endurance tests showing a peak increase in dynamic axial stiffness of 42%. Sub-component material tests on the core elastomer support the assembly tests, separately demonstrating that certain aspects of tether operation lead to increased material sample stiffness. The average increase in material radial compressive stiffness is 22% and 15% as a result of marine ageing and repeated mechanical compression respectively; these are the first results of this type to be published. The performance durability characterisation of the tether establishes the mooring design envelope for long-term deployment. This characterisation is crucial to ensure reliable and effective integration of novel mooring systems into offshore engineering projects.
[ "ENGI", "ENVI" ]
[ "Component testing", "Durability", "EPDM", "Elastomer", "Marine energy", "Mooring" ]
[ "2212", "2305" ]
[ "The highly dynamic nature of many marine renewable energy devices places new demands on mooring system design in contrast to conventional oil and gas floating offshore systems.", "In addition to reliably maintaining the station keeping of a device, for many device designs the mooring system must also provide the compliance required to harvest energy from the marine environment.", "Synthetic mooring ropes are now commonly used in this application (Weller et al., 2015a; Davies et al., 2014; Ridge et al., 2010) having greater compliance than wire rope or chain alternatives.", "For conventional synthetic ropes the compliance is correlated to the specified minimum breaking load (MBL).", "Hence, the compliance of the mooring system is frequently compromised in order to achieve the required MBL to survive the loads expected during operation.", "Additional compliance can be introduced through system architecture, utilising floats and weights in catenary mooring systems (Weller et al., 2014a).", "However, this introduces complexity, increases the risk of entanglement and increases the mooring footprint when ambitions for industry offshore deployments are striving for increasingly compact mooring footprints in array configurations.", "As a response to these new demands, several innovative mooring systems have been proposed to introduce greater compliance into the mooring system, and in some cases, provide customisation to the stiffness profile of the mooring (Parish and Johanning, 2012; McEvoy, 2012; Bengtsson and Ekström, 2010; Luxmoore et al., 2016).", "Studies have shown these systems have the potential to reduce peak loads observed in the mooring system (Thies et al., 2015; McEvoy, 2012; Parish et al., 2017; Luxmoore et al., 2016).", "Thies et al. (2015) demonstrate a maximum line tension reduction of 20% and a mean line tension reduction of 10%, while McEvoy (2012) shows an 80–90% load reduction in a storm scenario when substituting conventional mooring configurations for a novel elastomeric tether.", "It is anticipated that a reduction in mooring loads will allow the down rating of other mooring components leading to a downward spiral of reduced weight and further reduced loads, with the potential to reduce the structural requirements and mass of the floating body (Parish et al., 2017).", "Estimates suggest foundations and mooring systems account for 10% of device costs (Low Carbon Innovation Coordination Group, 2012) and given the strong correlation between MBL and cost of mooring components (Harris et al., 2004), the potential savings through use of these novel systems are significant.", "Ultimately, the resulting mooring system will be lighter, cheaper and easier to deploy.", "If these novel systems are to be successful, the long term performance must be proven and, given the harsh operating environment of marine renewable energy devices, this remains a key priority for these systems.", "The focus for the work presented here is a durability assessment of the Exeter Tether (Parish and Johanning, 2012), focusing on both the tether assembly and detailed material tests of the elastomeric core.", "The paper is organised into five parts.", "In this introduction the key features and operating principles of the tether are presented.", "Section 2, Methods, outlines the test facilities and techniques employed.", "Section 3, Results, presents key findings from the study followed by a Discussion in Section 4 which puts these results into the operating context of the tether and addresses the limitations of the work.", "Finally Section 5 details the conclusions and outlines next steps for this work.", "A detailed overview of the Exeter Tether and the P1 Prototype Series is presented in (Gordelier et al., 2015).", "A summary is provided here, based upon the specific prototype iteration utilising a core of seven solid elastomeric cords.", "The Exeter Tether comprises a hollow polyester rope braided around a core assembly of EPDM (Ethylene Propylene Diene Monomer) polymer strands wrapped in an anti-friction membrane as detailed in Fig. 1a.", "The polyester rope acts as the predominant load carrier and is terminated at either end with an eye splice.", "The key principles of operation to appreciate are:", "When under load, the tether extends along its length (axially) and simultaneously contracts across its diameter (diametrically), according to the helix angle of the braided rope.", "As the rope diameter contracts, the elastomer core is compressed radially.", "The core resists this compression and thereby acts to resist the axial extension of the rope.", "The compressibility of the elastomeric core dictates the resistance to diametric contraction, and in turn controls the axial extension.", "This compressibility is determined by both the cross-sectional form of the core and the material selection.", "The helix angle of the braided rope dictates the ratio of the force vectors that act axially to extend the rope and circumferentially to compress the core.", "Typical helix angles for the P1 Prototype Series are detailed in (Gordelier et al., 2015).", "Points (3) and (4) result in two distinct phases of tether extension as detailed in Fig. 2:", "Phase 1 - During the first phase of extension the elastomeric core strands are more easily deformed from seven individual round sections to eliminate the free space between the strands.", "At the same time, the helix angle is high, giving the braided rope considerable mechanical advantage in compressing the core.", "These factors combine to produce low axial stiffness during the first phase of extension.", "Phase 2 - As the free space in the core bundle tends towards zero, the resistance to further radial compression increases non-linearly.", "Simultaneously the extension of the tether also reduces the helix angle of the braid, thus reducing the rope's mechanical advantage over the core.", "These changes result in a second phase of extension which displays a marked increase in axial stiffness.", "Critically, the choice of core geometry and material can be selected completely independently of the hollow rope.", "Thus, a series of tethers with the same MBL can be designed with a range of compliance values selected specifically for a device or location.", "The working concept of the tether is presented in (Gordelier et al., 2015).", "This details the results from a Proof of Concept study with the P1 Series of 14 tether prototypes comprising a range of tether stiffness profiles achieved by varying the profile geometry and the Shore A hardness value of the elastomeric strands.", "The results presented in this paper focus on three of these tethers; the construction of these tethers is detailed in Table 1.", "It should be noted that the sheathing acts as an anti-friction membrane and to limit biofouling of the elastomeric core but will have minimal effect on the stiffness profile of the tether.", "If the benefits of increased compliance offered by the Exeter Tether are to be realised, the long term performance of the tether must be understood.", "The work presented in this paper outlines an investigation into the evolution of the stiffness profile of the tether when subjected to both sea trials and high load fatigue testing.", "It compares functional tether assembly test results to material tests which include analyses of the effects of marine ageing and cyclic compression on the EPDM core material.", "This work is of critical importance to the ongoing development of the Exeter Tether, and is also of interest to other mooring developers utilising elastomers in their proposed novel mooring systems (McEvoy, 2012; Bengtsson and Ekström, 2010).", "Mooring designers looking to exploit novel mooring systems will be interested in these findings for improved understanding of long-term performance and to inform the correct specification of mooring system configurations utilising these novel solutions.", "Finally, establishing the impact of marine ageing and compression testing on EPDM will be of interest to anyone utilising this material in these or other relevant applications.", "The work presented in this paper details a combined approach utilising both assembly test techniques and sub-component material tests to investigate the durability of the elastomeric tether in the proposed operating environment.", "To provide an overview of the test approaches applied, Table 2 is included, which outlines the assembly and sub-component investigations completed.", "Each of the listed test procedures has a dedicated section in the Method, Results and Discussion chapters that follow.", "The challenge for offshore technology innovations is validation during early development in order to demonstrate and eventually commercialise a novel concept.", "Whilst full-scale field tests are preferable and reduce potential scale-induced uncertainties (Mueller and Wallace, 2008), they are expensive and challenging to control.", "Laboratory based tests offer a high degree of control but can limit the scale and relevant factors that can be investigated (Salter, 2003).", "This work has chosen a combined approach.", "Based on realistic, large-scale field test exposure, the operational durability of the tether is assessed.", "The assessment is further enhanced through repeated, controlled experimental tests at assembly and sub-component level.", "The performance durability appraisal can thus be based on both test methods.", "This section initially describes facilities utilised before describing the test procedures adopted.", "Tether prototypes were exposed to realistic operational conditions through deployment on a mooring limb of the South West Mooring Test Facility (SWMTF).", "This is an at-sea test facility located just inside the harbour limits of Falmouth Bay, Cornwall, UK.", "A fully autonomous instrumented buoy is configured to enable mooring components under investigation to be deployed on mooring limbs and exposed to a typical mooring environment, with in-line and tri-axial load cells to monitor mooring loads.", "Further details of the facility are provided in (Johanning et al., 2008).", "For the purposes of this work, the SWMTF test facility was utilised to expose Exeter Tether prototypes to both marine ageing and simultaneous cyclical loading at typical mooring loads.", "Controlled testing of the tether assembly was conducted in the Dynamic Marine Component Test Facility (DMaC).", "This facility provides fully submersible freshwater testing of components up to 6m in length and 0.8m diameter, with a 1m stroke linear hydraulic actuator able to deliver tension or compression forces of 30t dynamically and 45t statically.", "Further details of the test facility are available in (Johanning et al., 2010).", "The 444 kN pancake load cell controlling the linear actuator is calibrated annually and upon re-positioning; a full calibration was conducted in advance of this work and is reported in (Gordelier et al., 2015).", "Material testing of the EPDM was conducted at the IFREMER1 Marine Structures Laboratory.", "ISO7743 tests were conducted using an Instron 5566 load frame with a 10 kN load cell.", "Compression fatigue testing of the samples was conducted using an MTS test machine with a 250 kN load cell.", "Immersion of material samples in natural sea water was conducted using a bespoke system developed at IFREMER comprising a 60 L fresh seawater tank.", "The water was maintained at a constant temperature of 60 °C whilst being continually renewed with fresh sea water from the Brest estuary at a replacement rate of 60 L/24hrs.", "All equipment in the IFREMER laboratory is annually calibrated as part of the ISO9001 Quality Management Systems accreditation of the laboratory.", "Four prototype tethers were deployed on mooring limb 1 of the SWMTF from 4th June 2013 to 26th November 2013.", "Mooring limb 1 and mooring limb 3, installed at 185° and 65° respectively, experience the highest mooring loads due to the dominant wave conditions on the site as observed during previous deployments (Harnois et al., 2013, 2016).", "The composition of a typical SWMTF mooring limb is detailed by Harnois et al. in (Harnois et al., 2012).", "The existing 20m section of nylon rope at the top of the mooring line was replaced with four tethers; the focus for the work presented in this paper is tethers P1-3 and P1-8, with EPDM core Shore A hardness values of 59A and 70A respectively.", "P1-8 was situated at the top of the mooring line connected into the tri-axial load cell of the SWMTF via shackles.", "P1-3 was connected into P1-8 via a lanyard hitch.", "This top zone of the mooring line where P1-3 and P1-8 were located was anticipated to be the most aggressive zone for tether deployment.", "A qualitative assessment of the tethers was conducted following the deployment to assess degradation of the tether and sub-components; this assessment is reported in (Gordelier et al., 2015).", "For the purposes of the work presented here, in order to quantitatively assess the change in dynamic axial stiffness of the tethers, the DMaC test facility was used to conduct tether functionality tests both before and after the six month sea deployment.", "Force driven conditioning tests were conducted, followed by a displacement driven test exercising the tether to a displacement of 0.9m for 5 cycles at an 8s period.", "The displacement of 0.9m was selected to maximise the extension available within the 1m stroke range of DMaC, and a cycle period of 8s was selected to represent a typical operating period.", "This is important due to the time dependant nature of the EPDM core material.", "The drive data for this test, ETT-08, are presented in (Gordelier et al., 2015) and the load-extension results from the fifth cycle of this test were extracted and processed to establish the stiffness profile of the tethers pre and post SWMTF deployment.", "The processing involved a deduction to account for the extension of the eye splices as measured and reported by (Gordelier et al., 2015).", "To account for the reduced eye splice extension following the sea deployment, an adjustment was made assuming that the extension of the eye splice reduces in accordance with a typical worked polyester rope.", "The Viking Braidline polyester rope was selected as the most appropriate reference rope, which over the full operating range demonstrates an average reduction in extension of approximately 25% upon working (Bridon, 2017).", "This approach was repeated for tether prototypes P1-3 and P1-8 before and after the SWMTF deployment.", "Further to the assessment of the tether stiffness profile, it was originally anticipated that an assessment of load data from the 69 kN axial load cells and 69 kN tri-axial load cells located at the top of each mooring line would allow for a quantitative assessment of the impact of the mooring tether on mooring loads and additionally log the load exposure profile of the tethers during the deployment.", "However, the load cells failed early during this deployment.", "Numerical modelling to estimate mooring loads utilising data collected from the SWMTF inertial measurement unit (IMU) and digital global positioning system (DGPS) is ongoing and will be the subject of future publications.", "Typical load profiles for SWMTF mooring limbs during previous deployments are detailed by (Harnois et al., 2016) and demonstrate peak mooring loads for Limb 1 exceeding 50 kN with typical operational mooring loads below 10 kN and typical peak periods ranging from 2 to 11 s.", "To establish a fatigue performance benchmark for the P1 Series a Thousand Cycle Load Limit (TCLL) test was conducted.", "This test, designed by the Oil Company's International Marine Forum (OCIMF), was established to quantify mooring hawser response to tension-tension fatigue (Oil Companies International Marine Forum, 2000).", "The test subjects the component to a series of 1000 tension cycles at increasing load levels; starting with 50% NWBS (new wet breaking strength) and increasing in 10% increments until the sample fails.", "The test is conducted with the tether fully submerged in fresh water.", "Full details of the test method, establishing the NWBS of the tether (222 kN) and the full calculation of the TCLL value for the tether are presented in (Gordelier et al., 2015).", "Of specific interest to the work presented here is the stiffness performance of the tether.", "The TCLL test was therefore analysed to quantify the evolution in dynamic axial stiffness of the tether throughout the TCLL testing series.", "The test failed during cycling at 70% NWBS so there is a full data set of 1000 cycles at 50% and 60% NWBS load levels, and a partial set of data available at 70% NWBS load level (the sample failed on 176th cycle of the 70% level).", "The tether utilised for the TCLL test was P1-16; this is a modified tether, with a reduced working length to enable the high extensions of the TCLL test to be achieved within the working constraints of DMaC.", "The tether core is manufactured from seven Ø20 mm 70 Shore A EPDM strands from supplier B. Due to the different construction of this tether, it is not possible to directly compare the results to those from the field test at the SWMTF.", "The results however, do provide useful insight into the evolving stiffness profile of the tether design concept when worked at high loads.", "A key principal of the tether is the control of axial stiffness by varying the core material and construction, with each core configuration providing a particular resistance to compression.", "The compression stress-strain properties of the core material are therefore critical to the operation of the tether; understanding how these material properties might be affected by ageing of the core material, or exposure to multiple compression fatigue cycles is important for ascertaining long term performance.", "ISO7743:2011 Rubber, vulcanized or thermoplastic determination of compression stress strain properties (ISO, 2011) provides a standard to calculate the compression modulus of a given polymer.", "Quantifying this property for virgin material can then be used as a benchmark to compare with further material tests following marine ageing or compression fatigue exposure.", "It should be noted the ISO7743 test is conducted in air.", "The ISO standard provides a choice of test pieces and methods.", "The results presented here are based on the procedure standard reference Test Piece B (Fig. 3a), Method C2 As the EPDM material was supplied in 25 mm extruded section, and the compression modulus is affected by both the material properties and the manufacturing process, the standard Test Piece B dimensions were scaled up, so that the test could be conducted on the 25 mm section; these dimensions are detailed in Fig. 3b. Results from these tests, compressing the sample axially (along the axis of the extruded EPDM), are referred to as the Axial Compression Modulus (ACM).", "For each test condition, three separate samples were tested.", "In addition to the ACM tests, of specific interest to the operation of the tether is the compressibility of the EPDM across the diameter of the cord.", "This is not a standard ISO test and, unlike the ACM tests, the cross section of the sample in the radial direction is not consistent and the stiffness in this direction is related to both the material and the geometry of the sample.", "As this is not a standard test it is not comparable to external sources, however, quantifying the stiffness of the sample in this direction allows for a direct comparison across the range of EPDM hardness values and an understanding of how fatigue compression exposure and marine ageing of the material affect this crucial property.", "The same procedure was followed as outlined for ACM.", "As this is not a direct calculation of material modulus, this result is referred to as the Radial Compressive Stiffness (RCS).", "The target dimensions for the RCS samples are detailed in Fig. 3c.", "It should be noted that in some of the extruded EPDM sections a 2–3 mm flat ridge was evident running along the length of the cord; this was a product of the manufacturing extrusion process.", "To minimise the effect of this ridge the sample was orientated ensuring the ridge was at the side and the sample was marked with a white dot to ensure consistency between sample measurements and test procedures.", "Three repeat tests were performed for both ACM and RCS across the range of Shore A hardness values specified: 54, 59, 70, 70.7 and 80.7.", "The durability aspect of this work was to ascertain whether these properties are affected by the operational requirements of the tether and a mid-range EPDM with Shore A hardness 70.7 was selected for further testing as detailed below.", "EPDM samples were cut to the dimensions detailed in Fig. 3c and immersed in natural sea water tanks as detailed in Section 2.1, heated to a temperature of 60 °C for a period approaching 13 months (15/05/2014–10/06/2015).", "Regular weight measurements were recorded to monitor sample saturation.", "Immediately following removal from tanks the ISO7743 test procedure was conducted as detailed in Section 2.4.1 to establish the RCS following marine ageing.", "During operation the tether extends and contracts to accommodate the wave induced motion of a floating body.", "This in turn exposes the EPDM core to repeated radial compression and axial extension cycles.", "This is a durability concern for the tether as the long term consequence of these actions is unknown.", "To investigate the potential effects of this process, samples of the EPDM core were subjected to repeated compression cycles and the ISO7743 procedure was used to calculate the RCS of the sample following this exposure.", "Material samples were prepared to the dimensions detailed in Fig. 3c and set up in the MTS compression test machine using a 250 kN load cell.", "A small dot of cyanoacrylate was used to secure the underside of the cord to the MTS load platen as during trials the sample had been observed slipping out of the test machine.", "A compromise was required in specifying a frequency that maximises the number of cycles in a given test window whilst limiting the self-heating of the sample.", "A preliminary investigation into the test parameters using an infra-red camera indicated that at a compressive diametral strain of 0.2 the frequency of compression cycles should be limited to 2 Hz.", "Frequencies greater than this led to excessive self-heating of the EPDM sample, with surface temperatures beyond 10 °C.", "The MTS machine was set to operate in displacement mode, with a diametral strain of 0.2 calculated from the average of three measurements of the cord diameter.", "The test sample was subjected to 707,000 compression cycles to a strain of 0.2 and a frequency of 2 Hz.", "The RCS test procedure (outlined in Section 2.4.1) was conducted immediately following the compressive testing and at subsequent intervals to observe any time dependency in the results.", "Conducting the ISO test both radially and axially on one single sample was not considered good practice as one procedure may affect the other and, as the critical property of interest in this instance was the radial compressive stiffness, the test was only conducted radially.", "Key observations from this assessment are a measurable increase in dynamic stiffness of the tethers following the sea deployment.", "The majority of the increase in stiffness is observed in Phase 1 of operation with an increase of 54% and 110% for tethers P1-3 and P1-8 respectively.", "Additionally, and perhaps more critically, the stiffness profiles of the worked tethers have become more aligned, settling on a similar stiffness profile as highlighted by Fig. 4.", "Pre-deployment during Phase 1, P1-3 demonstrated a stiffness 13% greater than P1-8, however, following deployment the stiffness during this phase was 15% less than P1-8.", "During Phase 2 the difference in stiffness between P1-3 and P1-8 dropped from 17% to 6% following deployment.", "Given the design philosophy behind the tether is to establish a range of stiffness profiles, this finding is very relevant and will be further addressed in the Discussion, Section 4.", "The same procedure was repeated for the 1–60% load level and the 1–70% load level.", "Fig. 6 provides a summary for the complete TCLL test series, capturing the dynamic axial stiffness calculated from the secants of Phase 1 and Phase 2 at cycles selected from all load levels.", "Plotting the data in this way highlights that the dominant change in stiffness during this high load fatigue test occurs in Phase 2 of the tether operation, where the functionality is dominated by the polyester rope rather than the EPDM core.", "The drop in stiffness observed between TCLL load levels is the result of adjustments required for the test set-up to accommodate recovery of the tether between load levels.", "Following completion of one load level the test ends at a load of 1% NWBS, however, as the tether recovers it attempts to contract in length.", "As it is fixed in the test machine, the result of this is an increase in load.", "To obtain a starting load of 1% NWBS for the subsequent load level, slack must therefore be introduced into the system, which results in the decrease in stiffness observed between the TCLL loading levels.", "The results in Fig. 6 also show a small reduction in stiffness occurring in Phase 2 at the start of the 1–60% load level.", "This could be due to the higher load level causing local damage in the rope construction or slippage within the tether construction where the hollow braided rope slips off the tether core.", "Following this initial drop however, a steady increase in stiffness is observed, though less pronounced than the 1–50% range with a maximum increase of 6% over the full 1000 cycles.", "The stiffness again stabilises at higher cycle numbers.", "Results from the final load level of 1–70% demonstrate the gradual reduction in tether stiffness in Phase 2 as the polyester rope yields further and approaches final failure.", "A typical load-strain plot from the ISO7743 test procedure is detailed in Fig. 7.", "The plot reveals the full extent of the Mullins effect that is observed in elastomers.", "It is characterised by the permanent softening of the stress-strain curve observed the first time a polymer is exposed to a compression or tension load (Cantournet et al., 2009).", "This can be seen in Fig. 7, where cycle 1 requires a greater load than any subsequent cycles.", "Utilising data from the fourth load cycle for the ISO calculations ensures the Mullins effect does not distort the results.", "Although this effect is clearly observable during the ISO7742 tests, the impact of this softening is minimal in comparison to the effects from marine exposure and radial compression of the material samples.", "As expected, a positive correlation can be seen between Shore A hardness and the calculated values for ACM and RCS.", "The observed range of stiffness values are a dominant factor controlling the variety of stiffness profiles observed in the tether assembly.", "It is also evident that at each Shore A Hardness value the ACM and RCS calculated at a strain of 0.2 is always higher than that calculated at a strain of 0.1.", "Again, this is intuitively expected; at the higher strain level of 0.2 the sample is under greater compression and it will therefore demonstrate increased resistance to any further compression (and thus a higher result for ACM or RCS).", "Having established baseline values of ACM and RCS across a range of Shore A hardness values, a mid-range EPDM material (Shore A hardness 70.7) was selected for a detailed analysis of the effect of marine ageing and fatigue compression cycles on key material properties.", "These results are presented in the next section.", "During the marine exposure testing, samples were periodically removed for weighing to observe the saturation of the samples.", "It was anticipated that the weight of the samples would stabilise over time, once fully saturated.", "At 13 months, although the rate had slowed, a small amount of weight gain was still occurring (Fig. 9).", "Due to time limitations however, the samples were removed at this point for testing.", "Following removal from the heated water the ISO7743 test was repeated on the aged samples of EPDM.", "Again the procedure was repeated on three samples from each range and Fig. 10 details the average and standard deviation of these samples in comparison with the virgin material.", "The ageing process has significantly increased the material resistance to compression with ACM increasing by 14% and 16% at strains of 0.1 and 0.2 respectively, whilst RCS increased by 22% at both values of strain.", "Given the compression of the EPDM core is a significant factor controlling the stiffness profile of the operating tether assembly as detailed in (Gordelier et al., 2015) this finding has significant implications for the operation of the tether which will be further explored in the Discussion.", "RCS results calculated using the ISO7743 approach following 707,000 compression cycles at a strain of 0.2 and a frequency of 2 Hz can be seen in Fig. 11.", "These results show that the immediate RCS of the sample, measured 12 min after the compression cycles, does not reflect the ultimate RCS once the sample has had time to recover.", "At the 12 min point, there is not a consistent change for RCS measured at 0.1 and 0.2 strain; at a strain of 0.1 RCS is 7% less than the control sample whilst at a strain of 0.2 it is 11% higher.", "Over time this difference diminishes; at 42h20 the RCS is 13% and 15% higher than the average control at strains of 0.1 and 0.2 respectively.", "ISO7743 is finally repeated 66h20 after the compression cycling, and provides RCS values 14% and 16% higher than the average control at strains of 0.1 and 0.2 respectively.", "Ultimately, these results show that exposure to extended compression cycling, at a strain of 0.2, can lead to a 15% average increased resistance to compression, in comparison to the un-fatigued control sample.", "On comparing the observed increase in tether stiffness following the sea deployment to the expected performance for a conventional fibre rope, the literature is inconclusive.", "Although different methods have been applied, Weller et al. (2015b) review the performance of a nylon seven parallel-stranded rope and suggest a decrease in axial stiffness following an 18 month deployment at the SWMTF; this change in performance is attributed to mild internal abrasion damage occurring in the rope construction due to yarn on yarn wear, accelerated by the ingress of particulate matter including biofouling, during deployment.", "This result is of interest in the context of synthetic ropes, however, it is not directly comparable to the tether polyester rope.", "The finding from (Weller et al., 2015b) is in contrast with results presented by (Bitting, 1980a) who observes an increase in axial stiffness for both nylon and polyester ropes when exposed to the ocean environment for 4–5 years.", "The polyester rope investigated is 8-strand plaited and double braided in construction and the dynamic stiffness increases 10–25% following the sea exposure.", "Further to this, data published by rope manufacturers generally indicate an increase in stiffness for worked ropes, with separate performance curves provided for new and worked ropes to inform system design.", "Typical worked graphs are published by rope manufacturer Bridon (Bridon, 2017) and demonstrate an average increase in stiffness of approximately 25% over the working range of a worked polyester rope (although the precise definition of ‘worked’ is not specified).", "Rope construction and material selection will significantly effect the change in stiffness observed, but overall it is anticipated that in the short to medium term synthetic ropes will get stiffer with use.", "As noted by (Weller et al., 2015b) a reduction in stiffness will be observed as the rope approaches failure, particularly if the failure mechanism is fatigue.", "Given the changes observed in the stiffness profiles of worked conventional mooring ropes it is unsurprising to observe a change to the operating profile of the Exeter Tether prototypes following sea deployment, as the outer load carrying jacket is a hollow braided polyester rope.", "It is interesting to note however, that the dominant change in the stiffness of the worked tethers following sea deployment occurs during Phase 1 of operation which is dominated by the compression of the EPDM core, as outlined in Section 1.1.", "The average increase in stiffness following deployment of both tethers for Phase 1 is 82% in contrast to Phase 2 with an average increase of 8%.", "During the Exeter Tether operation, although Phase 1 is dominated by the EPDM core material properties, the increase in stiffness of the load carrying polyester rope will have an impact on tether operation across both Phase 1 and Phase 2.", "The design concept of the P1 tether series utilises a standard braided rope jacket with a range of different polymer core specifications to achieve a variety of axial stiffness responses as demonstrated in (Gordelier et al., 2015).", "Therefore of interest, is the observed reduction in operating range of the post deployment tethers, given achieving this stiffness range was one of the key design intentions for the P1 Tether Series.", "Results from EPDM material testing indicate an increase in stiffness of the EPDM material which would certainly lead to an alteration in overall tether stiffness profile.", "These results are discussed in more detail in Sections 4.3 and 4.4.", "Of relevance to these tether assembly tests and the controlled assembly fatigue tests discussed in the next section, is the stiffness of standard polyester mooring lines and this behaviour in relation to average load and load amplitude.", "Expanding upon earlier work by (Bitting et al., 1980b), investigations with a 12 strand, parallel laid polyester rope presented by (Fernandes et al., 1999) demonstrate there is an increase in stiffness proportional to the average load applied, and a decrease in stiffness proportional to the load amplitude, with a limited effect from loading frequency.", "This work has been confirmed and further developed by (Davies et al., 2002) who also demonstrate a stiffness dependence on mean load and load range for both aramid and HMPE ropes.", "These observations are further supported by (Weller et al., 2014b) and (Weller et al., 2015b).", "To establish the change in tether stiffness observed pre and post SWMTF deployment, identical functionality test ETT-08 was repeated.", "This is a displacement driven test so small variations in the peak load of the fifth cycle are observable.", "However, the stiffness profiles are calculated at consistent loads for each sample, 5–10 kN for Phase 1 and 20–40 kN for Phase 2.", "Comparing the stiffness profiles in this respect should minimise the variation brought about by an increase in average load.", "The main limitation of the tether assembly test work is due to the limited number of samples available and the large investment required for sea deployments.", "With the high costs involved at the early stage of prototype development, the total number of tethers manufactured for the P1 Series was limited to 13 and no two tethers were identical.", "Given the scope of test work required from this first set of prototypes it was not possible to conduct repeat sea tests on multiple samples.", "Results should therefore be considered to give an indication of performance trends rather than definitive values for stiffness profiles.", "The TCLL fatigue endurance tests, performed in fresh water, highlight the increase in dynamic axial stiffness of the tether when subject to high load fatigue cycling.", "In addition to the increase of the individual cycle dynamic stiffness, an overall increase in sample length is measured.", "This extension is observed in conventional fibre ropes and is due to both the permanent bedding in of the rope and end terminations (material and constructional reorientation and stretch), creep and visco-elastic stretch which are accumulated during tension cycles (Flory et al., 2004).", "Work conducted on polyester fibres attributes an element of both the permanent and recoverable strain to the macromolecular rearrangement of entanglements and orientation of crystallites (Chailleux and Davies, 2005).", "Further to this (Davies et al., 2000), state that the majority of permanent bedding in strain is due to the material behaviour and not the rope construction for long lay length polyester mooring lines.", "Indeed the extent of the permanent stretch in polyester mooring ropes leads to a common practice of pre-stretching ropes during installation to minimise future adjustments required to the mooring system (DeAndrade and Duggal, 2010).", "Regarding the recoverable element of the strain, in conventional fibre rope testing recovery of the accumulated elastic stretch and visco-elastic stretch is anticipated when the tension cycles stop.", "However in the tether, the time dependency of this recovery is particularly pronounced due to the additional recovery of the EPDM core which takes on a temporary compression set during cyclical loading.", "To accommodate this recovery, adjustments to the DMaC set-up led to a perceived drop in axial stiffness as observed between load level cycles detailed in Fig. 6.", "The significance of the observed compression set is caused by the high loading levels used in the TCLL test (up to 70% MBL in this case).", "Critics of the TCLL test procedure claim the high loading levels render it unrepresentative of the final application (Davies et al., 2012); certainly this time dependant recovery would be much less perceptible at the typical operating range of the tether.", "It should also be noted that the high level of loading in the TCLL test procedure accelerates any wear mechanisms occurring.", "The reduction in stiffness and final failure of the tether observed at the end of the TCLL test in Fig. 6 would be more gradual under typical operating conditions.", "Weller et al. (2014b) analysed the performance of a conventional nylon rope with a parallel stranded sub-rope construction during cyclical loading and found that the axial stiffness stabilises from 25 cycles.", "In comparison to the results for the tether, significant evolution is still occurring at 25 cycles which suggests that due to the novel construction of the tether, establishing a stabilised axial stiffness profile during cyclical loading may take longer than for conventional fibre ropes.", "It should be noted however that the TCLL test presented here subjected the tether to significantly higher loading levels than testing presented in (Weller et al., 2014b).", "As discussed in Section 4.1, previous work has shown some stiffness dependency on both mean load and load amplitude for standard polyester ropes (Fernandes et al., 1999; Davies et al., 2002; Weller et al., 2014b, 2015b).", "Given the increase in mean load between the TCLL testing load levels it might be anticipated that an overall increase in stiffness would be observed between loading levels.", "However, due to a combination of the drop in stiffness due to adjustments between loading levels, the mild reduction in stiffness at the start of the 1–60% loading level, and the final failure occurring early in the 1–70% loading level, no such overall increase is observed.", "It is interesting to note however, that the significant increase in stiffness observed during the TCLL test series occurs in Phase 2 of operation as shown in Fig. 5 and Table 4, where the tether behaviour is dominated by the polyester rope as opposed to the EPDM core.", "Results from ISO7743 testing conducted following a period of marine ageing demonstrate an average increase in sample stiffness of 15% and 22% for ACM and RCS respectively.", "To put these results into context within the range of EPDM Shore A hardness values specified in the P1 Series, Fig. 12 has been included.", "This figure demonstrates that although the ACM and RCS have increased following marine ageing, the change has not been significant enough to alter the order of the specified EPDM, with the aged 70.7 still having ACM and RCS values higher than for EPDM 70 and lower than EPDM 80.", "There are various mechanisms which can contribute to changes in mechanical behaviour of elastomers after wet ageing: Reversible mechanisms include plasticization and swelling, irreversible effects include hydrolysis, leaching of additives such as plasticizers, and oxidation.", "These last two and possibly swelling will tend to result in increasing stiffness, while plasticization and hydrolysis may reduce it.", "More work including physico-chemical analysis is under way to identify the specific mechanisms which are involved here.", "It should be noted that although establishing the value for ACM followed the standard ISO test procedure with a scaled sample size, the quantification of RCS is not a standard material test; the results for this are affected by both the material property and the sample geometry.", "This test was created specifically for the tether work presented, as the diametric compression behaviour of the EPDM cord is of critical importance to operation of the tether, controlling the axial stiffness profile during Phase 1 of operation.", "Thus the RCS result is not a standard material property that can be specified, but the presented work provides a better understanding of how this key property changes when the material is subject to realistic operating parameters.", "Limitations of this work revolve around the assumption that immersing EPDM samples in heated sea water will accurately accelerate the natural ageing process in line with Arrhenius behaviour.", "Although this technique is widely used (Celina et al., 2005; Davies and Evrard, 2007; Le Saux et al., 2014; Le Gac et al., 2012; Scheirs, 2000; Wise et al., 1995), it does have limitations, with critics concerned that using temperature to accelerate the ageing process may introduce failure mechanisms that would not have occurred through natural ageing at a more representative operational temperature (Davies and Evrard, 2007).", "The work presented here uses one temperature, 60 °C, to accelerate the marine ageing process, assuming that an Arrhenius relationship applies for EPDM.", "Given the apparent implications for the material properties of the EPDM samples tested, an extension to this work is underway to conduct further testing over a range of different temperatures to confirm these findings and to accurately establish the Arrhenius relationship for EPDM.", "Further to this, fatigue testing of samples in natural seawater will be conducted to establish the implications of concurrent exposure to fatigue cycling and the marine environment.", "The long term effect of the fatigue compression cycling is an average 15% increase in RCS, which was measured following 66 h of recovery.", "However, it is interesting to note that immediately after cycling the RCS is lower, gradually increasing and stabilising over time, with minimal change occurring between 42 and 66 h.", "These compression test parameters represent a realistic, if conservative, loading scenario.", "Assembly tether testing observed radial compressive strains exceeding 0.35 during anticipated extreme conditions.", "Therefore exposing the EPDM cord to fatigue compression at a strain of 0.2 during this test schedule certainly represents a genuine operational demand.", "Further to this, assuming a typical wave period of 8 s, 707,000 cycles represent just 65 days of operation.", "To put this observed change of RCS into context within the range of specified EPDM hardness values Fig. 13 has been included.", "This figure demonstrates that despite increasing the RCS of the material, the fatigued 70.7 EPDM remains in consecutive order of increasing RCS with increasing Shore A Hardness.", "A potential limitation for this section of work revolves around the validity of these results due to the temperature increase of the samples when under compression cycling.", "To investigate this further detailed testing was conducted observing the temperature increase in samples when subjected to repeated compression cycles at a range of frequencies and to a varying degree of compressive strain.", "Measurable temperature increases were observed when subjecting samples to high frequency or high strain compression cycles.", "For example, at a strain of 0.2 and a frequency of 5 Hz, surface temperature increases peaking over 13 °C were observed.", "More significantly, at a strain of 0.5 and a frequency of 2 Hz surface temperature increases in excess of 35 °C were observed at the centre of the sample core cross section.", "The experimental parameters for the compression testing presented in this paper are based on a strain of 0.2 and a frequency of 2 Hz.", "Observed surface temperature differentials using these parameters remain below 9 °C and are therefore considered to have a minimal effect on results.", "This temperature increase may have affected the radial compressive stiffness calculated immediately following compression testing (Fig. 11) but should have limited effect on the longer term measured compressive stiffness values.", "Considering all these findings in the context of overall tether implications a pattern of increased stiffness when exposed to the marine environment and/or cyclical loading is established.", "Findings from the material tests are not directly comparable to the assembly scale tests however they do offer insights into what is causing the observed changes in tether assembly behaviour.", "The findings from the ISO7743 tests following either marine ageing or radial compression fatigue of the EPDM core material demonstrate an increased material resistance to compression.", "This supports the observed increase in tether stiffness during Phase 1 of tether operation following sea trials, where the core will have been subject to both the marine environment and radial compression fatigue.", "To understand the relative contribution of different aspects of the material testing to the tether assembly performance, the ISO7743 material findings were plotted within the context of the range of hardness values of the EPDM utilised in the P1 series prototype range in Figs. 12 and 13.", "These Figures demonstrate that, although significant, neither the marine ageing or radial compression fatigue testing shifted the order of the ACM or RCS across the range of EPDM Shore Hardness values tested.", "The conclusion from this is that there are multiple factors leading to the increase in stiffness observed in the aged tether assemblies following sea deployment.", "This includes changes to both the EPDM material radial compressive stiffness (caused by marine ageing and repeated radial compression of the core through the extension of the tether during operation), and the overall increase in stiffness of the polyester rope as observed in conventional polyester ropes (Bitting, 1980a; Bridon, 2017).", "The work presented in this paper seeks to establish an understanding of the long term behaviour of the tether under typical operating parameters and does not address final failure mechanisms or fatigue failure rates.", "To establish a specific T-N curve for the tether would require a substantial test programme beyond the scope of work presented here and of limited use at this prototype stage.", "The polyester rope is the predominant load carrier within the tether and existing T-N curves for polyester rope (Flory and Banfield, 2006) can be used for fatigue estimation and design of mooring systems (Thies et al., 2014).", "The improved operating stiffness profile offered by the Exeter Tether will reduce both peak and fatigue loads throughout the mooring system, with (Parish et al., 2017) estimating a peak load reduction of approximately 70%.", "These load reductions should be considered when designing a floating system incorporating the tether as there are benefits that go beyond the mooring system, including a potential reduction in the required size of the floating body.", "The P1-Series of tether prototypes were developed to prove the design concept of the Exeter Tether.", "The durability of this series was not a primary objective therefore the work presented in this paper offers some initial insights into key durability considerations for the P1 series in order to inform future design iterations.", "Controlled testing of the tether assembly following a sea deployment approaching 6 months demonstrates a measurable increase in dynamic axial stiffness, with an average 82% and 8% increase in Phase 1 and 2 of operation respectively.", "Critically, this field deployment demonstrates that, for the two samples investigated, the diversity in stiffness profiles observed in the virgin tethers is reduced following a period of sea deployment.", "As the range of stiffness profiles is a key operating principle for the tether design, further work is required to establish if this reduction is observed across the full suite of tethers.", "Work is also ongoing utilising numerical models to estimate the load exposure of the tether prototypes during the SWMTF deployment and to establish the reduction in peak mooring loads achieved through tether placement within the mooring system.", "The controlled assembly fatigue testing of tether P1-16 also highlighted the increase in dynamic axial stiffness upon working.", "The maximum increase of 42% was observed during the first loading level of 1–50% NWBS in the Phase 2 secant.", "This testing demonstrated the increase in stiffness stabilises over time reaching a natural limit.", "It is suggested that for each tether design a minimum of 500 cycles at typical operating loads is used to ascertain a working stiffness profile for use in mooring system design.", "The dynamics of the floating system (and the resulting peak and fatigue mooring loads) will be significantly affected by the mooring stiffness profile.", "Establishing the ‘worked’ operating profiles of the tether assembly is of central importance to inform mooring systems designed to incorporate the tether.", "In addition to tether assembly testing, detailed material tests of the EPDM core material established an increase in material stiffness when exposed to operational parameters including the marine environment and repeated compression cycles.", "Average increases in RCS of 22% and 15% were observed following marine ageing and exposure to fatigue compression cycles respectively.", "Prior to this work the effect of these parameters on EPDM was unknown.", "This information will therefore be useful to anyone intending to utilise EPDM in a marine environment or an application where it is repeatedly exposed to compressive strain.", "Breaking the overall assembly down and conducting sub-component tests as presented in this paper allows a greater understanding of causality regarding the observed changes in the tether assembly operation.", "Going forward, it also creates an opportunity to investigate alternative materials more efficiently, without the need to construct full tether assemblies for each iteration.", "This general approach, combining both field and laboratory experimentation to identify and investigate particular durability aspects of a novel component is particularly relevant for the offshore technology sector.", "Field work in this sector involves high operational costs, and although it is certainly necessary to prove components in the field, identifying opportunities for laboratory based testing to develop particular aspects of component investigation has shown to be an effective supplement to the field based work.", "Bringing all aspects of the work presented here together, the assembly tether testing and EPDM material testing corroborate one another, all indicating an increased tether axial stiffness as a result of exposure to operating conditions.", "Marine ageing and low level loading (from the sea deployment) is found to significantly affect the stiffness during Phase 1 of tether operation, whilst high level fatigue loading has a greater impact on Phase 2 of the tether stiffness profile.", "The key objectives for the tether development are to increase the compliance of the mooring system and allow the specification of an axial stiffness optimised for a specific device and location.", "Whilst the tether successfully increases the overall compliance of the system as demonstrated in (Gordelier et al., 2015) and (Parish et al., 2017), this work has shown that the evolution of the tether stiffness profile during operation must be taken into account when designing a mooring system incorporating the Exeter Tether.", "Following these findings, work has continued to further investigate the affect of marine ageing on EPDM.", "In addition, OPERA, a European Union Horizon 2020 project, has commissioned the development of up-scaled tether prototypes including the P3 series and the full scale P4 series which will be deployed in the Bay of Biscay, Spain.", "The P4 tether will replace a conventional polyester rope on selected limbs of the Karratu shared mooring system, securing the MARMOK-A-5 wave energy device (Weller et al., 2017).", "Mooring load data from this deployment will allow an assessment of the load reductions achievable with this novel mooring system and a further assessment of the operational durability of the tether assembly.", "This work was supported by the Engineering and Physical Sciences Research Council, via a PhD funded through the SuperGen UKCMER programme (Grant Numbers: EP/1027912/1 and EPN/M014738/1).", "Additional funding was provided by the European Commission through the MARINET FP7 programme which funded access to the IFREMER Materials in a Marine Environment Laboratory for two weeks (Grant Number: 262552).", "The P1 Tether series were developed with assistance from Lankhorst Ropes and HEFCE through the HE Innovation Fund.", "The P3 and P4 Tether series were developed with Lankhorst Ropes as part of the OPERA Project, funded by the European Commission's Horizon 2020 programme under grant agreement No 654.444.", "Due to the confidential nature of some of the research materials supporting this publication not all of the data can be made accessible to other researchers.", "Please contact [email protected] for more information." ]
[ "Assembly and sub-component durability tests of an elastomeric mooring tether.", "6 month field trials lead to an average 45% increase in tether stiffness profile.", "A 42% peak increase in tether stiffness observed during laboratory fatigue tests.", "Marine exposure of elastomer increases material compressive stiffness by 22%.", "Compression fatigue of elastomer increases material compressive stiffness by 15%." ]
An open data repository and a data processing software toolset of an equivalent Nordic grid model matched to historical electricity market data
This article presents an open data repository, the methodology to generate it and the associated data processing software developed to consolidate an hourly snapshot historical data set for the year 2015 to an equivalent Nordic power grid model (aka Nordic 44), the consolidation was achieved by matching the model׳s physical response w.r.t historical power flow records in the bidding regions of the Nordic grid that are available from the Nordic electricity market agent, Nord Pool. The model is made available in the form of CIM v14, Modelica and PSS/E (Siemens PTI) files. The Nordic 44 model in Modelica and PSS/E were first presented in the paper titled “iTesla Power Systems Library (iPSL): A Modelica library for phasor time-domain simulations” (Vanfretti et al., 2016) [1] for a single snapshot. In the digital repository being made available with the submission of this paper (SmarTSLab_Nordic44 Repository at Github, 2016) [2], a total of 8760 snapshots (for the year 2015) that can be used to initialize and execute dynamic simulations using tools compatible with CIM v14, the Modelica language and the proprietary PSS/E tool are provided. The Python scripts to generate the snapshots (processed data) are also available with all the data in the GitHub repository (SmarTSLab_Nordic44 Repository at Github, 2016) [2]. This Nordic 44 equivalent model was also used in iTesla project (iTesla) [3] to carry out simulations within a dynamic security assessment toolset (iTesla, 2016) [4], and has been further enhanced during the ITEA3 OpenCPS project (iTEA3) [5]. The raw, processed data and output models utilized within the iTesla platform (iTesla, 2016) [4] are also available in the repository. The CIM and Modelica snapshots of the “Nordic 44” model for the year 2015 are available in a Zenodo repository.
[ "MULT" ]
[ "Common Information Model (CIM)", "Dynamic simulations", "Electric power transmission", "Electrical power systems", "Historical market data", "Modelica", "Modeling", "Power flow", "Power system dynamics", "Power system modeling and simulation", "Simulation", "Smart grid" ]
[ "1000" ]
[ "Raw data from Nord Pool was consolidated with a physical model representation of the Nordic grid by matching the measurement records to the model׳s power flow results (processed data).", "This has resulted in thousands of representations of the “Nordic 44” model for the year 2015 that are made available in the GitHub repository with the submission of this article.", "These snapshots are provided in the form of CIM v14, Modelica and PSS/E (Siemens PTI) files.", "The Python scripts (i.e. software toolset) used to generate these snapshots (CIM v14, Modelica and PSS/E) are also made available in the repository.", "The pre-processed (raw) data was downloaded from Nord Pool webpage.", "For each hour, the records contain the active power production and consumption data in the bidding regions of the Nordic grid and the active power exchange between them.", "These records were matched to an electrical grid model steady-state response, as explained next.", "To match and consolidate the historical market data to a physical description of the power network, the Nordic 44 model was developed.", "Note that the aim here was to set a “base case”, from which multiple snapshots of actual measurements records could be mapped to the quantitative response from computations on a physical model of the grid that included both steady-state and dynamic analysis features.", "The development of this source model/“data” consisted of (a) obtaining editable files in the PSS/E form to, (b) extend the model to represent better the Norwegian portions of the Nordic grid and to adapt it to modeling limitations in both the iTesla platform and the iPSL library (i.e. lack of HVDC link models).", "Step (a) used source files which was stripped from user defined models and other equipment, and other additional modifications carried out by Emil Hillberg of STRI on behalf of Statnett SF.", "The resulting model of this step is archived in Models.zip in respective Zenodo repositories.", "Note that the starting model is an extension itself of the Nordic 23 bus model developed at SINTEF Energy Research in several steps [6].", "The Nordic 23 bus model was developed from a 15 bus Nordic power system model developed at NTNU and the details of this model are explained in [7].", "Step (b) included the assignment of bus bar names, grouping of busses according to the actual bidding region, and numerous other changes as described in the documentation available in.", "/…/ SmarTSLab_Nordic44/00_Documentation/N44_changes.docx.", "The model developed in Step (b) is used throughout the historical data matching and consolidation process to create snapshots of the actual operational conditions of the Nordic Grid for 2015.", "This base case can be found in the repository in the GitHub repository at.", "/nordic44/models/ and archived in Models.zip in respective Zenodo repositories.", "The pre-processed (raw) data was downloaded from Nord Pool webpage as MS-Excel files (Consumption_xx.xlsx, Exchange_xx.xlsx, and Production_xx.xlsx) were saved in a folder with corresponding date (e.g. N44_20150401 refers to the folder containing the hourly snapshots of the 1st April 2015).", "The MS-Excel files (Production_xx.xlsx and Consumption_xx.xlsx) contain the active power production and consumption data in the bidding regions of the Nordic grid for every hour as shown in Fig. 1.", "The MS-Excel file (Exchange_xx.xlsx) contains the active power exchange data between the bidding regions of the Nordic grid for every hour.", "Now, the toolset was updated to automatically download the data from the Nord Pool ftp server, inspired by the work in [11].", "The raw data is stored in a Python dictionary allowing for easy data manipulation in Python and integration with PSS/e through psspy.", "It is still possible to save the data to excel and to load in excel files to the dictionary ensuring both forward and backward compatibility.", "For each hour, an equivalent Nordic power system model (Nordic 44) was created, matched and consolidated with the load generation balance in the bidding regions and the active power exchange between the bidding regions.", "The buses in the Nordic 44 model were named according to the closest city or town corresponding to both the geographical location and the detailed grid structure available to Statnett SF.", "The work flow used to create the PSS/E snapshots is shown below in Fig. 2.", "The major steps of the data processing workflow include:", "The raw data for the year 2015 was downloaded from Nord Pool (http://www.nordpoolspot.com) to MS-Excel files through manual queries to the Nord Pool database.", "This procedure was automated by implementing a Python class for connecting to the Nord Pool ftp server and handling the Nord Pool data.", "An example on how to use the Python code to generate the cases is provided in the GitHub repository (.", "/examples/multiple_data_sets_from_nordpool.py /data_set_from_nordpool.py)", "The raw data from Nord Pool contains only active power flow measurements (generation, consumption and exchange) within the bidding regions and between the bidding regions, for every hour.", "Another Python script simulates/computes the power flow with the constraint of minimizing the error between the power through the lines between the bidding regions.", "The method implemented in the Python script performs several checks (e.g. convergence, limits etc.), and after completing these tasks, it computes the error between the Nord Pool measurement records and those obtained from the Python script computations on the Nordic 44 model.", "A summary of results is written in an MS-Excel file for each snapshot and named PSSE_in_out.xlsx.", "The obtained PSS/E snapshots (processed data) contain the power flow solutions that give the best match to the historical data from Nord Pool.", "These are necessary to initialize simulations, especially those needed for DSA.", "At the end of this work flow, MS-Excel files (PSSE_in_out.xlsx) are generated by the Python script for every snapshot with raw data from Nord Pool and the results from PSS/E.", "These MS-Excel files (PSSE_in_out.xlsx) include limit checking messages (branch overloading, bus voltage out of limits and generator overloading).", "A screenshot of the created Excel file is shown in Fig. 3.", "The PSS/E snapshots for each hour before solving the power flow (hx_before_PF.raw, unmatched processed data) and after solving the power flow (hx_after_PF.raw, matched processed data) were also made available in the repository.", "The Nordic 44 model and the PSS/E snapshots can be accessed from the repository as shown in Fig. 4.", "CIM v14 and Modelica snapshots were generated from the matched PSS/E solved power flow snapshots (processed data).", "The PSS/E snapshots and PSS/E dynamic model parameters data (.", "dyr) files were used by the Python script to generate the CIM v14 snapshots as shown in Fig. 5.", "The Python script uses the Application Programming Interface (API) of the Operational Database Management System (PSS/ODMS) software [12] to generate the snapshots.", "These generated CIM snapshots can be used for information exchange according to CIM and to perform analysis in CIM compliant tools [13,14].", "The generated CIM snapshots were placed in the folder corresponding to the day they refer to (e.g. N44_20150401 refers to the 1st of April 2015).", "In each folder there are three files that define individual CIM snapshots for each hour (N44_hx_EQ.xml, N44_hx_SV.xml and N44_hx_TP.xml).", "N44_noOL_RDFIDMAP.xml is the file with IDs mapping of those cases with fixed overloading problems.", "N44_RDFIDMAP_2015-1.xml and N44_RDFIDMAP_2015-2.xml are the files with IDs mapping of the remaining snapshots from 2015.", "The screenshot of the generated CIM snapshots in the GitHub repository is shown in Fig. 6.", "PSS/E snapshots and PSS/E dynamic model parameters data (.", "dyr) files were used by the Python script “Raw2Record” (.", "/…/SmarTS-Lab/Raw2Record) to generate the associated Modelica snapshots as shown in Fig. 7.", "These generated Modelica snapshots together with the OpenIPSL library can be used for simulation in the Modelica compliant tools.", "The generation of the record files using the python script is illustrated with an example (.", "/examples/multiple_data_sets_from_nordpool.py /data_set_from_nordpool.py) and is provided in the GitHub repository.", "During the iTesla project, another methodology to generate Modelica model snapshots using the iTesla platform [3] was attempted.", "Note that these snapshots were created using a different methodology reported in [15].", "The resulting snapshots available for this approach cover only from April 1, 2015 to July 31, 2015 and are archived in iTesla_Platform.zip in the Zenodo repository.", "This archive contains both the snapshot models together with the appropriate version of the iPSL library.", "The data records are stored in the /ModelicaSnapshots/ sub-folder of the Zenodo archive.", "They require the Modelica model that was manually implemented for Nordic 44 reported in [1] with the record structures corresponding to the PSS/E snapshots.", "The Python script (“torecord”) used to generate Modelica snapshots from PSS/E snapshots as described in Fig. 7.", "The OpenIPSL library used in generation of these snapshots can be found in.", "/…/SmarTS-Lab/OpenIPSL (Fig. 8).", "The CIM and Modelica snapshots (processed data) of the “Nordic 44” model for the year 2015 are available the aforementioned Zenodo repository (see [2])." ]
[]
Quantification and simulation of thermal decomposition reactions of Li-ion battery materials by simultaneous thermal analysis coupled with gas analysis
Differential Scanning Calorimetry (DSC) and Thermal Gravimetry (TGA) combined with gas analysis are used to identify the main decomposition processes and to develop reaction kinetic models for thermal runaway modelling of Graphite – Lithium Nickel–Manganese–Cobalt-Oxide (NMC 111) cells. Heating rates of 5, 10 and 15 °C min−1 with multiple replications are performed to determine the frequency factor, activation energy and the heat of reaction of the different sub-processes. It is found that both the anode and cathode decompose in multiple parallel and consecutive reactions between 5 °C and 600 °C. A double breakdown mechanism of the protecting Solid Electrolyte Interface (SEI) is suggested to describe the anode decomposition reactions. For the cathode, decomposition and evaporation of ethylene carbonate (EC), decomposition of NMC with the liberation of oxygen, combustion of EC with the liberated oxygen, decomposition of binder, decomposition of EC and combustion of carbon additive reactions are identified and modelled. The proposed model can be used to simulate thermal runaway initiation methods in a realistic way.
[ "CHEM", "ENER", "ENGI" ]
[ "Battery", "Li-ion", "Safety", "Simulation", "Simultaneous thermal analysis", "Thermal runaway" ]
[ "1606", "2102", "2105", "2208" ]
[ "Various standards and regulations include tests [1] for the characterization of safety performance of batteries during non-normal circumstances (e.g. overcharge, external short circuit, crash) [2].", "However, testing thermal propagation (TP) of batteries caused by internal short circuit during normal operation (e.g. Boeing 787 incident [3–6]) is still in the forefront of research and discussions [7].", "Standards and regulations have identified several possible methods [1,8–10] but there is no common agreement which one can be used for a fit-for-purpose regulation.", "The difficulty stems from the fact that the determination of suitable testing parameter combinations by experimentation requires an unmanageable number of iterations.", "Alternatively, simulation can be used [11] to design and optimize an initiation method.", "However, use of a 3D model can be prohibitively computational expensive to assess the effect of all combinations of possible parameters on thermal runaway.", "Our overall goal therefore is to develop a simplified model, able to assess the most significant parameters for triggering thermal runaway (TR) induced by internal short circuit and thereby, to narrow down the testing needs.", "However, published thermal decomposition models found are not fully suitable for this purpose.", "Consequently, as a first step, thermal data together with measurement uncertainty is determined from Differential Scanning Calorimetry (DSC), Thermal Gravimetric Analysis (TGA) and gas analysis.", "From these, an approximate kinetic model is developed, which is able to mimic the dynamics of TR in a realistic way.", "In a second (follow up) paper, this model is used to simulate TR and to calculate the probability of thermal runaway as a function of the parameters of a triggering method.", "In the next sub-sections, first a summary of experimental methods and their main conditions are given.", "Then, the key decomposition reactions are listed for graphite, NMC and electrolyte.", "Finally, different kinetic models, developed for the decomposition reactions, are summarized.", "Accelerated Rate Calorimetry (ARC), Differential Scanning Calorimetry (DSC), Differential Thermal Analysis (DTA), Thermal Gravimetric Analysis (TGA) coupled with gas analysis are the commonly used methods for the characterization of thermal decomposition reactions of Li-ion batteries and materials.", "McNeil and Dahn [12] compared ARC and DSC measurements and found that they are equivalent, however ARC is more sensitive to determine the reaction order, while DSC is more precise to determine the so-called thermal triplets (activation energy, frequency factor and heat of reaction).", "Furthermore, ARC is unfit to detect endothermic reactions [13].", "Simultaneous Thermal Analysis (STA), which consists of DSC and TGA, is also performed [14] and there, more information can be retrieved.", "The sample crucible is usually sealed for ARC and DSC tests [15,16].", "Obviously for TGA and STA tests, the crucible cannot be closed [17].", "For open systems, the size of the opening in the crucible is of utmost importance.", "Watanabe and Yamaki [18] investigated the effect of the size of the opening and showed that if the opening is small, the DTA signal is similar to tests with a sealed crucible.", "However, the DTA signal obtained by crucibles with large opening is considerably suppressed.", "The size of the opening also influences gas evolution and mass loss [18].", "In an open system, the solvent(s) evaporate(s) earlier, which may result in different reactions from those occurring in a closed or half closed system.", "Pressure and concentration of the substances may influence reaction rates, which in turn, may result in different products and yields, consequently, different heat production.", "It is difficult to determine which experimental setup – open or closed crucible – is the most appropriate for simulating a realistic scenario, because all batteries vent under certain but different abusive conditions.", "At least, three different sample preparation methods have been used in literature for thermal analysis of electrodes: a) washing followed by drying, b) drying (without prior washing) followed by adding electrolyte, c) drying only (without prior washing and without adding electrolyte).", "Zhuang used Attenuated Total Reflection Fourier-transform Infrared Spectroscopy (ATR FTIR) to identify the components of Solid Electrolyte Interphase (SEI) and found that washing modifies SEI considerably [19], however in this case, the effect of the electrolyte on the decomposition reaction and heat of active materials can be studied [17].", "MacNeil et al. [12] showed that electrode drying or exposure to the glove box (GB) atmosphere alters the decomposition heat of Li-Cobalt-Oxide (LCO).", "There is no general agreement on the optimal drying time, furthermore, there is no general agreement about the amount of the extra added electrolyte to the crucible.", "Often, the amount is not mentioned in reported experiments.", "Xuning et al. [20] found in their review that the amount of added electrolyte is crucial for DSC and ARC investigations and requires further study.", "Because the difference in the purpose of the experiment may justify the selection of different experimental and sample preparation techniques, the comparability of the outcome of different experiments is not straightforward.", "It is also crucial to understand which approach is the most representative for a real thermal runaway scenario.", "In this work, as gas analysis is used, we have chosen an open system, but the hole of the crucible is made as small as possible (see section 2.3).", "No washing and no extra electrolyte are applied to minimize SEI modification.", "The harvested samples are dried for 2 h before the STA study to reach a stable initial mass for TGA and to suppress the effect of endothermic evaporation heat on the measureable heat release.", "Depending on the studied material and the experimental conditions, several decomposition processes have been identified in the literature.", "Table 1 summarizes, comparable to our experimental conditions (see section 2.3), the decomposition processes, grouping them by components and evolved gases.", "Further descriptions about the reactions are given in supplementary materials.", "The gain factor K was determined by fitting and the best fit was obtained for K = 5 [20,33,34].", "The heat of reaction was assumed to be the same for the newly formed SEI, i.e. there is no distinction between the primary and secondary SEI.", "Other authors proposed the following mechanism [14,24,35]: the primary SEI (p-SEI) layer breaks down from around 100 °C (e.g. reaction 1–5 in Table 1), then Li starts to react with the electrolyte and builds up a secondary SEI layer (s-SEI) (e.g. reaction 5 in Table 1 producing metastable compounds).", "The s-SEI is stable enough to protect further EC-Li reactions.", "However, with increasing temperature, the metastable part of the secondary SEI layer breaks down together with imminent EC-Li reaction resulting in a sharp exothermic peak around 250 °C (e.g. reaction 6–10 in Table 1).", "No kinetic expression has been proposed yet, therefore, in this work, an approximate kinetic expression is formulated to be able to model TR in the full temperature range.", "The reviewed decomposition reaction of NMC (111) is summarized in Table 2.", "Further details about the reactions are given in the supplementary materials.", "The first kinetic model for LCO electrodes has been developed by MacNeil et al. [12] and Hatchard et al. [30], which is used for several simulation studies [11,20,31,32].", "An autocatalytic reaction mechanism was proposed and the reaction parameters were determined by ARC and DSC experiments.", "Later, the same autocatalytic reaction mechanism was suggested and used for other cathode materials, such as LCO, NMC(111), NCA, LMO, LFP, by Peng et al. [38].", "It is common to all models that only one decomposition reaction is taken into account, despite the electrode containing binder and the fact that oxygen is released from NMC(111) in a multistep process according to Li et al. [39].", "While Campion et al. [40] and Wang et al. [41] suggested that EC reacts with LiPF6 salt at around 200–230 °C, Yang et al. [42] did not find evidence for that reaction.", "The difference might originate from the different measurement conditions.", "Campion et al. [40] and Wang et al. [41] used sealed crucibles, where the reactive decomposition product of LiPF6 remained near the EC, therefore they were able to react.", "If TGA and online gas detection were applied, such as by Yang et al. [42], PF5 or EC was released from the crucible before they could react.", "Therefore, their reaction was suppressed or completely avoided.", "Consequently, depending on whether venting happens, the dominating decomposition reactions can be different which in turn may make the comparison of realistic scenarios to laboratory measurements more complicated.", "Freshly assembled, cycled and fully charged materials were used for all STA measurements with subsequent evolved gas analyses.", "Graphite (Gr), NMC (111), binders as pristine materials and the 18 mm discs electrodes made of the same pristine materials were supplied by Customcells (Germany).", "Further details of the materials are given in supplementary materials.", "The protocol for adjusting the SOC consisted of a constant current (CC) charge at C/20 rate up to a cut-off voltage of 4.2 V, followed by a resting period of 1 h (OCV), and a CC discharging at a C/20-rate to a cut-off voltage of 3 V.", "This cycling process was repeated twice in total, followed by charging to cut-off voltage of 4.2 V.", "This voltage threshold is considered full charge.", "The actual lithiation percentage of graphite and de-lithiation percentage of each NMC were calculated from the actual loading and rated capacity (further details about the testing conditions can be found in supplementary materials).", "After SOC adjustment, the electrochemical cell was transferred to the glove box for disassembly.", "The anode and cathode sample were then weighed and afterwards dried in the vacuum antechamber of the glove box at approximately 1 mbar for 2 h at room temperature.", "Under these conditions, DMC solvent is expected to evaporate fully (due to its low boiling point of 90 °C at 760 mm Hg [43]), whereas EC solvent, with a boiling point of 246.7 °C at 760 mmHg [44], is expected to mainly remain.", "Following the drying step, the electrode surface is carefully scratched by using a tweezer and spatula, in order to remove the active material from the current collector.", "The harvested material is further analyzed to characterize its thermal properties and the evolved gas during heating.", "DSC/TGA tests were carried out using a STA 449F3 – Jupiter® Simultaneous Thermal Analysis, instrument (Netzsch, Germany) with a silver furnace and a TGA-DSC sample carrier for evaluating the thermal behaviour of the collected materials.", "As shown in Fig. 1, this apparatus is located inside an Ar-filled glove box (MBraun MB 200B, H2O < 0.1 ppm; O2 < 0.1 ppm), which was connected to the glove box used for sample preparation.", "Therefore, the samples remain constantly under inert atmosphere and exposure to air is excluded.", "STA was operated using helium as a carrier gas for both the purge of the sample compartment (flow rate of 50 ml min−1) and as protective gas (flow rate of 20 ml min−1).", "After placing the crucible inside the sample chamber, the chamber was carefully evacuated and refilled with helium 2 times to flush argon originating from glove box atmosphere.", "In the glove box, the harvested anode or cathode material is loaded in a sealed aluminium crucible with laser-pierced lid (opening 5 μm diameter) for each Differential Scanning Calorimetry/Thermogravimetric Analysis (DSC/TGA) test.", "No extra electrolyte was added to the crucible.", "The actual weight of the sample and the crucible has been measured before placing in the STA.", "All measurements were carried out from 5 °C to 590 °C starting with an isothermal phase of 20 min at 5 °C then continuing with heating rates of 5, 10 and 15 °Cmin-1 with STC control.", "At the end of the dynamic heating phase, an isothermal stage of 10 min at 590 °C was applied before cooling to room temperature.", "The measurements were repeated at least 3 times for each heating rate.", "The TG and DSC data were analyzed by Netzsch Proteus® software.", "Gas analysis was not performed on all samples.", "When it was used, the flow rate of the process gas in the STA was increased to 100 ml min−1.", "This higher flow rate was necessary to be able to split the gases between the two pieces of analytical equipment.", "The sensitivity to gases released during the decomposition at smaller quantity can be increased by increasing the amount of material but in this case, the accuracy of heat flow measurement may be reduced.", "In the current experiments, however the sensitivity for the main reported gas compounds was satisfactory and the amount of sample was not increased.", "The gases released during thermal decomposition were constantly monitored using GC-MS (Agilent 7820A gas chromatograph equipped with an Agilent 5977E quadrupole MS) and FTIR (Bruker Vertex 70 V Fourier Transform Infrared spectrometer), both coupled with STA equipment via heated transfer lines (150 °C) (Fig. 1).", "The following parameters for the GC-MS were used: column type: HP-PLOT-U (Agilent, 35 m length, 0.32 mm internal diameter, 0.01 mm film thickness, max.", "temperature 180 °C), split ratio 10:1; helium flow rate: 1.5 ml min−1; temperature program: 100 °C (60 min), 10 °C min−1 up to 150 °C (10 min) and 10 °C min−1 to 100 °C.", "At the beginning and the end of the HP-PLOT-U column, 2 m of trap columns (Agilent, 0.32 mm siloxane stationary phase) were attached to protect the main column from HF and to protect the MS from the possibly detached particles, respectively.", "The MS detector was used in scan mode.", "Composition of evolved species was determined using Netzsch GC-MSD Data Analysis software.", "The 10 cm path IR gas-cell was heated to 200 °C.", "Together with the liquid nitrogen cooled detector, they were placed in the TG-IR Box (Netzsch, Germany) which was purged with Ar during the measurement.", "Even though the TG-IR box and the Vertex V70 were placed as close as possible to each other (<1 cm), the IR beam passed through a short non-evacuated path and might collect the signals of CO2 and H2O from the atmosphere.", "This effect was compensated by background correction after the measurement.", "The FTIR spectra were collected in absorbance mode from the wavelength range of 500–4500 cm−1 with 4 cm−1 spectral resolution and 18 spectra were collected.", "OPUS software version 7.2 (Bruker, Germany) was used to process the FTIR spectral data.", "The qualitative identification of gas compounds of MS spectra and FTIR graphs was performed based on online NIST Standard Reference Database 35 (a NIST/EPA Gas-Phase Infrared library).", "JMP software (SAS, USA) was used to calculate activation energy and frequency factor.", "Matlab 2018a (Mathworks, USA) was used to simulate DSC curves.", "The decompositions of the anode, cathode and the electrolyte are discussed separately.", "For establishment of all decomposition reactions, the following steps are taken:", "Identification of possible reaction mechanisms from Tables 1,2 and 3.", "Thermal analysis for the identified most important DSC peaks", "Calculation of thermal parameters: heat of reaction, frequency factor, activation energy and their uncertainty", "Simulation of the kinetic reactions and heat flow", "The main goal of the analysis is to find a model that describes the heat generation resulting from anode, cathode and electrolyte decomposition for the used chemistry and electrode design in the temperature range 20–590 °C.", "Table 4 shows the investigated cells' capacities and the calculated lithiation, delithiation levels for the Gr anode and NMC (111) cathode, respectively.", "According to the manufacturer, the anode electrode has been designed to have 10% higher capacity than the cathode to avoid Li plating on the graphite anode.", "According to our measurement, the anode has on average 11% higher capacity than the cathode.", "It should be noted that the NMC delithiation level in sample #10 and #11 is higher than it is expected by the manufacturer, which is most probably stemming from the variation of composition and non-uniform distribution of active electrode material on the current collector surface.", "Fig. 2 shows the cycling behaviour of the cell for sample #5 in Table 4.", "The anode potential in this configuration does not reach 0 V vs. Li, the theoretical maximum lithiation, but stops at around 50 mV vs. Li.", "Consequently, Li plating is not expected.", "Furthermore, the minimum delithiation is reached at ca. 400 mV.", "These narrow limits may have an impact on the SEI formation, but for realistic experimental results, it is important to keep the cycling potential range close to that in real cells.", "Fig. 3a shows a representative simultaneous thermal analysis result: heat flow (mW/mg), mass loss (wt%), and the relative FTIR intensity of CO2 (2346 cm−1) and EC (1863 cm−1), obtained for fully charged graphite anode active materials (Sample#5 from Table 4) as a function of the testing temperature from 5 °C to 590 °C at 10 °C min−1 heating rate.", "The decomposition profile can be split into 4 different regions (shown by Arabic numbers), which are discussed separately in the followings.", "Roman numbered peaks show the analyzed peaks due to their importance on heat flow.", "Fig. 3b and c show the FTIR spectra registered at 110 °C and 250 °C, respectively.", "The pristine CO2, ethylene, and water spectra reproduced from NIST are also shown for comparison.", "In region 1 below 100 °C, no mass loss or gas formation is detected, however the DSC signal shows a sharp endothermic peak around 70 °C.", "This peak is far from EC melting (ca. 36 °C) and from DMC evaporation (90 °C) [45].", "This endothermic peak can also be found on pristine graphite after contact with the electrolyte without cycling (see Fig. S1a in the supplementary material), therefore it is not considered to be related with the lithiated graphite.", "The origin of this peak is currently unknown, but some possible causes are discussed in the supplementary materials.", "This peak's influence on TR is minimal and therefore, neglected from further calculations and analysis.", "Region 2 is commonly identified as the breakdown of SEI followed by the reaction of Li with electrolyte according to reactions 3, 4 and 9 in Table 1.", "However, it is not necessary that SEI decomposes in one-step (further explanation is given in supplementary materials).", "In our system, no evidence is found for one-step SEI decomposition according to 3, 4, and 9.", "In the beginning of region 2 at 110 °C, the FTIR spectra in Fig. 3b indicates that mostly CO2 and water is present in the exhaust gas.", "Only a small increase of CO2 is detected at around 100–110 °C, but later its concentration drops (see CO2 FTIR signal intensity at 2346 cm−1 in Fig. 3a and 3b).", "A small peak of CO2 is also detected by the GC-MS (not shown) but no ethylene was found, as it is expected from 3, 4 and 9 in Table 1.", "The ca. 4% mass loss is negligible compared to the expected mass loss of reactions 3, 4, and 9 (in Table 1).", "Furthermore, it is mostly correlated with the beginning of EC evaporation, as shown by EC's 1863 cm−1 FTIR peak intensity in Fig. 3a and 3c, which starts at around 150 °C.", "Neither FTIR spectra (Fig. 3b) nor GC-MS show traces of other gases in region 2.", "Therefore, the most likely reactions from Table 1 are 1, 2 and 5.", "While CO2 release occurs only close to 100 °C, it is plausible that reaction 2 occurs as traces of water evaporates (Fig. 3b) from the pores or other parts of the system (e.g. crucible).", "In the current state of research, it is not possible to draw a final conclusion about the origin of this CO2 peak.", "Shortly after or in parallel with the CO2 peak, a mild heat generation starts and continues after the broad exothermic peak around 150°C–170 °C, identified as peak I.", "This mild heat generation, with the absence of major mass loss and gas formation, may also indicate that the SEI does not disintegrate but continuously thickens or changes its composition.", "Reaction 2 in Table 1, which is supposed to happen around 100 °C, may render the SEI more permeable for Li-ions or for EC but the new or transformed SEI keeps its protective nature.", "Furthermore, reactions 3, 4 and 9 in Table 1 should be accompanied by mass loss and more gas generation, especially ethylene [22,23,30], which was not found in region 2.", "Therefore, reaction 1 and 5 might be possible mechanisms of heat generation rather than 3, 4, and 9.", "Chen et al. [47] demonstrated, by means of XRD, that the content of lithium in graphite decreased gradually during thermal ramping from 110 °C to about 230 °C as a consequence of the formation of secondary SEI film.", "Reaction(s) in region 2 should not generate gas but should consume Li.", "Therefore, reaction 5 is the proposed mechanism rather than the decomposition of SEI followed by immediate Li-electrolyte reaction according to reactions 3 and 4 or SEI decomposition according to reaction 1.", "It must be noted that for other systems where the SEI is different, this scheme may be different.", "A small endothermic peak around 200 °C can be the consequence of the melting of LiPF6 or of plated Li [48,49] or graphite exfoliation [50].", "This reaction has minor impact on TR and will be excluded from further analysis.", "Region 3 starts with a sharp exothermic peak, large mass loss and gas evolution (Fig. 3a).", "The FTIR spectra in Fig. 3c is more complex than that of in region 2 (Fig. 3b) and there are several unidentified peaks, however the most pronounced compounds with the highest match are CO2 and EC.", "GC-MS analysis (not shown) shows the presence of ethylene and PF3 in addition to the characteristic peaks of CO2 and EC in the FTIR spectra in Fig. 3c.", "It indicates that in region 3, multiple reactions may occur at the same time: a) decomposition of SEI, b) Li electrolyte reaction (reactions 3, 4, 6, 7, 8 and 9 from Table 1), c) EC decomposition, LiPF6 decomposition and EC evaporation (Table 3).", "The STA of pristine graphite in contact with electrolyte shows (Fig. S1a in supplementary material) that EC evaporation is the fastest among these reactions under these circumstances.", "Therefore, reactions 16, 17 and 19 in Table 3 are less relevant.", "The minor CO2 generation (Fig. S1a in supplementary material) without HF and PF3 formation, also supports this assumption.", "Hence CO2, ethylene and PF3 (decomposition product of LiPF6) most probably originate from the graphite-EC decomposition (reaction 6 in Table 1).", "Even though PF3 is not a primary product of any reactions listed in Table 1-Table 3, it may be formed in the GC columns or in the heated lines.", "In region 3, peak II.", "is most probably the consequence of multiple consecutive and parallel reactions.", "Perhaps, it is governed by the slowest reaction; however, the measurable reaction heat is the sum of all reactions.", "Therefore, in spite of the complex scheme, the exothermic DSC peak II.", "in region 3 still can be approximated by the Kissinger method if the slowest reaction follows Arrhenius kinetic.", "Without speculating too much about the possible reaction mechanisms, the most important consideration is that the exothermic reactions in region 2 and in region 3 are different.", "Therefore, the SEI breakdown and Li-electrolyte reactions are not a single reaction mechanism as suggested by Refs. [22,23,30] and they must be modeled by different reaction mechanisms.", "In this work, we propose and formulate a double-break down mechanism.", "It must be noted that this is not a general decomposition reaction model and that may be different for other materials or for the same material tested in a different experimental setup.", "In region 3, the mass loss is mostly due to EC evaporation as that is faster than EC decomposition and combustion (as it is shown in supplementary materials Fig. 1a).", "In region 4, smaller, partly overlapping exothermic peaks are visible (see also Fig. 4a).", "The mass loss and gas production is minimal and detectable only at 15 °C min−1 heating rate.", "Traces of ethylene are found by GC-MS (not shown) together with C2H6, CH4, C3H6 (at 15 °C min−1).", "This region is in the same temperature range where the decomposition of the binder occurs (Fig. S1b in supplementary material).", "Evidence for the reaction between the electrolyte and CMC binder was found by El Ouatani et al. [51].", "The reactivity of CMC towards the electrolyte is most probably due to hydroxyl functional groups in CMC, which take part in the formation of the surface film and may decompose with higher heat of reaction than the binder alone.", "Since the fraction of binder is less than 2 wt%, its decomposition cannot explain the measured considerable heat generation.", "Most probably, though, some reaction products of the previous decomposition reactions decompose further here.", "Zhao et al. [25] found that at 330 °C and 430 °C the exothermic reaction is caused by the decomposition of two main SEI components, lithium alkyl carbonate and lithium oxalate.", "The EC evaporated and/or reacted fully, therefore possible reactions can be only 6, 7, 11 and 12 from Table 1 (which does not require EC).", "However, none of these reactions explains all the found gases or the absence of gases.", "Nevertheless, the processes in region 4 are different from the processes in the previous regions, however the reaction products from previous regions may affect reactions in region 4.", "As the current work's focus is the calculation of an approximate heat generation, Arrhenius type decomposition reaction is considered, in spite of the complex shape of peak III.", "However, the total heat generation in region 4 was determined and used in the model.", "The Kissinger analysis was performed on the DSC curves at three heating rates with several replications (Table 4) by using the following procedure:", "Fig. 4a shows the anode decomposition DSC curves at 5, 10 and 15 °C min−1 heating rates.", "As the heating rate increases, the intensity of peak II.", "becomes larger and shifts to higher temperatures, as expected for an Arrhenius-type reaction.", "Peak III.", "shows a higher variation in shape than peak II but the most pronounced peak (peak III.)", "seems to follow an Arrhenius-type behaviour.", "The shape of peak III.", "varies probably because it is affected by the reaction products from region 2 and 3.", "Peak I., however, shifts to lower temperature with increasing heating rate and becomes narrower; hence, it does not to follow an Arrhenius type reaction.", "Furthermore, at low heating rates, it is hard to identify the peak's maximum because of its broad nature, which makes the Kissinger analysis unreliable in this case.", "The Kissinger plots are shown in Fig. 4b and c for peak II.", "and III., respectively.", "The temperature at which the maximum depth of the peak is observed, is plotted against the normalized heating rates (5, 10, 15 °C min−1).", "Since NMC-Gr-23 in Fig. 4b for peak II is outside of the confidence of prediction of the other data, assuming normal distribution, this data is treated as an outlier.", "Therefore, sample NMC-Gr-23 is disregarded from the subsequent calculation of thermal triplet of peak II, but not from peak III.", "In the supplementary materials, all data were included and the thermal triplets were calculated accordingly.", "Even though the shape of peak III.", "indicates a more complex kinetic, its approximation by Arrhenius kinetic may be still feasible if the rate determining process or the decomposition of the main component follows an Arrhenius type kinetic.", "Peak I. could not be fitted with the Kissinger plot as it is mentioned previously and the literature values from Refs. [22,23,30] are used for the simulation and listed in Table 5.", "The standard errors are calculated by using Eqs. (4) and (5) and are listed in Table 5.", "In average, the thermal decomposition parameters show 10–20% relative error.", "In the literature, relative error data is hardly found.", "Wang et al. [54] reviewed that the total heat of decomposition of the graphite anode can reach 2000 Jg-1 ± 300 Jg-1, which shows similar variation to our data.", "As every electrode coin's composition and also every cell assembly (e.g. pressure applied), may slightly vary, the lithiation, delithiation level of anode and cathode are different.", "This, in turn, contributes to the uncertainty of DSC measurement results.", "While higher accuracy may be achieved by optimizing the experimental conditions, this is not the scope of this study.", "In our following work, sensitivity analysis is used to understand also the effect of uncertainty of measurement results on the overall outcome of TR initiation.", "The next step is to build an approximate reaction kinetic model to describe the heat generation associated to the decomposition of lithiated graphite during TR.", "As shown previously, the reactions in region 2 and region 3 are different.", "Therefore, neither Richard's [29] nor Feng's model [20] has been chosen, but the double breakdown mechanism suggested by Refs. [14,24,35].", "The p-SEI does not break down, but transforms to more stable compounds or a thicker layer (reaction 5 in Table 1) called secondary SEI (s-SEI), which compensates the enhanced reactivity between the more mobile Li and EC at elevated temperature.", "The formation of the secondary SEI is supposed to be the rate determining step, so this process governs the dynamics of the DSC curve.", "The consecutive SEI decomposition, if it happens, is supposed to be a fast Arrhenius type process, which may affect the total heat of the overall reaction, but not the dynamics.", "hi is the specific reaction heat (Jg−1), xi is the dimensionless concentration and i is the number of decomposition reactions (Eqs. (6)–(9)).", "Fig. 5a compares simulated heat release - shown at 10 °C min−1- but prevail for all other heating rates-for the single break-down mechanism and the proposed double breakdown mechanism.", "The single break down mechanism (blue curve) was calculated using data from Refs. [22,23,30].", "In the original work of Richard [23,29], it was stated that the model is more suitable to capture the on-set temperature and that for the simulation of TR, further processes need to be incorporated into the model.", "In their model, the diffusion layer continuously thickens by slowing down the decomposition reaction of graphite, which in turn, results in a broad peak ending well above 500 °C such as it is shown in Fig. 5a.", "In our system (Fig. 3), however the heat generation ends around 350 °C, therefore the introduction of a double break down procedure fits better to the experimental data.", "The blue curve in Fig. 5b shows the simulated dimensionless concentration of intercalated Li.", "It starts to decrease after the first breakdown but slows down as the secondary SEI forms simultaneously.", "The change in the slope at ca. 220 °C corresponds to the transition from diffusion-type to Arrhenius type reaction as the second breakdown of the SEI layer kicks in.", "The orange curve, in Fig. 5b, illustrates the simulated binder and s-SEI components consumption, as proposed in Eq. (9).", "Even though the proposed approximation does not reproduce all features of the DSC curve, it enables a suitable prediction of the heat generation for this specific system.", "Further generalization, e.g. to other initial lithiation states, active materials, electrolytes and binder, requires further studies, though.", "In supplementary materials, Fig. S2a shows the comparison of heat flow when the outlier (NMC-Gr-23) was taken into account, which only creates higher standard error with more overlap of simulated peaks.", "However, it does not modify the simulated heat flow considerably.", "A similar approach, used in the anode study, is employed for the cathode part: reaction mechanisms identification, followed by thermal analysis of important DSC peaks, calculation of thermal parameters and simulation of heat flow signals.", "Fig. 6 shows heat flow (mWmg−1), mass loss (wt%), and the relative FTIR intensity of CO2 (2346 cm−1) and EC (1863 cm−1) obtained for fully charged NMC(111) cathode active material, as a function of the testing temperature from 5 °C to 590 °C at 10 °C min−1 heating rate.", "An obvious observation is that the generated heat and the heat flow are smaller than that of the anode, which suggests that heat from NMC(111) decomposition is less significant for TR than heat from anode decomposition.", "A similar pattern of peaks was observed by Fleischammer et al. [14] for NMC-LMO blend cathode, except the first sharp exothermic peak in region 3.", "This difference is discussed later.", "The decomposition profile of delithiated NMC (Fig. 6) can be split into four regions (marked by Arabic numbers).", "As in the case of the analysis of anode decomposition, not all peaks are taken into account.", "Roman numbers marks the most significant ones.", "In region 1, the peak at ca. 70 °C may have the same origin as described in the anode region 1.", "Later, CO2 increase without significant heat release is also visible a bit above 100 °C, similar to the behavior of graphite in the same temperature range (CO2 intensity curve in Fig. 3).", "It might have the same reason that the evaporation of traces of water from the pores reacts with organic compounds or LiPF6 resulting in CO2 generation.", "As for the anode, this endothermic peak is neglected from further analysis and calculations.", "In region 2 in Fig. 6, an increase of EC characteristics FTIR signal accompanied with mass loss and a small endothermic heat flow are visible around 200 °C.", "Consequently, this small endothermic signal is associated with EC evaporation.", "The endothermic heat is more pronounced for heating rate 15 K min−1 in Fig. 7a.", "This endothermic peak overlaps with other exothermic process, therefore it is hard to analyse by Kissinger analysis and a different approach is applied in this work.", "In region 3, a sharp exothermic peak and simultaneous jump in CO2 and drop of EC are shown in Fig. 6 between 240 °C and 290 °C.", "The following options can describe the observed heat generation, mass loss and gaseous compounds:", "Reaction of HF from the decomposed LiPF6 with NMC according to reaction 15 in Table 2", "Electrolyte decomposition or the reaction of EC with LiPF6 (PF5) according to reaction 20, 19 in Table 3, respectively", "Burning of EC with the released O2 from NMC decomposition.", "The temperature range also matches the first O2 liberation found by Li et al. [39]", "Autocatalytic decomposition of NMC similar to LCO decomposition suggested by Refs. [22,23,30]", "Reaction 15 generates H2O, which was not found and does not generate CO2, therefore option 1 is not considered as the main reaction.", "Options 2–4 can be hard to separate therefore, they are all taken into account and the possible governing reaction is analyzed by simulation later.", "In region 4, three exothermic peaks can be found.", "The decomposition of NMC accelerates with temperature, resulting in more generated oxygen.", "The oxygen may further react with the carbon additive resulting in CO2 generation, as suggested by the increased CO2 FTIR signal in Fig. 6.", "Most probably, the heat generated in this region results from simultaneous NMC decomposition and burning of carbon additive.", "We may suppose that burning of carbon is faster than NMC decomposition, therefore the thermal parameters (e.g. frequency factor etc.) are characteristic to the NMC decomposition and oxygen liberation.", "Above 350 °C, heat generation slows down which corresponds with the finding by Li et al. [39] that physically absorbed oxygen should be released from NMC(111) below 350 °C.", "In the temperature range of 400–500 °C, Li et al. [39] did not find major oxygen release, in contrast to DSC and CO2 peaks (peak II.)", "found in Fig. 6.", "However, this temperature range is where the NMC binder decomposes (Fig. S1c in supplementary materials).", "The active material's weight loss in this temperature range varies between 2.97 and 3.54 wt%, which matches the expected weight loss associated with PVDF decomposition (the calculation is in supplementary materials).", "Therefore, peak II is attributed to the decomposition of the PVDF binder.", "Peak III.", "is in the temperature range 500–600 °C and follows the predicted release of chemically bound oxygen found by Li et al. [39].", "The same can be concluded as for peak I, i.e. the liberated oxygen burns the carbon or other organic compounds.", "In spite of the decomposition probably follows a more complex kinetics, the shift of the peaks with increasing heating rate is clearly visible in Fig. 7a.", "The thermal triplets of NMC peak I-III are calculated by the Kissinger analysis.", "The Kissinger plot of peak I. is shown in Fig. 7b. Data of sample NMC-Gr-30 is identified as an outlier, because it is outside of the confidence of prediction band of the other data, and it is therefore excluded from the analysis.", "However, in supplementary materials, the thermal data is also calculated by taking into account all samples for comparison purposes.", "The latter case shows similar results, but with more overlapping heat flow peaks (Fig. S2b in supplementary materials).", "The Kissinger analysis of peak II-III in Fig. 7c and d shows a good fit on all data.", "The calculated thermal parameters and their relative error (without the outlier) are listed in Table 6, assuming normal distribution of error.", "The thermal parameters of the simultaneous processes of evaporation, decomposition and combustion of EC in region 2 and 3 are approximated differently (i.e. not by Kissinger analysis).", "The thermal parameters of EC decomposition are taken from Peng et al. [55,56].", "The combustion of EC is approximated by data from Ref. [57] and fire calorimetry data assuming an average heat of the combustion of organic materials, which is usually used in other studies [58].", "The activation energy and the frequency factor of EC evaporation is determined from the differentiation of the mass loss (DTG) in time.", "Since no reaction between EC and NMC is assumed, the mass loss is supposed to be governed by only EC evaporation.", "Therefore, the maximum peak temperatures of the DTG signals at different heating rates are able to be analyzed by Kissinger's method (Fig. S3 in supplementary materials).", "The evaporation heat of EC is taken from the NIST database.", "In Table 7, the mean value of kinetic parameters are listed for EC evaporation, decomposition and combustion.", "In spite of the fact that oxygen release is expected in Eq. (15), it is considered to react immediately with the carbon additive to form CO2 (reaction 14, Table 2).", "Therefore, it is subsumed in Eq. (14)-(15) and not calculated separately to save computational time and preserve the stability of the model.", "The simulated heat flow is shown in Fig. 8a (parameters are taken from Tables 6 and 7).", "When electrolyte is added and both combustion and evaporation are considered, the simulation result matches the measured DSC curves in Fig. 6.", "Nevertheless, when EC combustion is not considered, the simulated DSC curve is closer to the experimental results of Fleischhammer et al. [14].", "They did not find a sharp exothermic peak around 250–290 °C in their DSC study on LMO/NMC blend cathode, however the other identified peaks match with our findings.", "The absence of the sharp peak might be related to the manually pierced crucible, used in Ref. [14], which had a bigger hole compared to our laser pierced crucible with 5 μm hole.", "The larger opening might have led to fast release of EC and O2 from the system and their mixture did not reach the flash point.", "Consequently, the sharp exothermic peak is not related with the autocatalytic decomposition of NMC but the combustion of electrolyte with the liberated oxygen from NMC.", "The simulated heat flow of the cathode decomposition is shown in Fig. 8a at a heating rate of 10 Kmin-1, but this model prevails for all other applied heating rates as it can be seen in supplementary materials (Fig. S4).", "The dimensionless concentrations are shown in Fig. 8b.", "The evaporated and the combusted EC are shown as negative concentration for the sake of better visibility.", "First, the EC starts to evaporate which results in a small endothermic heat in Fig. 8a and a drop in concertation of EC (EC, purple line) in Fig. 8b.", "Then the NMC-I decomposes and the physically adsorbed oxygen is released (O2).", "The oxygen concentration does not increase simultaneously with NMC-I decomposition because it immediately reacts with EC generating the sharp exothermic peak (Fig. 8a).", "When EC evaporated, the sharp exothermic peak ends abruptly.", "Later, oxygen concentration (O2 green line) increases in the simulation.", "In reality, O2 does not accumulate, but reacts most probably with the carbon additive immediately.", "In the current stage of model development, however it is not taken into account separately because carbon combustion heat is considered in the reaction heat of Eq. (14)-(15).", "EC decomposition in this system does not happen because EC evaporates faster.", "For modelling thermal runaway of Graphite – NMC (111) lithium-ion batteries, the thermal decomposition reactions and their parameters were measured and determined using Simultaneous Thermal Analysis (STA) coupled with gas analysis.", "DSC for both the anode and cathode decomposition showed that multiple reactions occur between 5 and 600 °C.", "In this work, all major heat sources (or sinks) were identified and their kinetics estimated.", "The thermal properties (reaction heat, frequency factor, activation energy) of each process and their standard error were determined.", "A double break down mechanism is proposed for describing decomposition of the anode.", "The simultaneous DSC, TGA and gas analysis showed that the primary SEI does not decompose fully in a single step, but with simultaneous formation of a secondary SEI layer.", "These reactions are modelled by diffusion type decomposition and formation mechanisms.", "Later a second breakdown occurs, decomposing the secondary SEI and consuming the intercalated Li.", "Meanwhile, the EC evaporates and decomposes simultaneously.", "In a third process, the more stable decomposition products and the binder decompose.", "During the decomposition of cathode, the following sub-processes were identified and are modelled: evaporation of EC, decomposition of NMC with liberation of oxygen, combustion of EC with the liberated oxygen, decomposition of binder, decomposition of EC and combustion of carbon additive.", "The simulated heat flow based on the experimentally deduced thermal parameters shows a good agreement with the STA signals in a broad temperature range.", "It can also be assumed that there is a coupling between the different processes, which may considerably alter the outcome of the simulated thermal runaway under different conditions.", "In the follow up paper, thermal runaway will be modelled with a broad range of thermal parameter combinations, derived from the measurement's uncertainty estimated here." ]
[ "Thermal decomposition of active materials in Li-ion cell is analyzed.", "Simultaneous Thermal Analysis coupled with gas analysis is used to identify the processes.", "Double break down mechanism is proposed for graphite decomposition.", "NMC decomposition coupled with electrolyte combustion are assumed.", "Mean and the variance of thermal triplets have been calculated and heat flow is simulated." ]
Environmental and health impacts of using food waste as animal feed: a comparative analysis of food waste management options
The disposal of food waste is a large environmental problem. In the United Kingdom (UK), approximately 15 million tonnes of food are wasted each year, mostly disposed of in landfill, via composting, or anaerobic digestion (AD). European Union (EU) guidelines state that food waste should preferentially be used as animal feed though for most food waste this practice is currently illegal, because of disease control concerns. Interest in the potential diversion of food waste for animal feed is however growing, with a number of East Asian states offering working examples of safe food waste recycling – based on tight regulation and rendering food waste safe through heat treatment. This study investigates the potential benefits of diverting food waste for pig feed in the UK. A hybrid, consequential life cycle assessment (LCA) was conducted to compare the environmental and health impacts of four technologies for food waste processing: two technologies of South Korean style-animal feed production (as a wet pig feed and a dry pig feed) were compared with two widespread UK disposal technologies: AD and composting. Results of 14 mid-point impact categories show that the processing of food waste as a wet pig feed and a dry pig feed have the best and second-best scores, respectively, for 13/14 and 12/14 environmental and health impacts. The low impact of food waste feed stems in large part from its substitution of conventional feed, the production of which has substantial environmental and health impacts. While the re-legalisation of the use of food waste as pig feed could offer environmental and public health benefits, this will require support from policy makers, the public, and the pig industry, as well as investment in separated food waste collection which currently occurs in only a minority of regions.
[ "BUSI", "ENER", "ENGI", "ENVI" ]
[ "Anaerobic digestion", "Animal feed", "Composting", "Food waste", "Hybrid life-cycle assessment", "Swill" ]
[ "1408", "2105", "2209", "2300" ]
[ "The disposal of food waste poses a large environmental problem.", "Food waste is abundant: in the UK, approximately 15 million tonnes are wasted annually (234 kg/person/year or 50% of food) (WRAP, 2015) and the available disposal options each have substantial environmental impacts.", "Landfilling produces large quantities of greenhouse gases (GHG) and is therefore being phased out under new EU regulation (EC, 2014), but is still the destination of up to 48% of food waste in parts of the UK (House of Lords, 2014).", "Incineration and composting also produce greenhouse gases, and wastewater from anaerobic digestion causes eutrophication and acidification of local ecosystems (Evangelisti et al., 2014; Salemdeeb and Al-Tabbaa, 2015; Whiting and Azapagic, 2014).", "To aid the selection of food waste disposal technologies, the EU provides guidelines on which disposal technologies are preferable (EC, 2014).", "This so-called food waste hierarchy (Fig. 1), stipulates that governments should prioritise efforts (in order of most to least preferable) to (i) reduce food waste, (ii) redistribute it (e.g. to the homeless), (iii) recycle it as animal feed and (iv) compost, (v) recover energy through anaerobic digestion, and finally, (vi) landfill the remainder.", "This legislation is, however, notably not applied with respect to the use of food waste as animal feed, because it is currently illegal to use most food waste as feed in the EU.", "Though food waste is the archetypal pig feed, if it contains meat wastes and is not heat-treated it can transmit diseases, such as foot-and-mouth disease and African swine fever.", "In 2001, a UK farmer illegally fed uncooked food waste to pigs, precipitating the foot-and-mouth disease epidemic, which cost the UK economy £8 billion (UK House of Commons report, 2002).", "As a result, the recycling of food waste as animal feed was banned across the EU (EC, 2002).", "The law still permits the feeding of some food wastes where it can be demonstrated that there is no risk of contamination with animal products, but this represents only a small proportion of all EU food waste.", "Currently, of the 89–100 million tonnes of food waste produced in the EU per year (Monier et al., 2010), only around 3 million tonnes are recycled as animal feed (zu Ermgassen et al., 2016).", "In other parts of the world, however, food waste continues to be commonly used as animal feed, including in modern systems of pig production.", "Heat treatment renders food waste safe for animal feed (Edwards, 2000; Garcıa et al., 2005; OIE, 2009), and in nations such as Japan and South Korea 35.9% and 42.5%, respectively, of food waste is recycled as feed.", "There, the use of food waste is closely regulated: legislation governs the heat treatment, storage, and transport of food waste feed (Sugiura et al., 2009; zu Ermgassen et al., 2016).", "Amid increases and volatility in the price of conventional feed (AHDB Market Intelligence, 2013; AHDB Market Intelligence, 2006), and concerns about the environmental impact of grain- and soybean-based feeds (Nguyen et al., 2012), there is growing interest in the potential relegalisation and promotion of the use of food waste as pig feed (The Economist, 2013; The Pig Idea, 2014).", "A recent survey of 1195 animal feed practitioners (from industry, academia, and NGOs) identified the use of food waste as a priority research area for sustainable animal nutrition (Makkar and Ankers, 2014).", "In this study we evaluate the environmental and health impacts of converting municipal food wastes into pig feed in the UK.", "We conducted a hybrid life cycle assessment (LCA) to compare the environmental impacts of two technologies for recycling municipal food waste as animal feed (as a dry or a wet pig feed), with two well established food waste management options: composting and anaerobic digestion (DEFRA, 2015a).", "In doing so, we address a gap in the literature.", "Few previous studies have evaluated the potential for recycling food waste as animal feed in the EU, even fewer consider environmental impacts other than greenhouse gas emissions or land use and, none, to the author's knowledge, have thus far specifically considered the use of municipal food wastes as animal feed. zu Ermgassen et al. (2016)", "suggest that if the EU were to recycle food waste as pig feed at similar rates to nations such as Japan and South Korea, this would provide enough feed to support 20% of EU pork production, reducing the land use of EU pork by 1.8 million hectares of farmland.", "Four European studies have evaluated environmental impacts beyond land use, though these considered only manufacturing or retail food wastes or agricultural co-products (such as beet tails or soybean meal) (Tufvesson et al., 2013; Vandermeersch et al., 2014; van Zanten et al., 2014; Eriksson et al., 2015).", "These studies each adopted a bottom-up life cycle assessment approach and therefore have several inherent drawbacks that lead to system incompleteness and underestimate environmental impacts (Bernstad and la Cour Jansen, 2012; Laurent et al., 2014a, 2014b).", "We overcome these methodological limitations by taking a more holistic, hybrid LCA approach (described in more detail below).", "This study focuses on municipal food wastes because they make up 66% of EU food waste (Monier et al., 2010) and are suitable for animal feed – they are currently used in both South Korea and China (Chen et al., 2015; Stuart, 2009) and have historically been used in the EU (Fairlie, 2010).", "We evaluated the environmental and health impacts of processing 1 tonne of municipal food waste in the UK using four different technologies: (a) conversion into dry pig feed, (b) conversion into wet pig feed, (c) anaerobic digestion, and (d) composting (Table 1).", "We used a hybrid, consequential life cycle approach, expanding the system boundary of the analysis to take into consideration the substituted processes.", "Product substitution operates as follows: if food waste is processed to produce dry pig feed, for example, this will lead to avoided emissions from the substitution of conventional pig feed, but also knock-on emissions from the composting or anaerobic digestion that did not take place.", "Similarly, the total emissions from composting are the sum of the emissions released during composting, minus the emissions from the production of fertiliser which compost replaces, plus the additional emissions from the conventional pig feed and electricity production, which result from the food waste not being recycled as pig feed or anaerobically digested.", "The hybrid LCA approach combines conventional process-based LCA and an input–output based LCA (Salemdeeb and Al-Tabbaa, submitted for publication).", "This approach is used to counter the limitations of conventional LCAs, which face a truncation problem: system boundaries are set a priori and typically cut off part of the product life cycle for the sake of simplicity (Bullard et al., 1978; Lenzen, 2001).", "Input–output approaches use data on the total project cost to estimate upstream-processes that are not modelled using traditional LCA, such as the manufacture of electronic products or technical consulting services, and thereby mitigate truncation error.", "The input–output component of the hybrid model was a single region model with a domestic technology assumption (i.e. economic activities in the country of origin of imports are the same as in the importing country; Appendix 1).", "The LCA component of the analysis was conducted in EASETECH, a LCA tool developed at the Technical University of Denmark (Clavreul et al., 2014).", "We characterised and normalised results for 14 mid-point impact categories (detailed in Table 1) for each of our four food waste recycling technologies; these impact categories include a diverse set of environmental and human health indicators to give a multi-criteria assessment of the impacts of our four food waste disposal technologies.", "Characterisation involves the calculation of each impact (for example, global warming potential requires the weighting of impacts from emissions of carbon dioxide, nitrous oxides and methane).", "Normalisation then permits comparison of the relative importance of each impact category, by expressing the process' emissions as a proportion of the total emissions (per capita) in the EU-27 in 2010.", "The global warming potential and particulate matter emissions from recycling 1 tonne of food waste are, for example, scaled relative to the per capita greenhouse gas and particulate matter emissions in the year 2010 (and are reported in units of milli-Person equivalents, mPE).", "Characterisation and normalisation followed ILCD methods (Benini et al., 2014; JRC, 2010).", "The four food waste disposal technologies and substituted products are depicted in Fig. 2.", "As all technologies require separate collection of food waste, food waste collection and transportation are excluded from this study.", "Food waste packaging is also excluded due to its insignificant impact (Lebersorger and Schneider, 2011).", "As the use of municipal food waste as animal feed is illegal in the EU, we used process-specific data from factories producing food waste feed in South Korea (Kim and Kim, 2010), where there were 259 registered feed manufacturers as of 2010 (Ministry of Environment, 2010).", "Food waste is loaded into a hopper, shredded and filtered for contaminants (Fig. 2).", "It is then sterilised and dehydrated by air-drying at 390 °C.", "Under South Korean law, food waste must be heat treated to a core temperature of >80 °C for a minimum of 30 min (National Institute of Environmental Research, 2012); in comparison, before the ban on using food waste as animal feed, EU law used to mandate heating food waste to 100 °C for 60 min (Stuart, 2009).", "The feed is sorted again before one more step of drying, producing 140 kg of dry feed per tonne of food waste (with a moisture content of 21.8%, i.e. 109.5 kg of feed on a dry matter basis).", "The food waste feed substitutes conventional feed 1:1 on a dry matter basis (zu Ermgassen et al., 2016).", "The ingredients of the substituted conventional feed (Appendix 2) are based on the weighted mean feed intake of all pigs in the pork production life cycle (sows, piglets, and slaughter pigs), taken from an LCA of UK pork production (Stephen, 2012).", "The impact of feed ingredients that are co-products was allocated according to their economic value.", "Soybeans, for example, are processed into both soybean meal, a common pig feed ingredient, and soy oil; soybean meal makes up 60% of soybean value, and soy oil the other 40% (USDA, 2012), and so 60% of the impact of soybean production was allocated to soybean meal.", "When calculating the environmental impact of soybean meal we use the most recent available inventory data on soybean production in Brazil (Nemecek et al., 2014); Brazil is the source of 88% of the UK soybean supply (FAO, 2014).", "In using data from South Korean processing plants, we assume that the municipal food waste used to generate animal feed in South Korea is comparable with municipal food waste in the UK.", "To check this assumption, we compared data for South Korean food waste with UK data, and found that the compositions are broadly similar (Table 2).", "When food waste is used as wet pig feed, it is injected into the hopper, shredded, and twice filtered for contaminants (Fig. 2).", "It is then partially dehydrated and heat-treated to 100 °C to sterilise it.", "It is mixed with 25 kg of ground maize before storage, to produce 430 kg of wet feed per tonne of food waste, with a mean dry matter content of 30.9%.", "The substitution of conventional feed is calculated as for dry feed.", "In this process, food waste is shredded, sieved, and sent to a digestion tank.", "The digestate has a dry solids content between 25 and 40% and is digested at a temperature between 50 and 55 °C (Hall et al., 2014).", "AD digestate utilization efficiencies are presented in Table 3.", "Biogas is then collected, purified and used to generate electricity (260 kwh/tonne of processed food waste), which substitutes electricity produced from the UK energy mix (Table 4).", "Finally, the remaining digestate undergoes dewatering and refinement producing a high-quality AD cake, which substitutes nitrogen, phosphorous and potassium fertilisers with an efficiency of 34.5%, 46% and 60%, respectively.", "Benefits from the contribution made by sulphur, magnesium, and other organic compounds in compost are excluded (Wallace, 2011).", "Incoming waste is shredded, mixed and aerated for 14–21 days at a minimum temperature of 60 °C for 48 h (Hall et al., 2014).", "The compost is then stored in windrows for a 56 day maturation phase.", "The matured material is removed, screened, and packed as compost.", "The compost utilization efficiencies used are: 20% for N, 100% for P, and 100% for K (Andersen et al., 2010) and the compost is considered to be applied on loam soil (see Appendix 3), where it substitutes synthetic fertilizers on a 1:1 basis.", "The composting process is assumed to be well managed, i.e. no failures occur that give rise to high emissions of methane and other products of anaerobic conditions.", "All leachate water is reused during the composting process for re-wetting in the reception area and the maturation pad.", "For each of our four food waste disposal technologies, we selected the eight parameters which had the highest sensitivity ratio, assigned them probability distributions and performed a Monte Carlo analysis (1000 simulations) to generate confidence intervals for our results.", "The selected parameters and probability distributions are listed in Appendix 4.", "For each metric, we tested for the significance of differences between technologies, also using Monte Carlo methods.", "We randomly sampled estimates of the mean for each technology, and calculated the difference between each technology, repeating this resampling 1000 times.", "We then tested to see if the difference between technologies overlapped with zero at the 99% confidence level.", "Each technology was then ranked (1–4; 1 = best, 4 = worst) for each of our 14 mid-point metrics and the mean ranking for each technology was calculated.", "The hybrid LCA analysis requires two datasets: process-based physical data, and input–output monetary data.", "Data, listed in Table 5, was either compiled or calculated based on information from project documents, literature, and the WRATE database (Hall et al., 2014).", "Upstream and downstream material flows and emissions were collected using existing databases, primarily the Swiss Ecoinvent database v2.2 (Ecoinvent, 2014).", "These processes include the acquisition of raw material and energy, production, on-site operation, and waste disposal (i.e. cradle to grave).", "Monetary data were obtained from two sources: data of animal feed technologies were obtained by direct communication with the Korean Ministry of Environment; expenditure data for 2014, available in South Korean Won (₩), was converted into British pound using purchasing power parity coefficients of the year 2014 (Appendix 5).", "Data of both AD and composting were obtained from UK industrial partners (Appendix 6).", "Appendix 1 lists sources of data and components for the IO-based element of the hybrid approach.", "The recycling of food waste as wet pig feed had the best score for 13 of 14 environmental and health impacts while dry feed had the second-best score for 12 of 14 impacts (Fig. 3 and Table 6).", "The mean ranking of the four technologies (1 = best, 4 = worst) were wet feed: 1.1, dry feed: 2.2, AD: 3.3, and composting: 3.4.", "Composting had the worst score for seven environmental indicators, anaerobic digestion the worst score for eight (including two joint-worst scores shared with composting), and dry feeding the worst score for one indicator (depletion of fossil fuels).", "After normalisation, composting and anaerobic digestion had disproportionate impacts through eutrophication (terrestrial, marine, and freshwater), environmental toxicity (including non-carcinogenic toxicity, NC-HT, and ecotoxicity, ET), and acidification (Fig. 3).", "The superiority of wet and dry feed in these impact categories stems in large part from their substitution of conventional animal feed (Appendix 7).", "All stages of conventional feed production, including the farming and transport of raw materials to the feed processing centre, the milling of the feed, and the storage of the feed mixes, contribute to the substantial emissions in these impact categories.", "For example, hotspot analysis shows that freshwater eutrophication impacts are principally caused by the use of phosphate-based fertilisers in the farming of feed crops, and marine eutrophication is principally caused by the energy consumption and fuel inputs involved in shipping feed ingredients (such as soybean meal).", "For non-carcinogenic toxicity, we found that the concentration of zinc during the growth of rapeseed (an ingredient in conventional pig feed) accounts for nearly 35% of the impact.", "This result agrees with other studies highlighting concerns that rapeseed may contain high concentrations of heavy metals (such as zinc and copper) and allergens.", "Heavy metals from the soil are known to accumulate in the roots, plant, and seeds of rapeseed (van der Spiegel et al., 2013).", "Our results support the diversion of food waste to animal feed, before composting or anaerobic digestion, as proposed under the food waste hierarchy.", "The difference between AD and composting is however less clear: composting rated better than AD for 7/14 indicators, including acidification, terrestrial eutrophication, and particulate matter; AD rated better for 5/14 indicators, including greenhouse gas emissions and ozone depletion, and there was no significant difference between them for 2/14 indicators (marine eutrophication and non-carcinogenic toxicity).", "While these results suggest that the re-legalisation of the use of municipal food waste as animal feed has potential to reduce the impact of food waste disposal in the UK, LCA results are often location- and assumption-dependent (Bernstad and la Cour Jansen, 2012).", "We therefore compare our results with previous LCAs of food waste recycling.", "Previous studies did not evaluate the same portfolio of environmental indicators as this study, but greenhouse gas consequences have been calculated for nine studies, shown in Fig. 4.", "Though the exact figures vary substantially between studies, some broad patterns emerge.", "Wet feed has lower emissions than AD (2/3 studies making this comparison); AD produces lower emissions than dry feed (4/5 studies); and wet and dry feed produce lower emissions than composting (4/4 and 4/6 studies, respectively).", "Some of the differences between studies may be due to particularities of the locations where the studies were performed and the waste stream analysed.", "Only two of these studies evaluated food waste recycling in Europe (Eriksson et al., 2015; Vandermeersch et al., 2014), and neither (as here) looked at municipal food waste (instead evaluating retail waste).", "Study assumptions also explain some of the differences: none of the studies in Fig. 4 include land use change and they therefore underestimate the avoided emissions from animal feed substitution.", "This truncated-boundary problem underestimates the GHG emissions from animal feed ingredients by up to nine-times (van Middelaar et al., 2013).", "For example, Eriksson et al. use a GHG emission for soybean meal of 0.66 kgCO2e/kg, while our study uses the most recent figure of 4.4 kgCO2e/kg (Nemecek et al., 2014).", "Eriksson et al. also report large avoided greenhouse gas emissions when food waste is anaerobically digested compared with its use as dry animal feed (−381.4 vs −40.84 kgCO2e/kg; figures for this study: 3.80 vs 3.96 kgCO2e/kg).", "This difference stems from assumptions about the yields of biogas during anaerobic digestion and the energy mix substituted.", "Eriksson et al. assume that the entire theoretical yield of biogas was produced, while our work is based on an actual AD plant figures; their study assumes biogas replaces diesel as a fuel for city buses, while this study assumes biogas substitutes UK electricity production (natural gas 61.46% and coal 38.54%).", "To better understand the uncertainty in our results, we tested the sensitivity of our results to the parameter values chosen in the model.", "Despite the large variability in some parameters (Appendix 4), the indicator values for all metrics are significantly different from one another (p < 0.01), except for the effect of composting and anaerobic digestion on marine eutrophication and non-carcinogenic toxicity.", "Land use change emissions are the largest source of greenhouse gas emissions associated with certain forms of animal feed, notably soybean meal (van Middelaar et al., 2013), yet are ignored in much of the literature on food waste disposal technologies (but see Tufvesson et al., 2013; van Zanten et al., 2014).", "In this study, we therefore used the most recent data available on land use change emissions for soybean meal (Nemecek et al., 2014), a major constituent of EU pig feed.", "This has a large effect on the modelled emissions from wet and dry feed (Fig. 5).", "This shows the importance of using updated data inventories for agricultural products, whose emissions vary over time and whose measurement is rapidly improving.", "We evaluated two different technologies for recycling food waste as animal feed.", "We find that the processing of food waste into a sterilized wet feed has lower environmental and health impacts for all indicators, compared with processing into a dry pig feed.", "The difference between wet and dry feed results in large part from the higher fossil fuel inputs required to dehydrate municipal food wastes (Fig. 6).", "Municipal food wastes have a high water content (typically 65–80%; zu Ermgassen et al., 2016), and their dehydration to make dry feed requires gas and electricity (Table 5).", "This result does not, however, suggest that food waste feed should always be fed as a wet feed, because these two technologies may be suitable for different pig production systems.", "In South Korea, for example, dry feed is often produced in centralised facilities before resale and transport to farmers, while wet feed has a much higher water content and is therefore more expensive to transport.", "It is typically produced on or near to pig farms in order to minimise post-processing transport costs.", "The suitability of dry or wet pig feed depends in part on the proximity of pig farms to sources of food waste.", "For this reason, wet food waste feed, or “swill”, has long been a favoured pig feed for smallholder farmers (Westendorf, 2000).", "Most industrial pig farms in the UK currently use dried feeds; wet feeding is more common in other EU nations, such as the Netherlands, where it is favoured because it permits the use of wet agricultural wastes, such as distillery wastes or beet tails (van Zanten et al., 2014), and because of reported nutritional benefits of wet feeding (Brooks et al., 2001; Missotten et al., 2015).", "Food waste can be fed to livestock other than pigs, including poultry, fish, and ruminants (Angulo et al., 2012; Boushy and van der Poel, 2000; Cheng et al., 2014).", "This study focussed on the use of municipal waste as pig feed because they have a long history of recycling waste into animal products (Fairlie, 2010), and because there are human health concerns with feeding food wastes which contain animal products to other livestock species, notably ruminants.", "The use of meat wastes in ruminant (cattle, goat and sheep) diets is banned in the EU because of concerns about Bovine Spongiform Encephalopathy (BSE), a disease that does not affect pigs, poultry, or fish (Andreoletti et al., 2007).", "Alternatively, food wastes can be fed to insects which may in turn be used as animal feed (van Zanten et al., 2015).", "This practice would be inherently less efficient than feeding food waste to pigs directly, and is also currently illegal, though there is an active campaign for the legalisation of the use of insects in animal feed (Searby, 2014).", "This study suggests that the use of municipal food waste as animal feed could reduce the impact of food waste disposal in the UK.", "This practice is currently illegal and there are a number of barriers to its adoption, both political and infrastructural.", "Animal feeds are of course not only selected on their environmental merit.", "The re-legalisation of swill would require the confidence and support of the public, pig industry, and policy makers.", "Though heat treatment renders food waste safe for pig feed, there is some concern that the re-legalisation of heat-treated food waste feed might increase the risk of uncooked food waste entering the feed supply, potentially leading to disease outbreaks in livestock.", "If re-legalised, however, the potential benefits of using food waste as feed include reduced impacts on the environment, improved profitability for many farmers, and high meat quality and taste (for more detailed discussion see zu Ermgassen et al., 2016).", "Food waste can only be used in animal feed if it is collected separately from other wastes and is sufficiently fresh.", "While this is the case in countries like South Korea and China (Chen et al., 2015), food waste collection in the UK is currently more variable.", "In 2013, separate food waste collection occurred in 95% of Wales, but only 34% of Scotland, 26% of England, and 4% of Northern Ireland (House of Lords, 2014).", "The potential use of food waste as animal feed is therefore not only a function of the availability of food waste, but its accessibility and quality.", "Where food waste is of poor quality or not adequately separated, it can be diverted to composting or anaerobic digestion, in line with the food waste hierarchy.", "It is promising that separate food waste collection in the UK increased from 15,000 tonnes in 2006 to nearly 350,000 tonnes in 2012 (DEFRA, 2015b).", "While feeding municipal food waste to livestock is currently illegal in the EU, it is a common practice in many parts of the world, and there is growing interest in its potential use as a replacement for high-impact, high-cost conventional pig feed.", "This is the first study to compare the environmental impacts of recycling municipal food waste as animal feed with alternative disposal options in the EU.", "We used a holistic, hybrid LCA approach to compare four food waste disposal technologies in terms of 14 different environmental and health impacts and found that converting municipal food wastes into pig feed would lead to lower environmental and health impacts than processing waste by composting or anaerobic digestion – the UK government's currently preferred disposal options (DEFRA, 2015a).", "The widespread use of food waste as animal feed in the EU will require consumer and industry support, policy change, and investment in food waste collection infrastructure.", "Our results suggest that if these barriers can be overcome, the re-legalisation of food waste in pig feed could lead to substantial environmental and health benefits." ]
[ "There is growing interest in the re-legalisation of food waste as animal feed in the EU.", "We compare environmental impacts of 4 food waste disposal options, including as pig feed.", "We use a hybrid LCA approach to overcome the limitations of conventional LCA.", "Recycling food waste as pig feed has lower environmental impact than AD or composting." ]
Quantification of mitochondrial DNA from peripheral tissues: Limitations in predicting the severity of neurometabolic disorders and proposal of a novel diagnostic test
Neurometabolic disorders stem from errors in metabolic processes yielding a neurological phenotype. A subset of those disorders encompasses mitochondrial abnormalities partially due to mitochondrial DNA (mtDNA) depletion. mtDNA depletion can be attributed to inheritance, spontaneous mutations or acquired from drug-related toxicities. In the armamentarium of diagnostic procedures, mtDNA quantification is a standard for disease classification. However, alterations in mtDNA obtained from peripheral tissues such as skin fibroblasts and blood cells do not often reflect the severity of the affected organ, in this case, the brain. The purpose of this review is to highlight the pitfalls of quantitating mtDNA from peripheral –and not limited to-tissues for diagnosing patients suffering from a variety of mtDNA depletion syndromes exhibiting neurologic abnormalities. In lieu, a qualitative test of mitochondrial substrate-level phosphorylation –even from peripheral tissues-reflecting the ability of mitochondria to rely on glutaminolysis in the presence of respiratory chain defects is proposed as a novel diagnostic assessment of mitochondrial functionality.
[ "BIOC" ]
[ "Blood", "Fibroblast", "Lymphocyte", "Neuropathology", "Oxidative phosphorylation", "Ragged-blue fibers" ]
[ "1303", "1308", "1312", "1313" ]
[ "Stated simply, neurometabolic disorders are those due to enzyme or cofactor deficiencies leading to significant alterations in metabolism to the extent of affecting the developing brain.", "Enzyme deficiencies may be caused by genetic mutations inherited from the parents or acquired during the fetal stage (Willemsen et al., 2016), (El-Hattab and Scaglia, 2013), (Suomalainen and Battersby, 2018).", "Even though mtDNA is maternally inherited (see section 2) the diseases mentioned below follow Mendelian inheritance since all mutations affect nuclear-encoded proteins.", "These disorders encompass a wide variety of conditions including organic acidurias, amino-acidopathies (including defects of the urea cycle), lipofuscinoses, leukodystrophies, and disorders of the peroxisomes, lysosomes and mitochondria.", "In the vast majority of cases there is no cure and treatment remains palliative, highly dependent on the diagnostic workup of patients.", "To this end, “five new things” have emerged in the literature reviewed in (Willemsen et al., 2016), specifically: i) next-generation sequencing combined with next-generation metabolic screening; ii) recognition of disease-specific MRI patterns; iii) realization that some neurometabolic disorders do not follow a classic autosomal recessive mode of inheritance in all families; iv) gene therapy may soon be an option for treatment; v) neurometabolic disorders also emerge in the adult life of some patients, thus future professionals should be aware and prepared.", "The above “five new things” not only shorten the time for definite diagnosis but also protect the patients from unnecessary, often invasive procedures as well as decreasing costs.", "There are a number of mitochondrial disorders that may yield a neurometabolic phenotype; among them, depletion of mitochondrial DNA (mtDNA) -due to a variety of reasons, see section 4 is a potential underlying cause which has rightfully earned own entries in the Mendelian inheritance in Man, termed mtDNA depletion syndromes (MDS).", "Before exploring this further, a background on mtDNA is warranted.", "In humans, mtDNA is a 16,589 base-pair long, double-stranded circular DNA sequence coding for 13 polypeptides of electron transport chain components, the 12S and 16S ribosomal RNAs and 22 transfer RNAs critical for mitochondrial protein synthesis.", "Its two strands exhibit different sedimentation rates due to enrichment of guanine in one of them, thus they are referred to as “heavy” and “light” strand.", "Nucleated cells harbor hundreds or thousands of mtDNA copies (Ylikallio and Suomalainen, 2012), (Miller et al., 2003); furthermore, mtDNA content may vary over time within the same cell (Moraes, 2001).", "It is a recurrent question how many mtDNA copies are present per mitochondrion, but it is best to consider mitochondria as a highly dynamic intracellular network (Chan, 2006); thus, it is perhaps more correct to report mtDNA copies per cell per some other mitochondrial marker rather than mitochondrial count number.", "These copies are bound to specific proteins forming so-called nucleoids in the shape of foci or puncta attached to the inner mitochondrial membrane (Hensen et al., 2014), (Spelbrink, 2010).", "The mtDNA/nucleoid molecule ratio in cultured human cells is 1.4 (Kukat et al., 2011), thus, it is probably safe to assume that one nucleoid binds to one mtDNA molecule (Johnston et al., 2015).", "In one end of the mtDNA content spectrum, the human oocyte contains hundreds of thousands of mtDNA copies.", "However, following fertilization, mtDNA replicates only after the blastocyst stage; until this occurs, zygote mtDNA copies are being distributed to all blastocyst cells.", "Consequently, each female primordial germ cell harbors a relatively small number of mtDNA copies.", "The reduction of mtDNA copies in the primordial germ cells is known as “bottleneck”, a phenomenon that we now know to exist in other cell types as well, reviewed in (Zhang et al., 2018).", "The number of mtDNAs transmitted through this bottleneck in humans has been estimated to vary from ~9 (Li et al., 2016) to ~30–35 (range 9–141) (Rebolledo-Jaramillo et al., 2014).", "The time elapsed for one round of mtDNA replication is about 1 h (Clayton, 1982) and it takes place independently from cell division, obviously also occurring in post-mitotic cells.", "During fetal life, mtDNA amount increases but not appreciably (Suomalainen and Isohanni, 2010); energy provision by glycolysis is more prevalent thus, even the most severe mitochondrial abnormalities do not yet manifest (Suomalainen and Isohanni, 2010).", "mtDNA content as well as respiratory chain subunits increase dramatically in the first year of life, a period coinciding with the manifestation of several mtDNA depletion syndromes (Morten et al., 2007).", "Up until recently it was believed that mtDNA is exclusively maternally inherited; however, it was recently shown that mtDNA can also be inherited from the father (Luo et al., 2018), although this seems to be a very rare event, and currently contested (Lutz-Bonengel and Parson, 2019).", "In any case, paternal contribution of mtDNA to the fertilized egg is expected to be negligible, because the number of mtDNA molecules in the oocyte outnumber those in the sperm by a factor of 10,000 (Pyle et al., 2015).", "Furthermore, it was postulated that the results of (Luo et al., 2018) could be explained by the presence of multicopies of mtDNA in the nuclear genome termed “Mega-NUMT” that segregate in an autosomal dominant manner regardless of parental origin (Balciuniene and Balciunas, 2019).", "Thus, in the vast majority –if not all-of cases the notion that male and female mtDNAs don't recombine imply that the mtDNA sequence can only be altered by accumulating mutations along the lineage.", "mtDNA is maintained by securing a sufficiently high number of healthy copies (a phenomenon termed “homoplasmy”, see below in this section), rather than proofreading a single copy; this is because mitochondria harbor only one DNA polymerase as opposed in the nucleus.", "This, and in addition to the fact that DNA in mitochondria are devoid of protective histones while being in proximity to reactive oxygen species emanating from the electron transport chain, substantiate the reasons why mtDNA is ~10 times more prone to uncorrected mutagenesis.", "It is not the subject of the present work to review mtDNA maintenance mechanisms, though some background is hereby outlined so as to assist the reader, focusing on processes that are associated with neurometabolic diseases.", "mtDNA maintenance mechanisms are exhaustively reviewed elsewhere (Gustafsson et al., 2016), (Suomalainen and Battersby, 2018), (El-Hattab et al., 2017), (Viscomi and Zeviani, 2017).", "Pathways known to be directly involved in mtDNA maintenance are shown in Fig. 1 (reproduced by permission from (El-Hattab et al., 2017)).", "From this figure it is apparent that mtDNA maintenance depends on many nuclear gene-encoded proteins.", "Furthermore, uninterrupted provision of nucleotides -achieved by intramitochondrial recycling as well as import from the cytosol-is required.", "In addition, mtDNA maintenance also depends on regulated exchange of intramitochondrial contents through mitochondrial fission/fusion and bioenergetic parameters that affect protein or other metabolite import, critical for mtDNA.", "Perturbations in one or more of the above mechanisms may impact mtDNA by conferring depletion or large fragment deletions.", "In rarer circumstances an excess of mtDNA can be observed to lead to disease, see section 4.", "Defects in mtDNA maintenance are due to mutations in nuclear genes coding for proteins involved in mtDNA synthesis, nucleotide pool maintenance, mitochondrial fission/fusion or regulating basic bioenergetic processes, rendering them among the most common of the inherited disorders of metabolism, reviewed in (Schaefer et al., 2004).", "Originally reported in 1988 (Holt et al., 1988) and (Wallace et al., 1988) and considered rare, we now know that the prevalence of mitochondrial diseases can be as high as 1 in 2,000 individuals (Suomalainen and Battersby, 2018).", "Secondary causes of mtDNA depletion are reviewed in (Nogueira et al., 2014), (Niyazov et al., 2016), (Rotig and Poulton, 2009).", "The mechanisms by which mtDNA gets depleted are incompletely understood: until recently, two theories have been formulated, i) the “slipped-strand model” (Shoffner et al., 1989) a consequence of the guanine-cytosine bias of the heavy and light strand, and ii) a second model proposing that deletions are caused by double strand breaks followed by DNA repair (Krishnan et al., 2008).", "Most recently though, evidence was put forward suggesting that mtDNA deletions are formed by copy-choice recombination during active DNA synthesis of the light strand (Persson et al., 2019), arguing against both previously proposed models.", "mtDNA depletion can also be drug-induced (Arnaudo et al., 1991), as it occurs during anti-retroviral treatment (Montaner et al., 2004), (Casula et al., 2007), (Morse et al., 2012), (Lewis et al., 2003), (Young, 2017).", "One-fifth of patients undergoing anti-retroviral treatment develop a phenotype stemming from mtDNA depletion, although in most cases this is reversible upon cessation of treatment (Lewis et al., 2003), (Young, 2017), (Dagan et al., 2002), (Lewis et al., 2001).", "Whatever the cause of damaging mtDNA maintenance, the result may be quantitative (mtDNA depletion) or qualitative (multiple mtDNA deletions).", "mtDNA deletion syndromes are caused by large-scale deletions of mtDNA on the order of several thousand base pairs and are usually sporadic (Holt et al., 1988).", "This author supports the classification by El-Hattab, Craigen and Scaglia, including the concept that defects in mtDNA maintenance genes result in mtDNA depletion and multiple mtDNA deletions, both representing the spectrum of a single disease group (El-Hattab et al., 2017), thus adopting the term of mitochondrial DNA maintenance defects (MDMDs).", "Rarely, mtDNA maintenance defects may be because of an excess: by supplementing HeLa cells with surplus thymidine for just 4 h, mitochondrial dTTP and dGTP pools increased to such an extent that multiple mtDNA deletions were apparent after 8 months of culture (Song et al., 2003); furthermore, a very high mtDNA copy number was associated with several pathologies associated with mtDNA maintenance defects in transgenic mice (Ylikallio et al., 2010); in humans, this possibility remains unexplored.", "When inherited, MDMDs do so in an autosomal recessive or dominant manner and can basically manifest in any organ at any time (Suomalainen and Battersby, 2018).", "Pathogenic variants for all nuclear genes depicted in Fig. 1 have been reported to yield an MDMD.", "mtDNA depletion syndromes are reported and periodically updated in the OMIM database; currently, the search string [\"mtDNA depletion” OR “mitochondrial DNA depletion\"] in OMIM database yields 65 entries.", "What is important to realize is that the knowledge of a specific defect in mtDNA due to one reason or another, does not afford the investigator with the capacity of predicting the severity of disease, simply because the latter depends on at least three parameters: i) the severity of mutation, partially dictated by its position in the affected gene, ii) percentage of variable mtDNA content, and iii) likelihood for disease manifestation (phenotype).", "The latter parameter also depends on the affected organ; furthermore, with lesser amount of mtDNA, chances for pathogenic mtDNA depletion increases.", "Thus, these three parameters are strongly interrelated.", "Despite that MDMDs may appear in any organ at any time, on the basis of an apparent preponderance for tissue-specific manifestation these disorders are usually classified as myopathic, encephalomyopathic, hepatocerebral or neurogastrointestinal (El-Hattab and Scaglia, 2013).", "In most cases it is thought that because adequate amount of mtDNA is required for the production of subunits of mitochondrial respiratory chain complexes and therefore energy-harnessing through oxidative phosphorylation (OXPHOS), mtDNA depletion results in energy-shortage (Moraes et al., 1991), (Sarzi et al., 2007), (Spinazzola et al., 2009).", "Notably though, MDMDs leading to respiratory chain dysfunction let subunits of complex II intact, simply because they are all nuclear-encoded.", "However, mindful that mitochondria participate in calcium homeostasis, biosynthesis of heme and steroid hormones, apoptosis, cell cycle regulation –to name a few-it must be pointed out that exactly because they do so many things, it is oversimplifying to consider that mtDNA depletion –or any mitochondrial problem for that matter-yields phenotypes exclusively from issues regarding OXPHOS.", "Indeed, mitochondria require ~1,500 proteins for proper operation (Pagliarini et al., 2008), (Calvo and Mootha, 2010) out of only ~100 of are directly involved in oxidative phosphorylation and the production of ATP (Gorman et al., 2016).", "This concept is critical for understanding the novel diagnostic test outlined below in which mitochondria are challenged to benefit from glutaminolysis in the absence of an active respiratory chain, see section 6.", "When a neurometabolic disease is suspected of having a mitochondrial origin a host of clinical assessments and multitude of investigations are at hand which differ for pediatric vs adult patients (Bernier et al., 2002), (Walker et al., 1996).", "Historically, serum creatine kinase, alanine and lactate levels have been used as indices of mitochondrial diseases, but these measures are known to be nonspecific and somewhat insensitive (Debray et al., 2007).", "Muscle biopsy followed by histologic and functional assessment are the “gold standard” (Mitochondrial Medicine Society's Committee on et al., 2008), (Suomalainen, 2011), (Viscomi and Zeviani, 2017), (Nishigaki et al., 2003), (Nishino et al., 1999), (Nolden et al., 2005), (Ostergaard et al., 2007a), (Chretien et al., 1998), however, carrying a number of uncertainties (Tucker et al., 2011).", "Functional assays encompass respiratory chain component activities including that of the Fo-F1 ATP synthase, blue-native gel electrophoresis, semi-quantitation of the various protein components within complexes and supercomplexes via western blots and gel electrophoresis, oxygen consumption rate estimations and their alterations in the presence of various specific inhibitors.", "However, a muscle biopsy is invasive, exhibits high false-negative and false-positive rates and a disappointingly high irreproducibility among laboratories evaluating respiratory chain enzyme activities (Rodenburg et al., 2013), (Medja et al., 2009).", "In addition, in muscle homogenate the activity of respiratory chain components may vary from normal to about 50% of the controls' mean (Servidei et al., 1991).", "Despite that MDMDs were discovered by measuring enzymatic activities of the respiratory chain complexes in affected tissues based on measurements prone to inaccuracies, exactly because of the complexity and high degree of irreproducibility, Viscomi and Zeviani have tactfully suggested that “these assays should be carried out in specialized centers, as they require ad hoc expertise and standardized, verified and complex technologies and diagnostic protocols” (Viscomi and Zeviani, 2017).", "Finally, the costs of surgical procedures especially for children are considerable, since they require anesthesia (Suomalainen, 2011).", "On the other hand, new biomarkers show promise in diagnosing mitochondrial diseases, such as measuring the serum levels of fibroblast growth factor 21 (FGF21) and growth/differentiation factor 15 (GDF15) (Suomalainen et al., 2011), (Davis et al., 2013), (Yatsuga et al., 2015).", "Mindful that normal respiratory chain activities in muscle do not exclude respiratory chain deficiency in another tissue, an underappreciated factor for diagnosis of mitochondrial diseases is tissue selection.", "As stated above, the amount of depleted mtDNA may vary among cells and therefore within and between tissues.", "In general, tissues harboring the highest levels of mtDNA depletion tend to be those that are difficult or almost impossible to obtain such as the brain, heart, retina and cochlea whereas more accessible tissues such as skin fibroblasts and blood cells exhibit low mutation levels (Sue et al., 1998).", "It is considered prudent to culture fibroblasts obtained from a skin biopsy from a patient who donated muscle for diagnostic procedures under the impression that fibroblasts may harbor the deficiency in oxidative phosphorylation as shown in skeletal muscle and serving as a much easier tissue for diagnostic workup regarding assigning pathogenicity to new mutations (Gorman et al., 2016).", "This may assist in the following: once an index case has been identified, relatives can also be tested in a much less invasive manner by providing buccal, urine and blood DNA samples (Gorman et al., 2016).", "However, reports of classical MDMDs are outlined below in which mtDNA was not found to be depleted in several tissues.", "In almost all cases, mtDNA was quantified by Southern-blot analysis or real-time PCR (Gourlain et al., 2003).", "In both approaches, mtDNA amount is compared to a specific nuclear reference gene, and always examined using age- and tissue-matched controls as well (Clay Montier et al., 2009), (Hakonen et al., 2007), (Gotz et al., 2008).", "An extent of mtDNA decrease of more than 35–40% compared to controls is deemed pathologic (Poulton and Holt, 2009).", "In (Tzoulis et al., 2014) it was shown that autosomal recessive mutations in POLG (see also Fig. 1) mtDNA depletion and secondary mtDNA mutations were cell- and tissue-specific.", "In (Nishino et al., 1999) and (Nishino et al., 2000), only half of the patients exhibiting mutations in TP causing mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome harbored mtDNA depletions.", "In patients with deoxyguanosine kinase (DGUOK) mutations, mtDNA depletion was observed in liver but not in muscle (Mandel et al., 2001), even though this disease affects the muscle as well (Freisinger et al., 2006).", "The effect of DGUOK mutations on mtDNA content obtained from fibroblasts is variable (Morten et al., 2007).", "In (Gauthier-Villars et al., 2001), it was shown that patients suffering from Alpers-Huttenlocher syndrome usually exhibit respiratory chain defects and mtDNA depletion in liver, but not always in skeletal muscle; fibroblasts were shown to be mosaic in nature in respect of their mtDNA content.", "Finally, mtDNA depletion was found to be brain-specific in patients with infantile-onset spinocerebellar ataxia (IOSCA) syndrome while no alterations were found in muscle (Hakonen et al., 2008).", "In (Ostergaard, 2008), it was shown that in patients with mutations in SUCLG1 and SUCLA2 a combined respiratory chain defect was observed in liver or muscle, but not in skin fibroblasts.", "Likewise, in muscle of SUCLA2-deficient individuals mtDNA content was reduced to 20–60% compared to age-matched controls (Elpeleg et al., 2005), (Carrozzo et al., 2007), (Ostergaard et al., 2007b), (Carrozzo et al., 2016).", "In patients suffering from TK2 deficiency, only 66% of those exhibited a greater than 30% mtDNA depletion (Garone et al., 2018).", "In the same line of thought, i.e. that mtDNA depletion may not be necessarily leading to mitochondrial pathology in humans, the MPV17−/− mouse with severe mtDNA depletion in liver failed to demonstrate an appreciable decrease in respiratory chain activities, not even overt liver failure with age, only grey hair early in adulthood and focal segmental glomerulosclerosis with massive proteinuria and cochlear lesions (Viscomi et al., 2009).", "Overall, mtDNA depletion in a tissue does not imply depletion in another; by the same token, mtDNA depletion is not necessarily associated with respiratory chain defects.", "It has been firmly established that when the respiratory chain is pharmacologically inhibited or in the presence of anoxia, mitochondria are still able to utilize downstream metabolites of glutamine (glutamate, α-ketoglutarate) using NAD+ obtained from sources other than complex I (Chinopoulos, 2019), (Ravasz et al., 2018), (Kiss et al., 2013), (Chinopoulos et al., 2010), a concept which is likely to be exploited in cancer cells thriving in adverse tumor microenvironments (Chinopoulos and Seyfried, 2018).", "This serves the purpose of generating high-energy phosphate nucleotides within the mitochondrial matrix through the process of mitochondrial substrate-level phosphorylation (mSLP) for i) fueling a reverse-operating F0–F1 ATP synthase (Chinopoulos, 2011b) and ii) maintaining the ANT in forward mode, thus un-straining glycolysis (Chinopoulos, 2011a).", "An mSLP test is qualitative (presence vs absence of mSLP, there is no parameter that needs to be quantified) and can be performed in skin fibroblasts requiring no specialized machinery (spectrofluorimetry in plate or cuvette format is standard laboratory equipment).", "The test that we have devised is based on an “interrogation” of the directionality of succinate-CoA ligase (SUCL), a citric acid cycle enzyme that interconverts succinyl-CoA and ADP (or GDP) to CoASH, succinate and ATP (or GTP) (Johnson et al., 1998), essentially performing mSLP.", "This enzyme is a heterodimer composed of an invariant α subunit encoded by SUCLG1 and a substrate-specific β subunit, encoded by either SUCLA2 or SUCLG2.", "This dimer combination results in either an ATP-forming (EC 6.2.1.5) or a GTP-forming SUCL (EC 6.2.1.4).", "The possibility of co-presence of an ATP- and GTP-forming SUCL in the same mitochondrion has only been recently investigated in human fibroblasts, where it was found that in these cells the majority of in situ mitochondria co-express both isoforms (Chinopoulos et al., 2019), see Fig. 2 (reproduced from (Chinopoulos et al., 2019) by permission).", "This seems redundant, mindful that an intramitochondrial diphosphate kinase (NME4) can transfer the high-energy bond between adenine and guanine nucleotides (Lacombe et al., 2018).", "In any case, mSLP supports the production of sufficient amounts of [ATP] in the mitochondrial matrix for both a reverse-operating F0–F1 ATP synthase (Chinopoulos, 2011b) and maintain the ANT in forward mode (Chinopoulos, 2011a).", "Presence of mSLP maintains ANT in forward mode for reasons outlined in (Chinopoulos, 2019), (Ravasz et al., 2018), (Kiss et al., 2013), (Chinopoulos et al., 2010), and this can be easily assessed by checking the effect of a highly-specific ANT inhibitor –carboxyatractyloside- in permeabilized cells with blocked complex I (using rotenone).", "Under these conditions, mSLP can only occur in the presence of suitable substrates specifically those stemming from glutaminolysis, but not succinate (Kiss et al., 2013).", "Such a test is depicted in Fig. 3 (obtained from (Tretter et al., 2016), by permission).", "As shown in figure panel 3A, the test relies on the principle that the ATP-ADP exchange through the ANT is electrogenic, since one molecule of ATP4 − is exchanged for one molecule of ADP3 − (Klingenberg, 2008).", "If the ANT operates in forward mode, abolition of its operation by carboxyatractyloside (cATR) will lead to a gain of ΔΨm.", "If the ANT operates in reverse, abolition of its operation by the inhibitor will lead to a loss of ΔΨm.", "Note that addition of the ANT inhibitor must take place after complex I is inhibited (i.e. by rotenone), thus, it cannot be overemphasized that the mSLP test addresses the extent of compensation (i.e. remaining mitochondrial functionality) when the electron transport chain is non-functional.", "It does not yield any information regarding the respiratory chain, it only provides information if the in situ mitochondria of the examined cells are able to harness energy from glutamine-derived metabolites when the respiratory chain is inhibited.", "In essence, this test will hint on the ability of mitochondria to cope with a defect in their respiratory chain.", "By employing this test, we have reported that defects in SUCLA2 and SUCLG1, respectively, yield an mSLP negative test, i.e. no mSLP present.", "As shown in figure panels 3B and 3C, in situ fibroblast mitochondria of a SUCLA2-deficient patient (B) or SUCLG1-deficient patient (C) exhibited no mSLP compared to age-matched controls.", "Note that patients with defects in SUCLA2 and SUCLG1 exhibit mtDNA depletion in some but not all tissues, while the phenotypes are debilitating leading to life termination at a young age (Ostergaard, 2008).", "Obviously, this test has been applied in fibroblasts from patients suffering from MDMDs due to mutations in the enzyme directly related to mSLP; efforts are ongoing in our laboratory to verify the validity of this test using fibroblasts from patients suffering from various mtDNA depletion syndromes.", "Finally, it may be relevant to ponder on the fact that mSLP relies on a sufficient clearance of succinate, which can occur by increasing succinate dehydrogenase (SDH) activity; it is thus not surprising that in several MDMDs, ragged-blue fibers are observed in histological examination of muscle tissue, which is none other than an increase in SDH activity (DiMauro et al., 1999).", "The mSLP protocol can be performed in cultured fibroblasts exactly as in (Kacso et al., 2016) and (Chinopoulos et al., 2019).", "It is best to compare cultured fibroblasts from the patient and at least three controls.", "It is not important to age- and gender-match the controls to the patient; the controls serve the purpose of ensuring that the mSLP protocol is being performed correctly; it is not a quantitative test, and for this reason it is also not necessary to normalize results to protein content or a housekeeping protein.", "Basically, fibroblasts [two 75 cm^2 (midi) or one 175 cm^2 (maxi) flask)] are cultured to approximately 80% confluence.", "On the day of the experiment, two buffers need to be prepared: 500 ml of ice-cold buffer “A”, containing 110 mM K-gluconate, 10 mM HEPES (free acid), 10 mM KH2PO4, 10 mM mannitol, 10 mM NaCl, 8 mM KCl, 1.5 mM MgCl2, 0.01 mM EGTA, 0.5 mg/ml BSA (essentially fatty acid-free), with the pH adjusted to 7.25 with KOH, and 5 ml of buffer “B” which in addition to the substances mentioned for buffer “A” it contains the following: 5 μM safranine O, 5 mM glutamate, 5 mM malate, 5 mM α-ketoglutarate and 175 μl of 2.5 mM digitonin (purity > 90%, dissolved in DMSO).", "For exact catalogue numbers of chemicals, the reader is referred to http://antactivity.com/Buffers_chemicals.html.", "Buffer “B” must be kept at 37 °C.", "Feeding media are aspirated and cells are washed once with ice-cold buffer “A”, then harvested by scraping in buffer “A” (no need for trypsinization, cells will be eventually permeabilized for the mSLP assay within a few minutes, it is redundant to maintain high viability of the cells).", "Pellet cells by centrifugation at 3,500 rpm for 2.5 min using an angular rotor (or at 1,100 rpm for 10 min if a swing-bucket rotor is used) in at 50 ml tubes containing at least 40 ml of buffer (to dilute calcium of the feeding media as much as possible).", "Wash cells once with buffer “A”, and repeat the centrifugation step.", "Resuspend final pellet in 0.7 ml of buffer “B”.", "Use a plate reader for recording safranine O fluorescence (495ex/585em).", "Split the 0.7 ml pellet in three 0.2 ml (record in triplicates) and add to individual wells of a 96 white plate suitable for fluorescence, and insert into the plate reader.", "The interior of the plate reader must be kept at 37 °C; possibility for automatic shaking of the plate is imperative (i.e. every 3 s).", "Acquisitions should be made every 10–15 s. Allow 5 min for adequate polarization of in situ mitochondria and cell membrane permeabilization by digitonin.", "Record baseline for 3 min, add 2 mM ADP (from a 6.9 pH-ed 200 mM stock, see http://antactivity.com/Buffers_chemicals.html), see Fig. 3.", "Record for 3 more minutes, then add 5 μM rotenone.", "Record for an additional 3 min, then add 1 μM carboxyatractyloside.", "Record for an additional 3 min, then add 250 nM of the uncoupler SF6847.", "The volume of each addition to each well should be not less than 1 μl and no more than 2 μl.", "Convert fluorescence traces to percentages and compare the 3 controls with that from the patient cells.", "If carboxyatractyloside leads to hyperpolarization in the controls but to a depolarization in the patient, mSLP in the patient is impaired.", "This means that the fibroblasts of the patient are unable to harness ATP from mitochondrial substrate-level phosphorylation from glutaminolysis, thus his/her mitochondria cannot compensate for a potential electron transport chain dysfunction to an appreciable degree.", "The present review does not make an attempt to indicate how much mtDNA depletion is required for disease manifestation; it merely discusses that quantitating mtDNA on peripheral –but not limited to-tissues is not pathognomonic.", "Furthermore, the concept of mSLP as a qualitative and not quantitative test for evaluating the extent of mitochondrial functionality in the absence of an electron transport chain is put forward.", "Having said that, it cannot be overemphasized that the mSLP test cannot be used to differentiate among the numerous defects described in mtDNA translation, or involving a single complex, or secondary to other diseases involving mitochondrial dysfunction; it can only provide an indication if the affected mitochondria are able to energetically compensate and maintain –albeit- minor- ATP production and not act as sinks of cytosolically made ATP.", "This work was supported by grants from NKFIH [FIKP-61822-64888-EATV], [VEKOP 2.3.3-15-2016-00012], [2017-2.3.4-TET-RU-2017-00003] and [KH129567] to C.C." ]
[]
Ethically minded consumer behavior: Scale review, development, and validation
This paper details the development and validation of a new research instrument called the ethically minded consumer behavior (EMCB) scale. The scale conceptualizes ethically minded consumer behavior as a variety of consumption choices pertaining to environmental issues and corporate social responsibility. Developed and extensively tested among consumers (n = 1278) in the UK, Germany, Hungary, and Japan, the scale demonstrates reliability, validity, and metric measurement invariance across these diverse nations. The study provides researchers and practitioners with a much-needed and easy-to-administer, valid, and reliable instrument pertaining to ethically minded consumer behavior.
[ "BUSI" ]
[ "Attitude-behavior gap", "Consumer ethics", "Ethical consumption", "Scale development" ]
[ "1406" ]
[ "More businesses realize the need to consider ecological and human welfare implications when adopting sustainable development principles (Chow and Chen, 2012).", "At the same time, ethical consumer behavior, which incorporates the consideration of ecological and human welfare issues, is increasing dramatically (Fairtrade International, 2013).", "Spurred partly by the Fairtrade movement that attracts attention from mainstream brands (Low and Davenport, 2007), ethical products are no longer the remit of niche markets in many nations (Carrington, Neville, and Whitwell, 2014; Doherty and Tranchell, 2007).", "Indeed, increasing numbers of products bear the marks of initiatives such as Fairtrade and Rainforest Alliance, or make a variety of social or environmental claims (Ethical Trading Initiative, 2010).", "Unsurprisingly, research pertaining to different aspects of ethical business practices is also increasing (Chow and Chen, 2012).", "Corporate social responsibility (CSR) is now one of the most prominent and important concepts in the literature (Lee, Park, Rapert, and Newman, 2012) with an abundance of recent papers focusing on different aspects of marketing and consuming ethical products (Andorfer and Liebe, 2012; Auger, Burke, Devinney, Louviere, and Burke, 2010; Autio, Heiskanen, and Heinonen, 2009; d'Astous and Legendre, 2008).", "Only as recently as the 1990s did research begin to focus more strongly on ethics from a consumer rather than a corporate perspective (Schlegelmilch and Öberseder, 2010), and it seems that measurement scales pertaining to consumer ethical purchases are rare, especially in comparison to the scales available to measure ethics in business decisions.", "Even when ethical research does focus on consumers, it tends to emphasize environmental issues, with fewer studies incorporating wider social issues (O'Rourke, 2011).", "Consequently, despite the fact that ethical consumers are no longer classified as fringe (Carrington et al., 2014), and ethical products and services now account for increasing shares of many different markets (Ethical Consumer Markets Report, 2012), it is still relatively unusual to find reliable and validated scales pertaining to ethical consumer behavior that incorporate both ecological and social issues.", "The need for such a scale is pressing, given the current “burgeoning social movement” (Carrington et al., 2014, p. 2759) that is ethical consumerism.", "Of course, observational research has an advantage over self-report measures as it analyses what people do rather than what they claim to do.", "However, while technology is allowing for easier use of observational data in terms of scanner-tracking and Internet purchases (Lee and Broderick, 2007), it is still not possible to collect accurate data for every individual purchase.", "Consequently, there remains a need for a psychometrically sound, reliable, and validated scale to use as a shorthand method to indicate the levels and types of ethical purchasing claimed by different individuals.", "Such a scale would enable the collection of valuable and timely information from large numbers of people in relatively short periods and reasonably cost effectively and be useful to businesses, researchers, and policy makers.", "Businesses need a valid and reliable instrument that is comparatively quick and easy to administer in order to gather quantifiable data to analyze and profile different groups, for planning and forecasting purposes and to develop segmentation models to design more accurate targeting and positioning strategies.", "Researchers need such an instrument to use in future studies to ascertain the different underlying motivations and antecedents for ethical purchasing, and just as importantly uncover and analyze the barriers to such purchasing because as Gleim et al. (2013) recently point out, well-grounded theoretical studies to explain why consumers do not engage in environmentally sustainable behavior are rare.", "Moreover, a standard scale, particularly one that exhibits measurement invariance, is a potentially valuable research tool for comparative and longitudinal research purposes in a variety of nations in order to create new theories and or test existing hypotheses (Ackroyd and Hughes, 1981).", "Finally, policy makers need to understand the reasons for not consuming ethically in order to begin to address and change behavior because despite phenomenal increases in recent years, sales of ethical goods and services still remain a small percentage of total sales; thus, sustainable solutions will require policy intervention (Ethical Consumer Markets Report, 2012).", "There are, of course, some scales pertaining to ethical consumption already available.", "However, many of the existing instruments measure attitudes, intentions, or utilize hypothetical scenarios (Trudel and Cotte, 2008), which are problematic because of the well-documented attitude–behavior gap (Carrington et al., 2014).", "Empirical evidence shows that stated ethical intentions rarely translate into actual ethical consumer choices (Carrigan et al., 2011).", "Of the few remaining instruments that do pertain to actual behavior, the older instruments tend to focus solely on environmental issues and omit wider social considerations (Schlegelmilch et al., 1996), while more recent ones tend to focus exclusively on a specific aspect of ethical consumption such as Fairtrade (Shaw, Shiu and Clarke, 2000) and do not consider a wide range of issues.", "A scale that considers both environmental and social issues is important because recently differences in consumer reactions to these different strands of ethical consumption have emerged (Ailawadi, Neslin, Luan, and Taylor, 2014).", "Yet only one scale, the socially responsible consumer behavior (SRCB) scale (Roberts 1993, 1995), utilizes wording that asks consumers to recall their actual ethical consumption (as opposed to intended, hypothetical, or attitudes toward ethical issues) from both environmental and CSR perspectives.", "The SRCB scale is however two decades old, and during these years, the world has changed dramatically.", "Harrison (2014) charts the rise of ethical consumers from the 1990s – the development period for the SRCB scale – to the present day.", "He finds that during the 1990s, surveys revealed 20–30% of people professed that they could not be bothered with any form of ethical consumption, while a further 60–75% were sometimes ethical but did not really work very hard to seek out ethical alternatives.", "He finds that it is not until the latter years of the first decade of this century that ethical consumption becomes truly mainstream in that it crosses cultures, classes, and geographical boundaries.", "In the UK alone, the sale of ethical goods and services has grown 360% since the turn of the century (Ethical Consumer Markets Report, 2012).", "Moreover, some of the scale items in the SRCB refer to practices that are now illegal (e.g., discrimination against minorities), while other items are no longer relevant and instead reflect the different political landscape of two decades ago (e.g., I do not buy products from companies that have investments in South Africa).", "The SRCB scale was clearly ahead of its time when it was developed.", "However, in its current format, it is no longer valid in a world that has changed so dramatically since its inception.", "Nevertheless, the SRCB instrument proved invaluable as a starting point to the current scale development study, from which emerges a new instrument called the ethically minded consumer behavior (EMCB) scale.", "The current study therefore fills a gap in that it develops a scale comprising questions pertaining to actual behavior rather than intentions or hypothetical situations.", "Of course, any self-report measure that depends on honesty and accuracy from respondents has limitations, but because it asks about actual behavior, it has advantages over those scales that measure ethical attitudes or intentions that are very poor indicators of what people actually do at the checkout (Cowe and Williams, 2000).", "The new scale comprises a range of ethically minded consumption choices, better reflecting contemporary ideas of what ethical consumption is.", "The study is also relatively unique in that it develops and validates the scale using consumers in 4 diverse nations (UK, Germany, Hungary, and Japan).", "This paper justifies and validates the new scale.", "It begins by conceptualizing ethical consumer behavior before reviewing the available instruments that measure it.", "It then justifies the samples and the chosen nations prior to explaining the development and validation of the new measurement instrument.", "It concludes with an evaluation of the new scale and discusses its implications for research and practice.", "This section has three major purposes: (1) to conceptualize ethical consumption from today's perspective in order to identify broad themes that needed to be included in the new scale, (2) to explain the implications of the attitude–behavior gap for scale development, and (3) to review the available instruments pertaining to ethical consumer choices.", "Historically, ethical consumption was viewed very much as the behavior of a relatively small group of principled consumers (Shaw, 2007), while ethical brands (e.g., The Body Shop) were easy to identify.", "However, as the numbers of ethical brands increase in conjunction with ease of access to data pertaining to ethical products (O'Connor, 2014), there is a marked diversity in terms of definitions of ethical consumer behavior, and some terms seem to be more fluid than before.", "The concept of Fairtrade, for example, has developed from a focus on marginalized producers to incorporate broader social justice issues (Becchetti and Costantino, 2010).", "Nevertheless, from a starting point that assumes ethical purchasing is conscious and based on a particular ethical or social issue (Ethical Consumerism Report, 2011; Gulyás, 2008) rather than based on taste, color, or design, it was possible to identify several important issues that needed to be included in the new scale.", "First, most definitions of ethical consumption encompass reference to environmental issues (Ethical Consumerism Report, 2011; European Commission, 2011; IGD, 2007; Trudel and Cotte, 2008).", "Interestingly, while a plethora of studies include a wide range of different environmentally friendly issues and behavior (Abdul-Muhmin, 2007; Gilg, Barr and Ford, 2005; Kim and Choi, 2005; Niva and Timonen, 2001), almost all mention recycling issues specifically (Autio et al., 2009; Laroche, Bergeron, and Barbaro-Forleo, 2001; Straughan and Roberts, 1999; Sudbury-Riley, 2014; Thøgersen, 1999; Vicente and Reis, 2007), perhaps because recycling of household waste is becoming a normal everyday behavior for many people, due in part to various recycling policies and programs in many countries (DEFRA, 2014; EPA, 2014; European Commission, 2014).", "Second, most definitions of ethical consumption include social justice and human rights issues (Auger et al., 2010; Ethical Consumerism Report, 2011; European Commission, 2011; Golding, 2009; Trudel and Cotte, 2008; Valor, 2007), often with a particular emphasis on involvement in worker exploitation (Brenton and Hacken, 2006; Eckhardt, Belk, and Devinney, 2010; Valor, 2007).", "Beyond these mainstream issues, wider-ranging definitions focus on animal welfare (Megicks, Memery and Williams, 2008) and local community initiatives (Carrigan et al., 2011; Grau and Garretsen Folse, 2007; Mattingly and Berman, 2006).", "Overlapping animal welfare and local community issues is the topic of organic food, and indeed many consumers purchase organic food for animal welfare reasons and/or to support their local communities (McEachern et al., 2007; Schröder and McEachern, 2004).", "The third theme pertains to consciously refusing to not buy products (Carrigan et al., 2004), or boycotting.", "Boycotting is a form of anti-consumption and, inter alia, can be targeted at particular products because they damage the environment (Papaoikonomou, Valverde, and Ryan, 2012) or because they are made by companies with a lack of corporate social responsibility (Creyer and Ross, 1997; Klein, Smith, and John, 2004; Kozinets and Handelman, 2004).", "Theoretically, the distinction between choosing an ethical product and refusing to purchase a nonethical product is an important one.", "Carrigan and Attalla (2001) explain that ethical and unethical actions have an asymmetrical influence on consumers in that many punish unethical behavior (boycott) but do not necessarily reward ethical behavior (purchasing over alternatives).", "Avoiding products and services deemed to have a negative impact on society or the environment is now a key part of ethical consumerism (IGD, 2007), and Megicks, Memery and Williams (2008) report that the number of consumers who boycott products due to a concern with one or more ethical issue is far greater than those who choose an ethical product over an alternative.", "Trudel and Cotte (2008) also find consumers to be more willing to reject unethical products than to select products for ethical reasons.", "Thus, Mattingly and Berman's (2006) contention that positive and negative social action are conceptually distinct constructs that research should not combine is particularly noteworthy.", "A fourth constant factor in the literature is price.", "Many consumers are unwilling to pay a large premium in order to translate their ethical beliefs into purchasing practice (McEachern, Warnaby, Carrigan and Szmigin, 2010; Sudbury and Böltner, 2010).", "This unwillingness does not necessarily result from an inability to pay a price premium, as ethical consumer behavior is unrelated to income or work status (Laroche, Bergeron, and Barbaro-Forleo, 2001; Loureiro et al., 2002; Pepper, Jackson, and Uzzell, 2009).", "Thus, an optimum scale would include questions about past behavior that demonstrates a willingness to pay more for an ethical product.", "Far more consumers profess to care about ethical issues than actually purchase ethical products (Cowe and Williams, 2000).", "Indeed, stated ethical intentions are rarely acted upon in terms of actual ethical consumer choices (Carrigan et al., 2011), a phenomenon referred to as the attitude–behavior gap (Auger and Devinney, 2007).", "To illustrate, recent surveys (Euromonitor, 2013) find between half and two-thirds of consumers across the US profess to care about ethical issues, yet ethical products account for 15% of all sales (Bertini, 2014).", "This attitude–behavior gap is recognizable across many nations (Eckhardt, Belk, & Devinney, 2010).", "Part of the attitude–behavior gap may be due to the type of survey instruments used in consumer research (Auger and Devinney, 2007).", "Many scales measure future intentions rather than ask questions pertaining to actual behavior (Andorfer and Leibe, 2012).", "Of course, there are acknowledged potential problems with any scale in that respondents may lie, exaggerate, or misremember actual behaviors, but those that do focus on intentions and attitudes tend to overestimate purchase likelihood due to social desirability reasons (Liebe et al., 2014).", "While Auger and Devinney (2007) do not go as far as Ulrich and Sarasin's (1995) suggestion that ethical consumption research is inherently unreliable, they do note that weaknesses in some previous survey methodologies cast doubts about the reliability of some results.", "Drawing attention to research across several disciplines that finds the presentation of survey questions can influence answers; they suggest several areas that need attention, including the issue of wording.", "Vague wording can lead to erroneous answers; thus, the wording of items is critical.", "They also suggest context creation, where specific questionnaire items closely resemble the context in which consumers make their decisions, can reduce some of the inherent problems in some previous surveys.", "In other words, question wording requires high degrees of specificity, an issue that researchers rarely consider when designing general attitudinal questionnaires.", "Additionally, they recommend cognitive pretests, involving a small number of respondents and focusing on question interpretation.", "Finally, the method of administration is important, with self-administration providing a sense of privacy.", "The attitude–behavior gap is not unique to ethical consumption.", "Across a range of non-consumption-related behaviors, it is well established that general attitudes, in a similar vein to broad personality traits, tend to be poor predictors of behavior in specific situations (Ajzen, 1991).", "However, aggregating a number of different behaviors across a variety of situations can better predict behavioral outcomes.", "Hence, the theory of planned behavior (Ajzen, 1985, 1987) suggests that attitudes toward the behavior, subjective norm, and perceived behavioral control all impact intentions, which can then predict behavior in a specific context.", "Attitude toward behavior is the degree to which a person has a favorable or unfavorable evaluation of the behavior.", "The subjective norm refers to the perceived social pressure to perform or not perform the behavior, and perceived behavioral control refers to the perceived ability in terms of ease or difficulty to perform the behavior across situations (Ajzen, 1991).", "The theory helps to understand the attitude–behavior gap in ethical consumption.", "Vermeir and Verbeke (2006, 2008) found that despite positive attitudes toward sustainable food products, intentions to buy such products remained low due to low perceived availability of such products.", "In other words, although the attitude component was present, attitudes alone failed to translate into behavior because many consumers found availability (the perceived behavior control element of the theory) to be lacking.", "Similarly, a reported lack of availability in shops, related to perceived behavioral control, was identified in Sparks and Shepherd's (1992) study that focused on purchasing organic vegetables, and in Shaw and Clarke's (1999) study of Fairtrade goods.", "The theory has been criticized and subsequently extended (Shaw, Shiu and Clarke, 2000; Shaw and Shiu, 2003) but is useful in the current context for two reasons.", "Methodologically, the theory provides a better insight into why the attitude–behavior gap may exist and points out very clearly that scales designed solely to measure attitudes toward ethical issues may be poor predictors of actual ethical consumption.", "From a practical perspective, the findings of these studies have implications for marketing practice because it is clear that high price premiums and low availability of ethical products will counteract positive attitudes toward ethical consumption for many consumers, driving home the message that attitudinal scales are not reliable indicators of ethical consumption.", "Increasingly, businesses are recognizing the need to “take note not only of what consumers believe in, but equally what they do and choose to act upon” (Fukukawa and Ennew, 2010: 49).", "A scale that at least asks questions about what people actually do – rather than solely measure attitudes or intentions – is therefore much needed and currently lacking.", "Indeed, an extensive review of available measures reveals a situation where most are concerned with attitudes and intentions, rather than a focus on recalling actual ethical consumption.", "A substantive body of research pertains to ethical issues, but the majority concentrates on the business side of the buyer/seller dyad (Vitell and Muncy, 2005).", "In contrast, studies that do focus on consumers often utilize the well-known Machiavellianism scale (Christie and Geis, 1970), Forsyth's (1980) Ethics Position Questionnaire (EPQ), the New Environmental Paradigm (NEP) developed by Dunlap and Van Liere in 1978 and modernized by Dunlap, Van Liere, Mertig, and Jones in 2000, or the Consumer Ethics Scale developed by Muncy and Vitell (1992) and modernized by Vitell and Muncy in 2005.", "The Machiavellianism scale, originally developed to measure an individual's tendency to deceive and manipulate others for personal gain, gives an insight into the ethical beliefs of an individual.", "Comprising 20 items and utilized in both consumer and psychology research, the Machiavellianism scale is a valid and reliable instrument (Auger and Devinney, 2007; Erffmeyer, Keillor, and LeClair, 1999; Vitell, Lumpkin, and Rawwas, 1991).", "The EPQ (Forsyth, 1980) measures an individual's moral philosophy in terms of an inclination toward idealism or relativism.", "It too comprises 20 items, is valid and reliable, and is a popular scale to measure the ethical stance of consumers (Erffmeyer et al., 1999; Swaidan, Vitell, and Rawwas, 2003; Vitell et al., 1991).", "The NEP scale (Dunlap et al., 2000), developed to measure the move away from the anti-ecological dominant social paradigm of the late 20th Century toward a fundamentally contrasting pro-ecological new environmental paradigm (NEP) that began to emerge in the 1970s, is a valid measure of the extent of an individual's ecological beliefs and is used in a diverse range of studies (Steg, Dreijerink, and Abrahamse, 2005).", "Finally, Muncy and Vitell's consumer ethics scale examines the extent to which consumers believe that certain questionable behaviors are ethical or unethical.", "Comprising 20 situations in which consumers may find themselves (for example, lying about a child's age to get a lower price or getting too much change and not saying anything), it is noteworthy as it is widely used in cross-cultural studies.", "While the scales outlined above, and indeed the studies that utilize them, unquestionably extend knowledge pertaining to consumer's ethical and environmental beliefs, none even attempt to reflect behavior as opposed to beliefs, attitudes, or intentions.", "Indeed, Carrigan et al. (2011, p. 154) note a significant limitation within the ethical consumerism literature in that “researchers have generally failed to consider that intentions are not a reliable proxy for actual behavior.”", "As discussed in the previous section, there are serious disconnections between the issues that consumers claim to care about and their actual purchasing behavior (Auger et al., 2003).", "Indeed, some attitudinal scales even fail to differentiate between those who actively support social causes and those that do not.", "The central question in Auger and Devinney's (2007:364) work is “How do responses to survey questions about the importance of ethical issues relate to purchase behavior?”", "Using Mori poll questions, they found that on several items many respondents who were not actively involved in any social causes claimed to be just as socially conscious as supporters of Amnesty International.", "In other words, many items failed to differentiate between those who do actively support causes and those that do not.", "Of course, this finding has important implications for scale development, as an optimal scale would demonstrate the ability to differentiate between these segments.", "Despite the recognition that firms are beginning to take note of what consumers choose to do, as opposed to solely what consumers believe (Fukukawa and Ennew, 2010), research has not yet filled the need for measurement instruments that question consumers about their actual ethical consumption choices as opposed to their attitudes or intentions.", "An extensive review of the literature reveals a situation where even when the goal of a previous study is to focus on actual behavior, the available scales tend to be limited to a particular type of ethical behavior.", "For example, Pieters, Bijmolt, van Raaij, and de Kruijk's (1998) study focuses on motivation, ability, and pro-environmental behavior, with the 7-item scale including questions about energy use, water use, recycling, and the disposal of vegetables, fruits, and waste, with little to measure actual purchasing behavior.", "Shaw, Shiu, and Clarke (2000) measure the likelihood of purchasing a Fairtrade product next time the consumer shops for groceries, but this study is limited to beliefs about Fairtrade rather than overall ethical consumption.", "Vlosky, Ozanne, and Fontenot's (1999) study contains only three items on actual behavior and is limited to wood products.", "Pepper et al. (2009) include some questions pertaining to specific Fairtrade products (bananas and tea/coffee) and the ethical reputations of businesses but do not include environmental issues.", "In contrast, Schlegelmilch et al. (1996) utilize a set of questions pertaining to environmentally friendly purchasing, but their scale does not include social issues.", "It seems that few previous studies view ethical consumption from the perspective taken here, which is that ethical consumption pertains to both environmental and social issues.", "Thus, while all of these studies make very important contributions to knowledge, none contain a research instrument that matches the broad ethical perspective taken here as well as the 26-item socially responsible consumer behavior (SRCB) scale, developed by Roberts and his colleagues (Roberts, 1993, 1995, 1996a 1996b), which clearly asks respondents to reflect upon and report their past and current behavior as opposed to their attitudes or likely future behavior.", "As Ajzen (1991: 202) observes, the dictum “past behavior is the best predictor of future behavior” is a good one to test the predictive ability of a model.", "Thus, in order to design a scale that better predicts future ethical consumption, there is a need to ask consumers about their past and current ethical consumption.", "Importantly, the SRCB scale appeals because it has two factors comprising ecologically conscious consumer behavior (ECCB) and socially conscious consumer behavior (SCCB).", "Accordingly, the instrument has advantages over alternatives because, as Roberts (1995: 104) candidly notes, “Assessing a person's SRCB on only a social or ecological dimension would be akin to trying to solve a puzzle with half of its pieces.”", "The literature review very clearly demonstrated that two major strands to ethical purchasing are environmentalism and social issues, which are even more important given Ailawadi et al.'s (2014) finding that consumers have different reactions to these different strands of ethical consumption.", "Finally, testimony of the SRCB scale's usefulness comes from its usefulness in scale development (Webb, Mohr, and. Harris, 2008) and its use across so many different countries.", "The preceding sections identified several major implications that need consideration in the design and development of the new scale.", "First, consciousness is important – questions must be worded in such a way as to ensure that if an ethical product is chosen, the choice is driven by ethical (rather than quality or esthetic) reasons.", "Second, an optimum scale is one that incorporates the conscious purchasing of products that are environmentally friendly, with a particular emphasis on recycling, as well as contain wider social issues.", "Additionally, refusal to purchase and price premiums are further important considerations.", "Third, questions need to be specific and pertain to actual behavior rather than attitudes or intentions and should have the ability to differentiate between groups based on their membership of different causes.", "Finally, cognitive pretests comprising small numbers of consumers should focus on question interpretation.", "Given the advantages of the SRCB instrument over many alternatives, the SRCB scale is used a starting point in the current study, which aims to fill a gap by developing a scale to measure proclivity toward a wide range of ethically minded consumption choices.", "In so doing, the study aims to design a scale that better reflects contemporary ideas, from the consumer perspective, of what conscious ethical consumption is, with advantages over alternative measures as it will not be based on intentions or hypothetical situations, and should demonstrate the ability to differentiate between activists and nonactivists.", "The study also aims to validate the scale across 4 diverse nations, in order to provide researchers and practitioners with a much-needed measurement instrument that can be used across nations and cultures.", "Ebbinghaus (2005) contends that nation–state formation, international cooperation, and ease of availability of data have resulted in some countries being over (or indeed under) represented in many analyses.", "Consequently, research conducted in one country (usually the US) is assumed to be relevant to other countries, irrespective of differences in cultural and social forces.", "The inclusion of disparate nations in the current study is therefore an important contribution to knowledge.", "The UK is the baseline sample because it leads the way on Fairtrade.", "For example, one in three bananas purchased in the UK is Fairtrade (Fairtrade Foundation, 2013, 2015).", "However, differences in ethical beliefs between consumers across areas of the European Union are apparent, with the UK criticized as Eurosceptic and having more in common with the US than the rest of Europe (Economist, 2012).", "Thus, Germany is included because it has no Anglo-Saxon roots, has the largest economy in continental Europe, and is more representative of Western Europe compared to the UK.", "In comparison to the UK, German Fairtrade ethics are less commercialized (Varul and Wilson-Kovacs, 2008), although environmentalism embeds itself in German politics, with a successful Green party and much evidence of lobbying for environmental reasons (Witkowski and Reddy, 2010).", "Few previous studies into ethical consumption include countries from the new Europe.", "Indeed, Andorfer and Liebe's (2012) review of all research pertaining to Fairtrade consumption, which includes 51 journal publications, contains not a single study on a country from the former Eastern Bloc.", "The current study therefore fills a research gap.", "Dombos' (2008) ethnographic study of ethical consumption among Hungarian activists and entrepreneurs interprets ethical consumption as a way of expressing “hopes, desires, and frustrations about the seemingly never ending process of post-socialist transition” (p. 123), suggesting that motives for ethical consumption may be very different in Hungary to those found in other countries.", "Moreover, in 2006 a Fairtrade shop opened in Budapest, there are several ecologically and socially aware food communities directly linking consumers and producers, and Gulyás (2008) reports that around 16% of the Hungarian population joined in 20 recent boycotts.", "Hungary is therefore an obvious choice for inclusion in the study.", "Finally, Ralston and Pearson (2010) note that despite growth in immigration and emigrations resulting in intermingling of cultures, cultural groups still differ substantially in their ethical perspectives.", "Even though the three European countries selected are very different to each other, the inclusion of an Asian country is still beneficial.", "While the Japanese Fairtrade market is still comparatively small, its tremendous growth rates in recent years point to a new consumption trend in Japan (Kohlbacher 2013).", "Moreover, the sample comprises older adults (see below) and Japan has the oldest population in the world (United Nations, 2012).", "For these reasons, Japan comprises the final sample nation.", "“One of the greatest social, economic, and political transformations of our time” is how the United Nations (2012) describes the changing demographic make-up of the world due to rapid population aging and a steady increase in human longevity.", "Moreover, in addition to typically holding more financial assets than younger generations across a large number of countries (Sudbury-Riley et al., 2012), research suggests that older consumers are “increasingly likely to be among the ranks of the ethically motivated and adventurous” (Szmigin, Maddock, and Carrigan, 2003: 548) and feel less cynical and more positive about the impact of their ethical purchasing compared to younger adults (Carrigan et al., 2004).", "De Pelsmacker, Janssens, Sterckx, and Mielants (2006, p. 134) lend support to these findings when they state, “Inclination to action, concern and buying behavior increased with age ... and older people appeared to be more positive about the price level of Fairtrade products.”", "When age differences are considered in larger studies, older adults are found to be more environmentally concerned (Sandahl and Robertson, 1989), have stronger ethical beliefs (Dodge, Edwards, and Fullerton, 1996; Kim and Choi, 2003; Pan and Sparkes, 2012; Swaidan et al., 2003; Vitell, Singh, and Paolillo, 2007), demonstrate a stronger moral orientation (Pratt, Golding, and Hunter, 1983), and consume more ethical products (Trendbüro, 2009) than their younger counterparts.", "Indeed, Carrigan, Szmigin, and Wright, (2004: 412–413) conclude that “older consumers are a significant force within the consumer resistance movement ... they are inherently diverse in nature, yet present some consistency in their attitudes toward certain aspects of ethical purchasing.”", "Consequently, Bray, Johns, and Kilburn (2011) call for age to be included in future models of ethical consumption.", "Yet a widespread literature review fails to uncover a single large-scale study that focuses on the actual ethical purchasing behavior of older consumers.", "Additionally, scales are often developed using mainly young samples (Brunk, 2012; Kim, Lee, and Park, 2010).", "Thus, an older (50 +) sample is the focus of the current study.", "Purchased lists of randomly selected people ages 50 + comprise the samples in each country.", "In the UK, Germany, and Japan, the questionnaire was administered via a postal survey, but piloting in Hungary demonstrated difficulties of self-completion among many rural Hungarian adults.", "Hungary has successfully moved to a fully transitioned market-based economy from a centrally planned economy.", "It is a member of the OECD, NATO, and the European Union, enjoys a strategic location, and has a highly skilled and educated workforce.", "All of these things make it an attractive market for overseas firms (US Commercial Service, 2014).", "Thus, it was felt that Hungary was too important to drop from the study.", "Consequently, the data collection method was adjusted.", "The sample list, which is representative in terms of gender, region, work status, income, and age, was purchased from the Hungarian Central Statistic Büro, and a professional market research company conducted interviews face to face.", "This different administration method is viewed as a strength of the study rather than a weakness because the study uses the optimum method for each country, taking into consideration social and cultural factors.", "Face-to-face methods would have been prohibitively expensive in the more advanced countries.", "Moreover, social desirability bias tends to be higher when respondents talk to researchers directly, so a postal survey is preferable to interviews or a telephone survey where possible.", "Further, it was felt that nonresponse bias may be higher in interviews or telephone surveys due again for reasons pertaining to the ethical questions.", "Of course, the consideration of a trade-off between social desirability concerns and the feasibility of the study had to be made in the case of Hungary.", "However, as the analysis will show, social desirability bias did not emerge as a major concern.", "Response rates were 9.2% in UK, 3.8% in Germany, and 42% in Japan.", "Some of these response rates are lower than many attained in other consumer behavior studies, but surveys comprising older adults tend to be lower than average, in part because some older people are afraid of scams and junk-mail (Sudbury and Simcock, 2010) while survey response rates are declining overall (Tourangeau, 2004).", "Moreover, the survey also contained questions of a personal and ethical nature; thus, low response rates were expected.", "Research shows that surveys with relatively low response rates can be just as accurate as surveys with high response rates (Cook, Heath and Thompson, 2000).", "Reminders were sent out, and comparisons were made of early versus late respondents (i.e., before and after reminder letter).", "Armstrong and Overton's (1977) classic review of methods to measure nonresponse bias include a review of extrapolation methods, which are based on the assumption that respondents who answer later or only after prodding are more like nonrespondents.", "Analysis of the socio-demographic profiles of early (before reminder letters) and later (after reminder) respondents revealed differences in terms of education and work status.", "In common with many postal surveys (Armstrong and Overton, 1977), better educated respondents tended to reply early.", "For this reason, education and work status were not included in any further data analysis.", "Less than 1.5% of all cases contained missing data, which were deleted (Byrne, 2010), resulting in a usable sample of 1278 respondents; details are provided in Table 1.", "Diamantopoulos, Reynolds, and Schlegelmilch (1994) recommend a pre-pilot stage, which involved a review of the original 26-item SRCB scale by 2 British academic colleagues experienced in ethical research but not involved in the current study.", "The appendix provides the original SRCB scale.", "It became clear that the world has changed significantly in the twenty years since Roberts first developed the instrument.", "Consequently, several items referring to discrimination of women and minorities (items 19, 20, and 22) now outlawed, and an outdated item referring to South Africa (item 21), were removed.", "A further item was removed because it describes maven-related behavior (item 11), and another (item 6), although important to ethical/environmental beliefs, did not relate to consumption-related behavior.", "In total, 6 items were removed.", "No additions were made at this stage because it was felt that additions should come from feedback at the full piloting stage rather than from the academics involved in the pre-pilot stage.", "Teams in Germany, Hungary, and Japan then translated and back translated the remaining 20-item scale prior to piloting among respondents in each of the 4 nations.", "Fink (1995) considers 10 population members to be sufficient to trial such an instrument; however, piloting utilized 22 consumers from the UK, 14 from Germany, 25 from Hungary, and 30 from Japan.", "The piloting exercise only stopped when saturation was reached (i.e., no new issues were emerging).", "The debriefing method (Webb, 2002) is an important stage in scale development and modification, and its use in the redesign of the CPS (Current Population Survey) has led to great improvements to the quality of that research instrument (DeMaio, Rothgeb, and Hess, 2002).", "Thus, researchers in each country delivered the questionnaire to respondents and explained it was a pre-testing exercise, and therefore in addition to completing the questionnaire, they were to be critical and note any ambiguities, layout or order issues, or any other improvements they wished to suggest (Fowler, 1995).", "Respondents also noted the length of time taken to complete the questionnaire.", "Personal interviews then took place to debrief respondents.", "The members of each pilot test sample were representative of the final sample and included extreme respondents (Webb, 2002) in terms of age and location because research in Hungary has shown vast differences between rural and urban consumers (Hofmeister Toth and Neulinger, 2011).", "Consequently, each piloting sample included a wide range of ages and in the case of Hungary, comprised people from small towns as well as from Budapest.", "The piloting exercise uncovered a range of important issues, many of which emerged multiple times across different nations.", "Debriefings discovered unnecessary questions, revealed confusion or concern with several existing items, and revealed the need for additional items to better operationalize the concept (DeMaio et al., 2002) of ethical consumer behavior.", "First, item 6 was removed because many participants perceived it as failing to relating directly to actual consumer purchasing behavior, expressing the belief that if they do drive their cars at little as possible, it is for a variety of reasons, including saving money.", "Across all nations, feedback revealed perceptions of too much similarity between several groups of items relating to general recycling (items 5 and 8) and paper products (items 3, 14, 16), so these were merged into two new questions.", "Additionally, a large proportion of respondents wondered why there were questions relating to detergent and aerosols (items 10 and 13) when there is such a wide choice of environmentally friendly products available.", "Thus, reference to these items were dropped on the basis that environmentally friendly products are now widespread and more freely available than in the 1990s when the SRCB scale was first developed.", "The removal of item 23 was due to large numbers of respondents complaining that they do not always know which companies use deceptive advertising.", "Likewise, the removal of item 26 was due to the emergence of much skepticism about charitable donations, with many expressing the feeling that sometimes such donations are to cover up wrongdoing by corporations, while others felt that so many firms claim to help charities now that it is too difficult to be aware of all of them.", "High levels of agreement pertaining to far too much overlap between items relating to conscious choices (items 1, 2, 4, 7, 17) and brand switching (items 10 and 18) led to 7 original statements being merged into 2 new items.", "Feedback also uncovered a range of missing topics.", "Many respondents enquired about pricing issues when asked to select between alternatives; thus, 2 items relating to price were included.", "Clearly, these are important because the piloting exercise reinforced findings from the literature review pertaining to the fact that some consumers are unwilling to pay a price premium for an ethical product.", "At the same time, respondents pointed to an omission regarding other socially irresponsible behavior, and mentioned practices such as child labor and sweatshops, querying if these things came under the remit of general poor working conditions.", "Such practices have received increasing media attention in recent years (Sudbury and Böltner, 2010); thus, an item pertaining to such CSR practices was included.", "Few respondents mentioned local issues or organic produce, although, interestingly, UK consumers also mentioned oppressive regimes, freedom food, and animal testing.", "However, their Japanese counterparts complained that they do not always know which companies are investing in oppressive regimes; they did not understand the concept of freedom food, while they were rather mystified as to why using animals in testing is considered unethical.", "Overall, then, while there was a great deal of consistency across nations, some cultural differences also emerged.", "Noteworthy is the fact that many more respondents mentioned refusing to buy a product based on social irresponsibility (anti-consumption) as opposed to choosing a product produced by a company with a good CSR reputation over alternatives.", "Finally, feedback suggested the need for amendments to some terminology.", "Consequently, environmental damage replaces pollution and environmental replaces ecological.", "These noteworthy changes appear to go beyond mere semantics or language preferences, with respondents viewing the term environmental as more encompassing and ecological as too specific and narrow.", "Both Roberts (1995) and later Webb et al. (2008) note that socially responsible consumption needs continual refinements of its measurement due to its dynamic nature and the fact that consumers knowledge of CSR and environmental issues is changing substantially.", "The resulting short (10-item) scale is shown in Table 2.", "In terms of construct definition, the sheer number of quite severe modifications is perhaps indicative of the ways in which the concept of ethical consumption has evolved over the years since Roberts' work.", "The suggested name for the scale in its current 10-item form is the ethically minded consumer behavior (EMCB) scale in order to differentiate it from the original from which it evolved.", "The scale uses Roberts' original scoring method comprising 1 = never true, 2 = rarely true, 3 = sometimes true, 4 = mostly true, and 5 = always true.", "A higher score is indicative of a greater level of reported ethically minded consumer behavior.", "Bearing in mind Auger and Devinney's (2007) criticism that some scales fail to differentiate between social activists and nonactivists, the instrument contained questions relating to whether or not respondents take action (such as participate in demonstrations, write to an organization, use an Internet forum, or attend events, etc.) about any ethical, environmental, or conservation issue they feel strongly about.", "Additionally, the instrument asked respondents whether or not they (a) are members of any environmental group or cause or (b) regularly donate to any environmental charities.", "Finally, questionnaires also contained the short version (Strahan and Gerbasi, 1972) of the Marlowe–Crowne social desirability scale (Crowne and Marlowe, 1960).", "Across all single items in all 4 nations, responses ranged from a minimum of 1 to a maximum of 5, suggesting that ethical consumers are best viewed as a continuum.", "Clearly, there is a group of consumers in every country that always makes every effort to make a conscious ethical choice, and at the same time, there is a group that never makes a conscious ethical choice.", "In between lay other consumers who sometimes make ethical choices to varying degrees.", "Table 3 presents normative information such as item means, which may be useful to future researchers who wish to use the scale.", "This information is also useful when discussing the issue of measurement invariance across disparate nations, as Table 3 demonstrates some differences between countries.", "The scale measures propensity to demonstrate ethically minded consumer behavior by summing the 10 remaining items, with higher scores indicative of a greater propensity.", "Table 3 also shows the results of reliability analysis using Cronbach's alpha, and all alpha scores are acceptable.", "Moreover, corrected item-total correlations are all above .4 and in most cases even above .6.", "By comparing genders within each sample, known groups validity is tested.", "A body of empirical research suggests females are more likely to engage in EMCB than are their male counterparts (Laroche et al., 2001; Loureiro et al., 2002; Roberts, 1996b).", "As expected, independent t-tests show females score higher than males on the EMCB scale in all countries, although these differences only reached statistical significance in the UK (t = − 5.5, df = 417.5, p < .001) and Germany (t = 2.9, df = 206.3, p < .01).", "A preliminary analysis of construct validity comes from measuring the relationship between the scale and behaviors relating to environmentalism.", "Theoretically, those people who take action (such as take part in a demonstration, write to an organization, use an Internet forum, or attend events, etc.) about any ethical, environmental, or conservation issue that they feel strongly about should demonstrate higher EMCB than do those who have never taken any action.", "This predicted outcome emerges in all countries, and t-tests reveal these differences to be significant in the UK (t = 6.2, df = 401.1, p < .001), Germany (t = 3.4, df = 208, p < .001), and Hungary (t = 2.0, df = 198, p < .05).", "Significant differences in EMCB are also found between members and nonmembers of ethical or environmental/conservation groups or causes (UK: t = 3.8, df = 92.7, p < .001; Germany: t = 3.2, df = 210, p < .001; Japan: t = 3.1, df = 409, p < .01; Hungary: t = 4.0, df = 30.5, p < .001) and between those who regularly donate to environmental or conservation charities and those who do not (UK: t = 6.8, df = 323.1, p < .001; Germany: t = 4.3, df = 208, p < .001; Japan: t = 3.6, df = 410, p < .001; Hungary: t = 2.7, df = 198, p < .01).", "Confirmatory factor analysis, using the UK data and AMOS version 20, checked the dimensionality and factorial validity of the new scale.", "Fig. 1 depicts the hypothesized model.", "The model comprises 5 latent constructs, all of which are based on the literature review and later confirmed during pilot testing.", "Recall that the literature identifies 5 major strands of EMCB:", "The deliberate selection of environmentally friendly products over their less friendly alternatives (cf. Autio et al. 2009), comprising items 1 and 2 and labeled ECOBUY", "Specific recycling issues (cf. Sudbury-Riley, 2014) comprising items 5 and 6 and labeled RECYCLE", "Refusal to purchase a product based on environmental issues (cf. Klein et al. 2004), comprising items 3 and 4 and labeled ECOBOYCOTT", "Refusal to purchase a product based on social issues (cf. Pepper et al., 2009), comprising items 7 and 8 and labeled CSRBOYCOTT", "A willingness to pay more for an ethical product comprising items 9 and 10 and labeled PAYMORE", "Because SEM explicitly takes into account measurement error and hence reliability, it is permissible to have latent constructs, comprising two items (Xie, Bagozzi, & Østli, 2012).", "As Kline (2005) advises, the first step in testing for discriminant validity of a model structure with multiple latent factors is to reject the possibility of a single factor structure.", "For this reason, the model was tested against an alternative with a single factor structure.", "Table 4 details the CFA results.", "The data do not fit the one-dimensional model well.", "In addition to a significant chi-square value (χ2 = 474.8, df = 35, p < .05), the RMSEA value of .167 is outside the guidelines (Browne and Cudeck, 1993; MacCallum, Browne, and Sugawara, 1996) that propose values less than .05 indicate good fit, values ranging from .05 to .08 reflect reasonable fit, values between .08 and .10 indicate mediocre fit, while values greater than .10 reflect poor fit.", "Likewise, the comparative fit index (CFI), the normed fit index (NFI), and the Tucker–Lewis index (TLI), all of which should be close to .95 (Hu and Bentler, 1999), fall well below the cutoff points suggested for these indices.", "Finally, the relative fit index (RFI) and the incremental fit index (IFI) are below the recommended values of close to .95 (Byrne, 2010).", "The AIC and the CAIC are reported in Table 4 in order to compare the subsequent model, with smaller values representing better fit (Hu and Bentler, 1995).", "Clearly, ethically minded consumer behavior is not one-dimensional.", "In contrast, using the guidelines outlined above, all the goodness of fit indices shown in Table 4 suggest the hypothesized 5-factor model to be a good fit, with the AIC and the CAIC figures showing dramatic improvement on the previous models.", "Moreover, the RMSEA closeness of fit (PCLOSE = .813) far exceeds the p-value of at least .50 recommended by Jöreskog and Sörbom (1996), and examination of Hoelter's critical N at both the .05 and .01 CN values were greater than 200 (408 and 480, respectively).", "Examination of the standardized residuals reveals none to exceed the threshold of 2.58 (Jöreskog and Sörbom, 1987); indeed, the highest standardized residual is −.804 between EMCB2 and EMCB6, with other standardized residuals typically falling below .5.", "In sum, the hypothesized 5-factor model fits the UK data well.", "For the scale to be useful in multinational research, measurement equivalence is needed; without evidence of invariance conclusions based on the scale “are at best ambiguous and at worst erroneous” (Steenkamp and Baumgartner, 1998: 78).", "The next goal, then, is to examine the basic meaning and structure of the construct cross-nationally in order to establish whether or not the new scale can be conceptualized in the same way across countries.", "Before moving to analysis of multinational invariance, however, Byrne (2010) recommends testing the model separately in each group as the first step toward multigroup confirmatory factor analysis.", "Table 5 gives the goodness of fit indices for each nation (including the UK data for comparative purposes).", "All samples demonstrate all indices falling within the boundaries outlined above and RMSEA values of .065, .087, and .037 for Germany, Hungary, and Japan, respectively.", "Therefore, the model fit is acceptable for all countries.", "Steenkamp and Baumgartner (1998) contend that multigroup confirmatory factor analysis (CFA) is the most powerful and versatile approach to testing for cross-national invariance in consumer research and offer a sequential testing procedure for doing so.", "Consequently, a multigroup measurement model (based on the hypothesized 5-factor model) is constructed and tested first for configural invariance, which provides a baseline model for comparisons of subsequent tests for invariance.", "Testing the pattern of salient (nonzero) and nonsalient (zero or near zero) loadings defines the structure of the measurement instrument (Steenkamp and Baumgartner, 1998).", "Table 6 shows the results of this and subsequent analyses.", "The fit indices of the configural model (χ2 = 190.7, df = 100, p = .000), RMSEA = .027, CFI = .988, indicate that the model cannot be rejected, which leads to the conclusion that the specification of the items that index the 5 factors of EMCB are configurally invariant for the 4 nations under study.", "Configural invariance does not however mean that the respondents in the 4 different nations are reacting to the scale items in the same way.", "In order to compare item scores meaningfully across nations, and thus have confidence in observed item differences being indicative of cross-national differences in the underlying construct, Steenkamp and Baumgartner (1998) explain that metric invariance is required.", "The results of the metric invariance analysis, when all factor loadings are constrained equal across all 4 groups, are presented in Table 6.", "Despite the fact that metric invariance is often difficult to achieve (Chen and Tang, 2006), based on the chi-square change between the configural and the metric model, it can be seen that evidence of equivalence emerges.", "Indeed, both the “excessively stringent” (Byrne, 2010: 220) test of invariance resulting in a significant value in the change in χ2 (13.2, ∆ df = 15, p = .585) and the ∆ CFI of zero are well below the .01 cutoff point proposed by Cheung and Rensvold (2002).", "This means that the scale is ready to use, with a degree of confidence, in the different countries under study.", "In practice, most researchers focus on the two preceding and most fundamental steps, which are tests of configural and metric invariance (Chen and Tang, 2006).", "Indeed, the scale is now ready for exploring and testing structural relationships, which is the most important application for most researchers.", "Of lesser concern is the need to make direct comparisons of means scores of both the observed and the latent variables across nations.", "There may be some projects, however, where researchers want to compare means and in order to do this the scale needs to exhibit scalar invariance.", "Scalar invariance implies that cross-national differences in the means of the observed items are due to differences in the means of the underlying constructs (Hu and Cheung, 2008; Steenkamp and Baumgartner, 1998).", "As Table 6 demonstrates, the scale does not exhibit scalar invariance; thus, the analysis omits direct comparisons of mean scores.", "Both the change in χ2 and the change in CFI are outside the thresholds that demonstrate equivalence, suggesting that social and/or cultural differences affect the scale.", "Nevertheless, the establishment of configural and metric invariance are sufficient for confident use of the scale in most research projects.", "Indeed, several research studies across different nations used the original SRCB scale without this level of confidence.", "Noteworthy is the fact that the data indicate no departure from normality, as evidenced by no rescaled β2 values exceeding 7 (West, Finch, and Curran, 1995).", "However, there is some suggestion of multivariate kurtosis.", "Consequently, bootstrapping using 2000 bootstrap samples, none of which was unused, reveals only very small differences between the maximum likelihood-based estimates and the bootstrap-based estimates, and the bias is very low.", "Moreover, no confidence intervals include zero.", "Thus, there are no substantial discrepancies between the results of the bootstrap analysis and the original analysis, and the interpretation of the results presented earlier is without fear that departure from multivariate normality has biased the calculation of parameters (Garson, 2012).", "A simple regression analysis, using the scores for the EMCB scale as the outcome variable and the Marlowe–Crowne scores as the predictor variable, reveals the effects of social desirability to be very small, accounting for only 1.2% of the variance in the British sample, 4.3% in the German sample, 6.3% in the Japanese sample, and 8.2% in the Hungarian sample.", "The Hungarian result, although theoretically expected to be higher given the administration method used (Reed, Vidaver-Cohen, and Colwell, 2011), nevertheless suggests that social desirability effects are minimal.", "Finally, convergent validity was tested.", "First, inspection of the factor loadings presented in Table 7 reveals that all exceed the ideal of .7, with the exception of EMCB1 ← ECOBUY for Japan, which is .69 and is therefore well above the minimum of .5.", "Moreover, all factor loadings are positive and significant.", "Additionally, Table 8 presents the average variance extracted (AVE) and the construct reliability (CR) results for each nation.", "All AVEs exceed the cutoff of .5 (Ping, 2004), indicating convergent validity and all CRs exceed .7, again indicating good reliability.", "Taken together, the evidence provides support for the convergent validity of the 5-construct EMCB measurement model.", "Finally, discriminant validity was tested and fulfilled because the square root of AVE of each latent variable exceeds the correlation between this and all other latent variables (Fornell and Larcker, 1981).", "The finding that EMCB comprises 5 distinct factors is a novel and important one.", "Few previous studies investigate the factorial properties of ethical consumption, but interestingly a decade after Roberts (1993, 1995) identified 2 factors (CSR and environmental) Webb et al. (2008) discovered a third factor comprising recycling items.", "The current study adds two further factors to this knowledge, with one dimension pertaining to paying a price premium for ethical products and another relating to boycotting for ethical reasons.", "By identifying propensity to pay a price premium for ethical products, the new scale incorporates an important dimension to ethically minded consumer behavior, which is often cited as one of the explanations for the attitude–behavior gap (Loureiro et al., 2002; McEachern et al., 2010 Sudbury and Böltner, 2010); thus, the inclusion of this important dimension is a strength of the new scale.", "Second, the new scale highlights the fact that refusal to purchase a product or service based on social irresponsibility or the potential to cause environmental damage (boycotting) is distinct from choosing one product over alternatives for ethical reasons.", "Indeed, Carrigan and Atalla (2001) suggest many consumers punish firms for unethical behavior via temporary boycotts or longer term anti-consumption behavior, but fewer will reward ethical behavior by purchasing an ethical brand over alternatives.", "This behavior has come to the forefront in the design of the new EMCB scale.", "A third very important finding is the scale's ability to differentiate statistically between (1) members and nonmembers of an environmental group or cause and (2) those who regularly donate to environmental charities and those who do not.", "This finding not only demonstrates construct validity but also responds to Auger and Devinney's (2007) important question pertaining to how responses to surveys about ethical issues relate to actual behavior.", "This question is of central importance given the attitude–behavior gap.", "While no scale can claim to measure actual behavior, its demonstrable ability to differentiate between these different groups gives confidence to researchers in the ability of the EMCB scale to segment different behavioral clusters.", "Based on a comprehensive analysis of 131 relevant international studies, Diamantopoulos, Schlegelmilch, Sinkovics, and Bohlen (2003) conclude that insufficient tests of dimensionality, reliability, and validity mar many past research studies.", "Robinson, Shaver, and Wrightsman (1991) provide comprehensive criteria for scale evaluation, which, they suggest, should cover three criteria: scale construction, response set, and psychometrics.", "In terms of scale construction, the EMCB scale is based on a review exercise comprising extensive pilot testing using debriefing interviews in 4 different nations, giving it an advantage over scales developed in single nations.", "The simplicity of item wording is also an important feature of the EMCB scale.", "Robinson et al. (1991) note that early instruments comprise question wording that is far more complex and esoteric than contemporary measures, and the extensive piloting and feedback, resulting in elimination, revision, and addition of so many items, suggests the scale is now easy to understand and administer.", "The extensive interviews that took place in each country ensure that the wording and the context, which are so crucial in such scales (Auger and Devinney, 2007), are clear and meaningful to respondents.", "Moreover, the terminology preferred by respondents (for example, the preference for environmental over ecological) is utilized.", "This modernized terminology lends a major advantage to the scale because phrases come in and out of fashion, and to use the terms preferred by respondents when constructing items will ensure clear understanding by future respondents (Robinson et al., 1991).", "Response set occurs due to fatigue and/or noncooperation (Bearden and Netemeyer, 1999), which is likely to increase if the instrument is perceived as dull or unpleasant (DeMaio et al., 2002; Robinson et al., 1991).", "Reduction of the scale from 26 to 10 items provides a parsimonious scale that greatly reduces the chances of boredom.", "Consideration of acquiescence led to different wording of items (e.g., items 4, 7, and 8 refer to behavior avoidance while other items refer to actions that are accomplished).", "No scale can guarantee to measure actual purchase behavior.", "Instead, scales rely on accurate recollection and reporting on the part of the consumer.", "The fact that that scale is able to differentiate between activists and nonactivists suggests that the new EMCB scale does at least overcome some of the limitations found in attitudinal measures.", "Moreover, Nederhof (1985) clarifies that social desirability can originate from either self-deception or other-deception and explains that if, as is the case here, its effects are found to be small, a potential source of bias can actually be considered to be eliminated.", "The first of Robinson et al.'s (1991) psychometric criteria against which to judge a scale is the sample of respondents.", "All the samples used here are random, and samples are from four disparate nations.", "All too often, marketing and consumer behavior scales are developed using college students in the first instance (Bearden and Netemyer, 1999); thus, the choice of an older sample is seen as an advantage that can be justified on three major grounds.", "First, older adults have been shown to be more ethical than their younger counterparts (Pan and Sparkes, 2012).", "Second, older adults are less cynical of ethical purchasing (Carrigan et al., 2004).", "Finally, older adults are more willing to pay the higher price tag that comes with some ethical products (De Pelsmacker et al., 2006).", "Noteworthy is the fact that across every nation and indeed every individual item as well as the EMCB scale as whole, responses ranged from the minimum to the maximum possible scores.", "In other words, the scale is able to differentiate between consumers even using samples that have been shown to be relatively more ethically minded that younger samples.", "From a practical perspective, this finding suggests the scale has potential for segmentation purposes.", "Moreover, the use of a different data collection method in Hungary is an advantage because it is the correct method for that country.", "Based on the original pilot study in Hungary, obdurately adhering to a self-report methodology would likely have produced a host of problems with missing data.", "Cronbach's alpha scores and the corrected item-total correlations reported here are “exemplary” (Robinson et al. 1991: 13).", "Particularly noteworthy are the alpha scores because as Robinson et al. (1991) assert, internal consistency is relatively easy to achieve by including individual items that mean the same thing and writing them in much the same way.", "The fact that the new scale actually merges many of the original items and still achieves these high results across all 4 countries lends support to its internal consistency.", "Further, several measures of validity have also been utilized, all of which give confidence in the new EMCB scale.", "Steenkamp and Baumgartner (1998: 82) note, “When the purpose of the study is to relate the focal construct to other constructs in a nomological net, full or partial metric invariance has to be satisfied.”", "Clearly, the level of measurement invariance required for the purposes of investigating ethical consumer behavior in a variety of disparate nations is established.", "Moreover, further structural equation modeling can use the scale confidently to identify the antecedents of EMCB – in other words, the scale results are now ready for inclusion in a nomological net by future researchers.", "Furthermore, even partial metric invariance allows for valid comparisons of relationships between samples (Lopez, Babin and Chung, 2009).", "Consequently, because results assure full metric invariance, an increase of one unit in the latent variable would have the same meaning for all samples being compared (Freitag and Bauer, 2013); thus, the scale is ready for use across nations.", "The scale does not, however, exhibit scalar invariance, and Finney and Davis (2003) suggest several possible underlying reasons why this may be the case.", "First, there may be group differences in the levels of extreme response styles (ERS).", "ERS is the tendency to use the extreme points on a scale (very high and very low).", "While there was no clear evidence of this when examining the data, the psychological mid-point on a scale may differ across cultures (McDonald, 2000), and there is a tendency for Japanese respondents to give average or noncommittal answers (Dore, 1973), which may explain the smaller standard deviation found in the Japanese sample.", "Second is the possibility of acquiescence response styles (ARS), which is the tendency for one group to systematically give higher or lower responses on a scale than another group (Cheung and Rensvold, 2000).", "The German sample gave systematically higher scores than both British and Hungarian respondents, although this is not the case in comparison to Japanese respondents.", "Whether this is because German consumers are more ethically minded than the others, or whether it is a trait in German response styles, remains an unanswered question.", "Certainly, the standard deviation for the German sample is not as large as for the UK and Hungary.", "One final potential reason pertains to the relevance of the items to the construct.", "One group may endorse an item as much higher than another group because it “is more salient as a marker” (Chan, 2000: 77).", "In other words, social and cultural differences may account for the salience of different items across the samples, thus making scalar invariance impossible.", "What is ethical varies from one society to another (Forsyth, O'Boyle, and McDaniel, 2008), cultural differences affect decision making (Kim and Johnson 2013), and nationality has been found to explain more variation in unethical consumer behavior than have socio-demographic variables (Babakus, Cornwell, Mitchell, and Schlegelmilch, 2004).", "In other words, ethically minded consumer behavior does not happen in a vacuum, it takes place in social and cultural environments governed by complex and different sets laws, policies, rules and regulations, values, and norms (Scholtens and Dam (2007).", "As Schwarz (2003: 588) notes, “any observed difference between … cultures may therefore reflect a meaningful difference in attitudes and behaviors, a difference in the response process, or an unknown mix of both.”", "Robinson et al. (1991) argue that if a scale covers a construct for which instruments are already available, the new scale should demonstrate sound improvements over previous measures.", "The new EMCB scale does indeed exhibit many advantages over alternative self-report measures and therefore fills a gap for such instruments.", "While the current scale does not claim to be able to measure actual behavior in the same way as observation can, it does at least ask questions relating to actual behavior rather than intentions or attitudes, which is a common problem with many currently available scales.", "Further, the EMCB scale has demonstrated its ability to differentiate between people based on their group membership and charitable donations, giving the scale an added advantage of some existing measures.", "Other advantages of the EMBC scale include its brevity, ease of administration, and proven validly across nations.", "All self-report measures do of course suffer with a number of mutual limitations.", "Due to social desirability bias, individuals may lie or exaggerate.", "There may be genuine errors made by some respondents due to memory lapses or inattention to detail (Kolb, 2008), or due to the fact that questions can only ask about purchases in a generalized manner rather than scrutinize each individual purchase situation.", "Specific limitations to the research include the fact that scalar measurement invariance is not established.", "Thus, the new scale may not be suitable for research projects where there is a need to compare mean scores across nations or cultures.", "Future research needs to utilize the scale in the same nations as the ones used here, and in different countries.", "Future research should also attempt to establish scalar invariance.", "Finally, the scale emerged from an existing instrument, rather than the traditional scale development model that would utilize qualitative research as the foundation for the study.", "Ethnography in the form of accompanying people while they make consumption choices, or in-depth interviews pertaining to what is perceived as important from an environmental and ethical perspective, may well have uncovered additional items or issues that the current scale has missed.", "The new scale incorporates choosing a product over alternatives for environmental reasons (eco buy) but is limited in that the only reference to purchasing for CSR reasons is encompassed in the pay more factor: perhaps an explicit item referring solely to choosing on the basis of CSR reasons – as well as boycotting on the basis of environmental reasons – would strengthen the scale.", "If the scale had been developed using solely UK respondents, then it may have incorporated items pertaining to food (organic, freedom foods) and an animal welfare dimension.", "Reference to animal welfare, dropped because Japanese respondents did not understand the need for such a dimension, should perhaps have remained because interesting cultural differences would have emerged – although of course this would have compromised measurement invariance.", "Certainly, there is a need for further research into the societal concern for animal welfare (McEachern et al., 2007).", "Finally, the scale does not incorporate any economic dimensions in terms of buying locally produced goods, which is of great importance from an ethical perspective (McEachern et al., 2010).", "Perhaps if the scale had utilized qualitative research as its foundation and been developed from scratch rather than using an existing scale, these issues would have emerged.", "Conversely, however, it should be noted that studying organic food purchases purely from an ethical perspective is problematic because many people purchase organic food because they perceive it to be healthier, safer, or to taste better (Davies, Titterington, and Cochrane, 1995; Shaw Hughner et al., 2007).", "Clearly, researchers in different nations will still need additional measures in order to encompass a wider range of issues specific to different cultures and countries.", "Hopefully, other important facets to ethical consumer behavior, such as ethical logos and food miles, are incorporated implicitly in the scale, although perhaps explicit and direct items pertaining to such dimensions would further strengthen it.", "Nevertheless, previous studies have already acknowledged that ethical consumption needs continual refinements due to its dynamic nature (Roberts, 1995; Webb et al., 2008) and increasing importance (Ethical Consumerism Report, 2011), and this study has taken steps to refine and modernize ethically minded consumer behavior.", "Future research should utilize a qualitative phase of research in order to ensure that any future improvements or iterations of the scale are as comprehensive as possible.", "These limitations notwithstanding, the new scale does fill an important gap in that it provides future researchers with a measurement instrument ready to use in a variety of nations and in studies with a variety of research objectives, including the modeling of complex relationships among variables.", "Previously, researchers needed to use a variety of different scales to cover different facets of ethical consumption or to adapt existing scales such as the SRCB scale, which was no longer fully applicable in today's environment.", "The new EMCB scale is valid, reliable, easy to understand, and easy to administer.", "The scale is contemporary, and it views ethical consumption from a wide perspective.", "The choices of nations and the demographic of the samples therein are also strengths of the study: all too often scales development comprises young (often student and often US) samples.", "The scale demonstrated the maximum possible ranges across all nations; thus, it is assumed that as it can differentiate between consumers drawn from “the ranks of the ethically motivated” (Szmigin et al., 2003: 548).", "There is no obvious reason why the scale is not suitable for use with younger consumers, although further testing of the scale to ensure its use is appropriate with all ages is a recommendation for future research.", "Certainly, the wide range of scores found within every nation suggests that further investigation of the scale in order to identify its usefulness for segmentation purposes could be a productive research avenue.", "Moreover, studies often use scales developed in a different country or culture without checking that the measure is equivalent.", "This paper has demonstrated that the EMCB scale can be used with confidence across a variety of nations and cultures, providing future researchers and practitioners with a scale that measures the degree to which an individual perceives themselves as ethically minded when making consumption choices.", "Of course, as is the case with any consumer behavior scale, the instrument relies on the ability of respondents recollect their behavior and to tell the truth about that behavior.", "This caveat notwithstanding, the scale has advantages over many alternatives because it does at least ask questions about actual purchasing habits as opposed to attitudes and intentions, which is important given that ethical intentions rarely translate into actual purchasing behavior (Carrington et al., 2014).", "Looking ahead, ethical consumption may have the potential to become a mass-market phenomenon (Devinney et al., 2010).", "If a propensity toward ethically minded consumer behavior is to be considered a major asset for marketing and indeed for a society, it becomes absolutely necessary to have a valid measure for it.", "The EMCB provides researchers and practitioners from diverse countries with such a measure." ]
[]
Cyclic-AMP and bacterial cyclic-AMP receptor proteins revisited: Adaptation for different ecological niches
Escherichia coli cyclic-AMP receptor protein (CRP) represents one of the paradigms of bacterial gene regulation. Yet despite decades of intensive study, new information continues to emerge that prompts reassessment of this classic regulatory system. Moreover, in recent years CRPs from several other bacterial species have been characterized, allowing the general applicability of the CRP paradigm to be tested. Here the properties of the E. coli, Mycobacterium tuberculosis and Pseudomonas putida CRPs are considered in the context of the ecological niches occupied by these bacteria. It appears that the cyclic-AMP-CRP regulatory system has been adapted to respond to distinct external and internal inputs across a broad sensitivity range that is, at least in part, determined by bacterial lifestyles. © 2014 The Authors.
[ "IMMU", "MEDI" ]
[]
[ "2404", "2725", "2726" ]
[ "The cyclic-AMP receptor protein (CRP) and its effector cyclic-AMP (cAMP) were discovered in E. coli during investigations to explain the phenomenon of diauxic growth more than 40 years ago.", "Since then, E. coli CRP–cAMP has become a paradigm of gene regulation, providing insights into signal perception and transduction, DNA recognition by regulatory proteins, regulator–polymerase interactions and promoter architecture [1].", "The formation of the second messenger cAMP from ATP is catalyzed by a group of enzymes known as adenylyl cyclases.", "These enzymes are classified into six groups based on their primary structures.", "E. coli possesses a single Class I adenylyl cyclase (Cya) whose activity is controlled by glucose availability, such that growth at micromolar concentrations of glucose increases intracellular cAMP concentrations ∼10-fold (∼20 μm to ∼180 μm) compared to excess glucose conditions [2].", "The consensus view has been that when the preferred carbon source glucose is available, it is transported into the cell by the glucose phosphotransferase system (PTS) and glucose enters the cytoplasm as glucose-6-phosphate [3,4].", "The phosphorylation state of the PTS thus acts as a reporter of glucose availability — the phosphorylation state of the PTS is lower when the glucose transporter is saturated, whereas when glucose is absent, phosphorylated PTS proteins accumulate.", "The phosphorylated PTS interacts with Cya to enhance adenylyl cyclase activity [3].", "Thus, when the bacteria are glucose-starved, the intracellular cAMP concentration increases as a consequence of the altered phosphorylation state of the PTS; however this is difficult to reconcile with observations that the glucose PTS is still saturated when intracellular cAMP concentrations increase [2].", "Consequently, it has been suggested that cAMP concentrations increase in response to low energy charge, such that when ATP is low, cAMP is high, promoting catabolism and inhibiting anabolism by CRP–cAMP-mediated gene regulation to bridge the perceived energy deficit [5].", "Degradation of cAMP is mediated by a phosphodiesterase, CpdA, but this enzyme has a rather high Km for cAMP (∼500 μm) relative to the concentration of cAMP in the cell, and a cpdA mutant exhibited only a twofold increase in intracellular cAMP concentration [6].", "Consequently, the observation that cAMP is often found extracellularly (0.03–0.5 μm) led to the finding that E. coli can quench intracellular levels of cAMP by TolC-mediated efflux, although the cAMP transporter(s) that links to the outer-membrane pore TolC has not yet been identified [7•].", "Changes in intracellular cAMP concentration are perceived by the transcription factor CRP.", "CRP is a homodimer in which each subunit possesses three major structural features (Figure 1).", "The N-terminal region houses the high-affinity cAMP-binding domain and the C-terminal region consists of a DNA-binding domain with a canonical helix-turn-helix motif.", "These two domains are connected by a long helix (C-helix) that forms a coiled-coil at the dimer interface and a short linker followed by another helix (D-helix) (Figure 1).", "Cyclic-AMP binding to the sensory domain is initially exothermic (ΔH1 = −16.3 kJ mol−1; ΔS1 = 41 J K−1 mol−1) followed by an endothermic phase (ΔH2 = 25.1 kJ mol−1; ΔS2 = 176 J K−1 mol−1) and cAMP interactions with the two protomers that make up the CRP dimer are cooperative (ΔG2 − ΔG1 = 2.7 kJ mol−1) with binding constants of 8 × 104 m−1 for site 1 and 6 × 104 m−1 for site 2 [8•].", "In the apo-CRP dimer, the two DNA-binding domains interact to form a rigid body in which the DNA-recognition helices are buried such that they cannot interact with DNA [9].", "Binding of cAMP to CRP initiates structural rearrangements about a ‘hinge’ region allowing the DNA-binding domains to relocate relative to the cAMP-binding domain in a process mediated through hydrogen bonds between the N(6) position of adenosine with Ser-128 of the dimerization helices (C-helices; Figure 1) [9,10].", "This allosteric conversion critically involves extension of the C-helices by six residues and shortening of the D-helices by four residues, such that Asp138 becomes the N-terminal capping residue of the D-helix in CRP–cAMP (Figure 1), but is an internal part of the longer D-helix in apo-CRP (Figure 2a and b).", "The helix-capping propensity of residue 138 is correlated to the degree of co-operative cAMP-binding and hence this property of Asp138 is a key feature of the interdomain conformational changes that modulate the apo-CRP ↔ CRP–cAMP equilibrium [8•].", "NMR spectroscopy and thermodynamic analyses of several CRP variants revealed how changes in conformational entropy modulate DNA-binding activity [11••].", "These structural rearrangements expose the DNA-recognition helices (highlighted in green in Figure 1) such that they are able to participate in sequence specific (consensus sequence TGTGAnnnnnnTCACA) binding at two adjacent major grooves of DNA (Figure 2b) [12].", "Cyclic-AMP binding has a biphasic effect on site-specific DNA-binding by CRP.", "High-affinity binding of cAMP in the anti-conformation at the sensory domains of the CRP subunits (1:1 cAMP/CRP protomer) enhances DNA-binding ∼1000-fold.", "This is followed by decreased DNA-binding when cAMP in the syn-conformation interacts with weak binding sites formed by components of the helix-turn-helix, a β-hairpin from the regulatory domain and DNA (2:1 cAMP/CRP protomer) [13].", "However, proof that cAMP-binding to these low affinity sites is of physiological significance has not yet been provided.", "CRP–cAMP binding to intergenic CRP sites is associated with classical gene regulation [14,15•]; however, it is now recognized that CRP–cAMP also binds at many intragenic sites where it is thought to fulfill a role as a chromosome organizer or nucleoid associated protein (NAP) [14].", "Genome SELEX screening identified at least 254 CRP-binding sites across the E. coli genome and because CRP is capable of controlling expression of divergent promoters from a single binding site it is estimated that CRP–cAMP directly controls a minimum of 378 promoters, and perhaps >500 genes in E. coli [15•].", "Amongst these operons, CRP–cAMP acts as the ‘master’ regulator for 70 ‘slave’ transcription factors further expanding the profound influence of CRP on global gene expression in E. coli, in which it plays a key role in managing catabolism, including the transport of substrates, glycolysis, the Krebs cycle and aerobic respiration [15•,16].", "Unlike E. coli, which has only one adenylyl cyclase, M. tuberculosis H37Rv possesses at least 16 Class III adenylyl cyclase-like proteins, including soluble and membrane-associated multidomain proteins, suggesting that their catalytic activities (10 of the 16 have been shown to act as adenylyl cyclases) can be regulated by extracellular and/or intracellular signals, reviewed by Chakraborti [17].", "Accordingly, adenylyl cyclase activity of M. tuberculosis is affected by pH, CO2, and fatty acids.", "It has long been recognized that mycobacteria secrete cAMP, but it is only more recently that the capacity to intoxicate macrophages with cAMP has been recognized as a contributor to virulence [18,19].", "Thus, the synthesis (in particular by Rv0386) and secretion of cAMP are central features of M. tuberculosis pathogenesis and result in the bacterium being exposed to relatively high concentrations of cAMP; there are reports of intracellular concentration of cAMP as high as 4 mm for M. tuberculosis H37Rv and ∼3 mm for Mycobacterium smegmatis, which far exceed values reported for E. coli [20,21].", "However, it is wise to offer a note of caution here; because the different growth conditions and methods used to measure cAMP, it is difficult to make direct comparisons.", "Nevertheless, the important role that cAMP plays in tuberculosis pathogenesis exposes the need for careful investigation of both intracellular and extracellular cAMP concentrations using modern approaches.", "The relatively high concentrations of cAMP reported for mycobacteria are consistent with the presence of multiple adenylyl cyclases but of only one cAMP phosphodiesterase (Rv0805) in M. tuberculosis H37Rv.", "Moreover, the cAMP phosphodiesterase activity of Rv0805 is poor, and like its E. coli counterpart, it has a relatively high Km for cAMP (∼150 μm) [22].", "This rather poor in vitro activity is reflected in vivo, where overproduction of Rv0805 resulted in only a ∼30% decrease in cAMP (a ∼90% decrease was observed for overproduction of CpdA in E. coli), perhaps indicating alternative roles for this enzyme, which also possesses the ability to hydrolyze a range of cNMP and linear phosphodiesters [23•].", "In the light of these data, it has been suggested that intracellular cAMP levels might be controlled by excretion rather than conversion to AMP but, as is the case for E. coli, there is a need to establish the mechanism(s) of cAMP secretion and how this might be regulated.", "The M. tuberculosis CRP (Rv3676; 32% amino acid identity to E. coli CRP over 189 amino acids, including four of the six key cAMP-interacting residues in the sensory domain of E. coli CRP; Table 1) differs from the E. coli paradigm at several levels.", "The Rv3676 homodimer exhibits relatively weak (Kb = 1.7 × 104 m−1) binding of cAMP to two independent sites (1:1 cAMP/protomer).", "Furthermore, cAMP-binding is endothermic (ΔH = 30.7 kJ mol−1; ΔS = 183 J K−1 mol−1; ΔG = −23.7 kJ mol−1) and thus binding is entropically driven [24].", "The independent nature of cAMP-binding to Rv3676 compared to E. coli CRP was accounted for by the replacement of a single amino acid residue (Ser-128 of CRP, which is required for the dramatic conformational changes that occur upon cAMP-binding, is replaced by Asn in Rv3676) that has the effect of reorganizing a hydrogen-bonding network involving cAMP such that the cAMP-binding sites in Rv3676 are uncoupled [24].", "It has been argued that the relatively weak and independent binding of cAMP at the sensory domain of Rv3676 has evolved to allow the protein to be at least partially cAMP-responsive against the background of high cAMP concentrations required to intoxicate the host during infection.", "The crystal structures of apo-Rv3676 and Rv3676-cAMP reveal that cAMP-binding is associated with much less dramatic structural rearrangements than those observed for E. coli CRP [25–27] (Figure 2c and d).", "The major alteration that occurs upon cAMP-binding is weakening of the interactions between the DNA-binding and sensory domains, resulting in increased spatial freedom of the DNA-binding domain that is apparently sufficient to permit binding to target DNA sequences by an induced fit mechanism [27].", "Consistent with the relatively mild structural rearrangements that occur upon cAMP-binding by Rv3676, the formation of the Rv3676–cAMP complex has a relatively small effect (∼2-fold) on DNA-binding to a consensus sequence that is very similar to that of E. coli (GTGnnAnnnnnCACA) [28].", "Furthermore, unlike E. coli CRP, apo-Rv3676 is capable of site-specific DNA-binding and transcription regulation [24].", "These observations are consistent with the limited overlap between genes dysregulated in the crp mutant and those affected by Rv0805 overproduction [23•] and suggests that the primary role of Rv0805 might not be to act as a cAMP phosphodiesterase and/or that Rv3676 can significantly influence gene expression without the need to bind cAMP.", "Like E. coli CRP, M. tuberculosis Rv3676 is a global regulator but, perhaps unsurprisingly in the context of the very different lifestyles of these two bacteria, the corresponding CRP regulons differ.", "Thus Rv3676 appears to be involved in regulating the transition between replicating and nonreplicating states by exerting influence over virulence-critical functions, including phthiocerol dimycocerosate (DIM) synthesis, resuscitation promoting factors, the ESX-1 type VII secretion system, carbon metabolism, energy conservation and ‘slave’ transcription factors, such as the nitric oxide-responsive regulator WhiB1 [28,29].", "This degree of control over the transcriptome is consistent with the attenuated state of the M. tuberculosis crp mutant in models of infection [28].", "P. putida possesses a CyaA-type adenylyl cyclase capable of cAMP synthesis (and there is also a second protein PP_5187 annotated as an adenylyl cyclase), but nevertheless cAMP concentrations are below the level of detection in bioassays [30•].", "P. putida KT2440 possesses a cAMP phosphodiesterase (PP_4917) equivalent to the E. coli CpdA protein.", "Thus, the very low levels of cAMP in P. putida could arise from poor synthesis or rapid degradation, but based on complementation experiments with E. coli cya mutants the former is the more likely.", "Thus, P. putida seems to represent the opposite end of the ‘cAMP spectrum’ to M. tuberculosis.", "The P. putida CRP is 63% amino acid identical to E. coli CRP over 208 amino acids, including five out of the six cAMP-interacting residues in the sensory domain of E. coli CRP — interestingly the mismatch is again located at the position equivalent to 128 in E. coli CRP (Table 1).", "Consistent with the very low concentrations of cAMP, the P. putida CRP exhibits very high affinity (Kb = 4.4 × 107 m−1) binding of cAMP to two independent sites (1:1 cAMP/protomer).", "Furthermore, cAMP-binding is exothermic (ΔH = −25 kJ mol−1; ΔS = 63 J K−1 mol−1; ΔG = −10.5 kJ mol−1), and binding is both enthalpy and entropy driven [31••].", "Although detailed structural information is not yet available, this hypersensitive binding of cAMP invokes large conformational changes that can be detected by size exclusion chromatography and result in enhanced DNA-binding to a typical CRP inverted repeat sequence by >10-fold [31••].", "Although P. putida exhibits catabolite repression, this behavior is not mediated by CRP–cAMP, as a crp mutant was unaffected in its ability to utilize a full range of sugars as carbon sources [32].", "Rather, CRP–cAMP appears to control the utilization of l-phenylalanine and of various dipeptides as nitrogen sources in P. putida [30•,33].", "The full range of genes regulated by CRP in P. putida has not been established but a conservative analysis of the genome sequence for likely binding sites indicates that >30 genes might be regulatory targets, few of which appear to have a metabolic role [30•].", "These observations led to the suggestion that P. putida and M. tuberculosis CRPs have evolved to control different biological processes compared to the E. coli paradigm, a possible example of regulatory exaptation [30•].", "In recent years many aspects of the cAMP-signaling CRP-regulatory paradigm that has emerged from intensive studies of catabolite repression in E. coli have come under closer scrutiny.", "Almost every step from the relationship between the activity of the glucose PTS and cAMP synthesis to the role of cAMP–CRP in gene regulation and chromosome organization has been reassessed.", "The picture that is emerging is one in which intracellular and extracellular cAMP concentrations are modulated by adenylyl cyclases, phosphodiesterases and cAMP efflux systems some of which respond to external and/or internal signals.", "Changes in intracellular cAMP concentration are perceived by CRP proteins that react with different sensitivities related to the niches occupied by the bacteria.", "Thus, the pathogen M. tuberculosis Rv3676 is a low sensitivity CRP evolved to maintain some degree of responsiveness at the high cAMP concentrations used to intoxicate host macrophages; the commensal enteric bacterium E. coli possesses a mid-sensitivity CRP to regulate catabolite repression and chromosome structure, probably in response to energy charge; and the soil bacterium P. putida has a hypersensitive CRP, reflecting the very low concentrations of cAMP produced by this bacterium.", "Nevertheless, despite decades of study there are still many outstanding questions that need to be addressed to complete our understanding of cAMP-signaling in E. coli and other bacteria.", "For example, at the root of cAMP-mediated signaling in bacteria is the ability to synthesize and degrade cAMP in response to environmental and metabolic signals.", "To place investigations of cAMP-signaling on a sound footing there is a need to apply the latest metabolite quenching, extraction and analysis techniques to accurately measure intracellular and extracellular cAMP concentrations under diverse growth conditions.", "Further characterization of the phosphodiesterases involved in cAMP degradation and the processes required for cAMP excretion is required.", "Signal-dependent synthesis of cAMP is only one component in controlling bacterial responses to this second messenger; there have to be mechanisms for removing cAMP from the system.", "It appears that the cAMP phosphodiesterases are rather poor enzymes, leading to the suggestion that secretion of cAMP might be the major route to lowering cAMP in the cell.", "However, thus far no cAMP efflux systems have been identified beyond the recognition that TolC is involved in facilitating cAMP crossing the outer membrane of E. coli.", "Identifying cAMP secretion systems and defining their role in bacterial signaling and pathogenesis will fill a major deficit in our current knowledge.", "It will be informative to seek physiological and evolutionary explanations for the differences in cAMP-binding to the regulatory CRP proteins in different bacteria, in particular, establishing whether CRPs, like that of M. tuberculosis, which exhibits only a mild enhancement in DNA-binding in response to cAMP, control cAMP-independent and cAMP-dependent regulons.", "Similarly, mechanistic explanations for the extremely avid cAMP-binding by CRPs, as exemplified by the P. putida CRP, should be sought.", "At present it appears that responding to the second messenger cAMP allows CRP to be co-opted to control different regulons in bacteria that occupy distinct niches.", "Thus, because cAMP intoxication of the host is an important component of M. tuberculosis pathogenicity, its CRP has become desensitized to cAMP, whereas the CRP of the soil bacterium P. putida has become hypersensitive to cAMP.", "Hence through a common mechanism of CRP-mediated RNA polymerase recruitment and signal-dependent cAMP synthesis/degradation (e.g. glucose availability in E. coli; pH and other virulence-related signals in M. tuberculosis; unknown signals possibly related to the utilization of aromatic amino acids and nitrogen sources by P. putida), CRP has been co-opted to control distinct regulons according to the particular niches occupied by the bacteria.", "Developing a mechanistic framework that accounts for the shifts in cAMP-binding affinities observed in different CRPs would then allow questions about the physiological roles of cAMP–CRP complexes with alternative stoichiometries to be addressed.", "Papers of particular interest, published within the period of review, have been highlighted as:", "• of special interest", "•• of outstanding interest" ]
[ "E. coli cyclic-AMP receptor protein (CRP) is a paradigm of gene regulation.", "Comparison of CRPs reveals differences in their affinity of cAMP.", "A range of dependency on cAMP for DNA-binding exists.", "CRPs have adapted to function in the specific niches occupied by the bacteria." ]
Emerging perceptions of teacher quality and teacher development in China
This paper focuses on the work of senior high school teachers in three illustrative local authority regions of mainland China. It discusses interview and focus group data collected as part of ESRC/DfID-funded research which examined notions of quality as experienced by key stakeholders (national and local authority policy makers, teachers, head teachers and students). Building on previous international literature and current Chinese education policy, this paper examines aspects of teachers' work as experienced within the context of a fast developing emerging economy, which emphasises a clear link between individual and national development. Barriers identified as impacting on the provision of good quality teaching arose, largely, from the pressures due to changing societal patterns and the demands of far reaching curriculum reform, which highlighted tensions between a traditional reliance on the primacy of exam results and a newer demand for all round development and lifelong learning. In addition, there were common concerns with various structural and funding inequalities, both across different regions and between urban and rural schools, which could lead to differential student experience, shortages of specialised teachers, and a lack of opportunity for good quality professional development. © 2013 The Authors.
[ "SOCI" ]
[ "Chinese schooling", "Educational administration", "Educational policy", "Equity", "Teacher development", "Teacher quality" ]
[ "3303", "3304", "3312" ]
[ "The search for evidence to develop high quality, sustainable education systems continues to intensify as national economies seek to compete globally.", "The opening Foreword to the findings of a recent Teaching and Learning Survey (TALIS), which reported on the views of teachers and principals in 23 countries, explained the task in the following way:", "The challenges facing education systems and teachers continue to intensify.", "In modern knowledge-based economies, where the demand for high-level skills will continue to grow substantially, the task in many countries is to transform traditional models of schooling, which have been effective at distinguishing those who are more academically talented from those who are less so, into customised learning systems that identify and develop the talents of all students.", "This will require the creation of ‘knowledge-rich’, evidence-based education systems, in which school leaders and teachers act as a professional community with the authority to act, the necessary information to do so wisely, and the access to effective support systems to assist them in implementing change.", "(OECD, 2009, p. 3)", "Such concerns are not limited to economically developed economies but are also the focus of attention in those countries where economic development is less advanced, together with those newly emerging economies such as mainland China.", "International agreements, like the Millennium Development Goals (United Nations, 2002) incorporating the principles of relevance, equity and rights, also focus on the need to improve the quality of teaching and learning for all children (UNESCO, 2004).", "However, concepts of quality are recognised to be both complex and contested, especially in relation to disadvantaged communities.", "Previous work has drawn attention to the powerful discourses of human capital theory and a contrasting human rights approach, arguing for the importance of inclusivity (with regard to access and the achievement of desired outcomes for all), relevance (in relation to what is important for different individuals, communities and nations) and democracy (in terms of who can define what constitutes quality) in education (Tikly, 2011; Tikly and Barrett, 2011).", "Central to such debates are questions about what constitutes good quality teaching, and how to evaluate and develop the teaching workforce (Anderson, 2004; Leu and Price-Rom, 2006; Mpokosa and Ndaruhutse, 2008).", "This paper seeks to contribute to the debate on teacher quality and development by drawing on selected findings from research conducted in collaboration with the China National Institute for Educational Research (now the National Institute of Education Sciences) in Beijing.", "While findings from the main studies have been extensively reported elsewhere (Thomas and Peng, 2011; Thomas et al., 2012, 2013), the purpose of this paper is discuss some relevant findings which help to contribute to debate on the work of teachers and their ability to provide conditions for good quality learning.", "Following a short review of both Western and Chinese literature on aspects of teacher quality and effectiveness, and a discussion of the current policy context in China, a range of findings will be used to illustrate and discuss common stakeholder expectations, as well as teacher values, beliefs and practice.", "Concerns regarding barriers to further development are also highlighted and these are discussed in relation to current Chinese policy to redress disparities in educational provision, especially between urban and rural settings.", "Over the last 30 years, Western research has consistently identified differences in teacher behaviour at the classroom level, rather than differences at the school level, as ultimately more important in explaining variance in student outcomes (Scheerens and Bosker, 1997; Kyriakides et al., 2000; Muijs and Reynolds, 2010).", "For this reason, there is a growing body of literature which seeks to identify the dimensions of teachers’ work that might be identified as being more likely to produce the desired outcomes for pupils.", "While limitations of space preclude a detailed summary such a large body of research, much of this work identifies three broad areas which have been the focus of more detailed study.", "For example, a recent survey of schools and teachers within the OECD (TALIS) focuses on teacher professional competence and related beliefs and attitudes; teacher classroom practice and professional activities; and classroom and school level environment (OECD, 2009).", "This has similarities with the three domains of teacher background characteristics, teaching processes, and classroom ecology and climate identified as teaching variables by Scheerens (2007, cited in OECD, 2010, p. 30) and the professional characteristics, teaching skills and classroom climate identified by Hay McBer (2000).", "In a recent review of the literature discussing teaching skills and classroom practice, Creemers and Kyriakides (2012) draw on their own work and that of others (for example, Stallings, 1985; Brophy and Good, 1986; Creemers, 1994; Doyle, 1986; Galton, 1987; Muijs and Reynolds, 2000; Creemers and Reezigt, 1996; Creemers and Kyriakides, 2008) to explain those micro-behaviours which research has shown to be positively associated with student achievement.", "These include the quantity and pacing of instruction, providing work at an appropriate level for students to succeed, the smooth organisation and management of the classroom environment, systematic evaluation and reflective inquiry to improve practice, clarity of presentation and good communication with students, as well as the judicious use of asking questions and giving feedback to gauge understanding.", "Moreover, development of pedagogical content knowledge has also been identified as a dominant strand in effective teaching skills in TALIS survey and this is closely linked with the collaborative activities that teachers engage in beyond their individual classrooms.", "Research has also shown the importance of the classroom environment to good quality learning.", "The quality of teacher–student interaction, student–student interaction, students’ treatment by the teacher, competition between students, and classroom disorder has all been identified as important (Creemers and Kyriakides, 2008; Kyriakides and Christoforou, 2011).", "For Scheerens, classroom ecology and climate also includes elements such as class size and composition, teacher expectation and the match of teachers’ abilities with classes.", "For the TALIS framework it is closely linked to the school level environment and teacher–student relations.", "While for Hay McBer this category is described as ‘the collective perceptions by pupils, of what it feels like to be a pupil in any particular teacher's classroom, where those perceptions influence every student's motivation to learn and perform to the best of his or her ability’ (Hay McBer, 2000, p. 27).", "Sammons and Ko (2008) found similar evidence that effective teachers are more likely to demonstrate a supportive lesson climate, proactive lesson management, well organised lessons with clear objectives, and environmental and teacher support.", "There are also links to other research which has looked at the perceptions of students in relation to effective teachers (for example Rudduck and Flutter, 2000; McNess, 2006; Klieme, 2012).", "McNess and colleagues found that students in England, France and Denmark all identified being fair, explaining things well, and making work interesting as the three most important aspects of effective teachers.", "The more generalised area of teacher beliefs and values has also been the focus of much research (Lortie, 1975; Nias, 1989; Menter et al., 1997; Acker, 1999; Hargreaves, 2001; Day et al., 2007).", "Such work emphasises the importance of the social, historical, cultural and policy context in which teachers work (Broadfoot et al., 1993; Alexander, 2000; Osborn et al., 2003; McNess, 2004, 2009), and draws attention to the need to look behind the more obvious aspects of teacher practice to seek an understanding of the underlying beliefs and values of society in defining policy and the working environment of teachers.", "This argues against a universal or fixed concept of teacher effectiveness, or what Campbell et al. (2003, p. 352) have referred to as a ‘platonic ideal free of contextual realities’.", "This idea is taken further by some who call for an extension of the levels of analysis to include everything from the supranational to the individual (Bray and Thomas, 1995).", "Creemers and Kyriakides (2012) propose a ‘Dynamic Model of Educational Effectiveness’ which takes into account student, classroom, school and system, while Cheng et al. (2002) put forward a ‘Framework of Total Teacher Effectiveness’ where levels of effectiveness (individual, group, school) are linked to domains of effectiveness (behavioural, affective, cognitive).", "Such models are helpful in identifying possible areas of effectiveness though, it can be argued, that none can capture the full complexity of teaching in all contexts.", "The qualitative data presented later in this paper do not claim to cover all the aspects of teachers’ work identified above, nor are such models systematically applied.", "The authors recognise the limited nature of the data in both scale and scope, and so the intention is to reveal some common understandings and concerns that arose spontaneously in the interviews.", "But to help in contextualising these perceptions it is first necessary to discuss both current Chinese literature on the quality of teaching, and set this in the current Chinese policy context.", "Generally speaking, within the Chinese context, the term ‘teacher quality’ is used more broadly than in Western literature.", "It is used interchangeably with the concept of education quality but recognises the central role of teachers, where poor quality has been associated with less than adequate outcomes (Guan, 2004).", "More specifically, Li and Liu (1999) consider that teachers and students must be considered together as the two essential contributors to the learning process, and argue that, fundamental to education quality, are the personal attributes that they bring to the learning situation.", "These include physical attributes, knowledge, skills, cognitive ability, feelings and emotions, as well as underlying values.", "This links with Western ideas of teacher values and beliefs which have particular resonance in a Confucian heritage context where the teacher has traditionally been regarded as the keeper of moral values.", "Confucian tradition puts great emphasis on the morality of teachers and this continues to be an important aspect of teacher quality, in the form of teacher professional ethics (Ren, 2009; Jin, 2010; Tang, 2010).", "The most recent educational development plan within China emphasises the promotion of professional ethics among teachers to such an extent that it concludes that, ‘moral conduct shall become the foremost factor in testing, appointing, employing and evaluating teachers’ (Chinese Ministry of Education, 2010, p. 36).", "Much research on effective teaching, within mainland China, is relatively new and has been prompted, largely, by the new curriculum reform initiated in 2001.", "This reform essentially signals a shift in government policy towards strongly promoting new teaching methods and more active learner centred-approaches, in order to develop what is referred to as students’ all round development or sù zhì jiào yù.", "This term encompasses their moral, intellectual, physical and aesthetic education, and is sometimes translated as ‘quality’ education.", "This idea is also linked to the broader need to develop active citizens as the foundation for a prosperous society able to sustain the economic growth required of the new China (CCCCP, 1985; Xia and Song, 2009; Wang, 2011; Wu, 2009).", "Hence not only are the values and attitudes of the people (teachers, school leaders, curriculum designers, pupils and parents) important, but also an enabling learning environment (Li and Liu, 1999; Xia and Song, 2009).", "Such developments have led to a series of curriculum reforms which have brought ongoing tensions within the management of education (Dello-Iacovo, 2009).", "Gu (2011) argues that in order for teachers to successfully address this requirement, they themselves need to be practical, serious, diligent, rigorous, responsible, and demonstrate a high moral and ethical code so as to act as role models for their students.", "Some research evidence reveals a concern with a perceived reduction in teacher morals and professional ethics, and calls for a renewal of this through the efforts of both teachers and society (Jin, 2010; Guo, 2011).", "Yu (2008) argues that being aware of their responsibilities and pursuing successful outcomes for themselves and their pupils should serve as the critical driving force for teachers to undertake self-reflection on action to improve their teaching.", "Rather than other types of occupations, Nie (2007) argues that teachers should regard themselves as professionals, similar to doctors, lawyers and engineers.", "Much thought has also been given to why, despite the hard work of teachers, some students still find it difficult to learn, and how best to meet the challenges brought about by the recent curriculum reforms in an information rich and globalising world (Lu, 2009).", "More recent evidence (Jiang and Yao, 2012; Zhuang, 2012) argues that effective teaching is a process which enables students to both want to, and know how to, learn and employs question-driven instruction and activity-based learning to guide students in learning how to think.", "More importantly, it is argued that it is necessary to leave space within class time to encourage students to discover problems for themselves and to find their own personal meaning in knowledge, so that they can be the masters of their own learning.", "This links to previous research in the USA (Dweck and Leggett, 1988) and newer Western concepts of the teacher as facilitator.", "Tu and Pan (2011) meanwhile draw attention to the dimensions of efficiency in relation to time and results, in the context of large classes.", "There is also survey evidence indicating that, from the teacher's point of view, effective teaching consists of three major stages: (i) pre-lesson preparation where teachers think through the details of a lesson plan, identify teaching objectives and materials, and predicting difficult learning points, (ii) in-lesson teaching, which involves creating contexts for students to experience the exploration of new knowledge and thinking activity, adopting multiple approaches, including the use of ICT, to accommodate all students’ learning styles, while monitoring students’ learning, and (iii) post-lesson activity, which includes teachers engaging in self reflection on how the lesson went, and providing homework feedback to students (Huang, 2011).", "Another strong theme within Chinese research relating to quality is the concern with issues of inequality of educational access and provision (Fang and Feng, 2005; Zhu and Zhou, 2006; Liu, 2004; Yu, 2010).", "Despite the 1986 Compulsory Education Law which requires all six-year-olds to enrol in nine years of compulsory education, regardless of gender, ethnicity or race, significant inequalities remain.", "State Guidelines (Chinese Ministry of Education, 2010) maintain that the government's aim is to achieve education equity through the preferential allocation of resources to rural, remote and deprived ethnic minority areas, thereby narrowing the education gap.", "The guidelines set targets for achieving 95% retention rates for compulsory school children, and 90% and 40% gross enrolment rates for senior high and higher education (including vocational education), respectively, by 2020.", "Since the opening up of the Chinese state, there is an increasing focus on international responsibilities and some evidence that mainland China is influenced by a responsibility to meet UNESCO's initiative requiring education for all (Jing and Hu, 2007).", "There is also some evidence that policy is being influenced by international tests such as the OECD Programme for International Student Assessment.", "This has brought concerns with the traditional school test and entrance examination formats which have been criticised for being highly subject knowledge-based, leading to instrumental teaching methods (Zhou, 2010).", "Attention is now being focused on tests which look beyond knowledge processing and retention, to use of knowledge and problem-solving (Kong et al., 2005; Li, 2009).", "So, similar to themes identified in the Western literature, teacher quality within the Chinese context includes such dimensions as teacher motivation, affective attributes (such as passion and respect), underlying beliefs in the value of change and innovation, as well as specific personality traits, such as confidence, diligence, devotion, independence, reflection, openness and tolerance (Wang et al., 2010).", "Though there is some overlap in the conceptual models in both the Western and Chinese contexts, there is still no common definition.", "A definition proposed by Campbell et al. (2003) suggests that being an effective teacher should include: ‘the power to realise socially valued objectives agreed for teachers’ work, especially, but not exclusively, the work concerned with enabling pupils to learn’.", "They recognise that this definition is ‘not perfect’, but it is useful in that it helps to problematise a previous focus on narrowly defined student outcomes, raising issues of the underlying values of teachers’ work and how these may influence the broader goals of schooling.", "It is with these ideas in mind that the next section looks at the policy perspective of teacher effectiveness and development within mainland China.", "As the world's most populous country, China regards the development of a high quality teacher workforce as essential to the future prosperity of its people.", "The most recent Outline of China's National Plan for Medium and Long-term Education Reform and Development (2010–2020) opens with the following paragraph:", "A nation's enduring future hinges on education.", "Education is the cornerstone of national rejuvenation and social progress, and a fundamental way to improve citizens’ quality and promote their all-round development, bearing the hope of millions of families for a better life.", "Education must be enhanced if a nation is to prosper.", "(Chinese Ministry of Education, 2010, p. 5)", "But, the document goes on to express concern that:", "Our concept of education and our teaching contents and methodology are relatively outdated, schoolwork burdens on primary and middle school students too heavy, the promotion of quality education arrested, our students weak in their adaptability to society, and innovative, practical and versatile professionals in acute shortage.", "(Chinese Ministry of Education, 2010, p. 6)", "The situation is regarded as particularly acute in impoverished rural areas, and a growing body of literature, published both within and outside China, identifies a deficiency in both the quality and quantity of teachers as a key drawback in raising the quality of compulsory education provision, ensuring equity and enabling the government's aspiration of education for all (Liu, 2004; Jing and Hu, 2007; Robinson and Yi, 2008; Yi et al., 2012).", "Evidence from the 2008 National Inspection Report on teacher quality, based on a survey sample of approximately 320,000 teachers, field research in seven provinces/autonomous regions, and an analysis of the annual education statistics from all counties in mainland China from 2002 to 2007, clearly showed that there was still a significant gap between schools, especially between compulsory phase rural and urban schools in the career infrastructure, qualifications, rights and interests of teachers (National Education Inspection Team, 2009).", "Some rural schools, particularly those in remote areas, were found to have insufficient numbers of teachers, compounded by a lack of certain subject expertise (for example, foreign languages, physical education, music, the arts and ICT).", "There were also big differences between schools in the percentage of teachers with senior academic titles, especially between urban and rural schools, revealing an uneven distribution of resources, due in part to the negative effects of school choice where parents are willing to pay additional fees for some schools.", "On average, 20% of teachers in urban schools and more than 40% of teachers in rural schools had a mismatch between their professional qualification and the subjects that they were required to teach.", "The report also revealed poorer salary, medical insurance and working conditions for teachers in rural areas and limited budgets for professional development.", "With regard to accessing additional resources to address these challenges, senior high school education is currently vying with pre-school provision to be included as part of the planned extension of compulsory education provision to provide 12 years of schooling, the outcome of which is still undecided.", "As mentioned previously, since 2001, teachers have also been facing new challenges from the ongoing implementation of nationwide curriculum reform.", "This seeks to ensure the all-round development of students by requiring teachers to move outside a transmission model of teaching and learning in order to encourage students to be active, creative and responsible for their own learning, creating a democratic environment where students’ voices can be heard (The State Council of the People's Republic of China, 2001; Halpin, 2010; OECD, 2011).", "For example, the reform calls for teachers to develop optional courses which enrich students’ knowledge, skills and practice, by fostering students’ social responsibility and narrowing the gap between individual, employer and society priorities so as to meet local economic needs, particularly in rural areas (Chinese Ministry of Education, 2001).", "Specifically, in relation to the process of teaching, it demands that teachers have to actively interact with, and meet the needs of, individual students, guide students to question and explore as well as investigate, and use ICT to support their work.", "This is a fundamental change which calls for teachers’ use of constructivist models of teaching and learning, and links student learning to real life issues rather than their more common traditional transmission approaches to teaching, learning and the distribution of knowledge.", "Hence there is an increasing demand on teachers to professionalise their work, and to continuously enhance their level of teaching (Yu, 2010).", "Such concerns were highlighted and prioritised in the 2009 National People's Congress (National People's Congress, 2009) and the current National Plan (2010–2020) refers to the following strategies in the development of high quality teachers:", "It is therefore essential to improve the status of teachers, safeguard their rights and interests, raise their salaries and benefits, and turn teaching into a respected occupation.", "It is important to be strict with teachers’ credentials, enhance virtue building among them, and strive to establish a well-structured and dynamic contingent of professional teachers of high calibre and imbued with moral integrity and teaching expertise.", "(Chinese Ministry of Education, 2010, p. 36)", "The Teachers’ Law, within mainland China, states that teachers should be seen as professionals who are charged with the duty to impart knowledge, educate people and provide guidance and evaluation in relation to students’ study and development.", "They should enjoy the rights of being included in discussions about the reform in teaching, be able to engage in research and academic exchange, receive salaries on schedule, benefit from prescribed welfare, and gain opportunities for further professional training (National People's Congress, 1993).", "While being respected by society, the law further specifies that teachers have an obligation to abide by the constitution and act as a role model of virtue and learning, put educational policies into practice, fulfil their teaching contracts, educate students based on the basic principles defined in the constitution, care for all students with love and respect, promote students’ all-round development, stop acts that are harmful to students, and continuously improve their professional competence in education and teaching.", "Historically, education within China has been respected and seen as a means of escape from poor socio-economic conditions.", "Academic achievement has for thousands of years been a way of achieving status and honour, both for the individual and the family.", "In the past, the Chinese government took sole responsibility for defining quality and allocating resources.", "More recently, the process of drafting the recent guidelines has taken two years and is the result of two rounds of consultation.", "The data reported here relate to individual and focus group interviews collected in eight Chinese senior secondary schools located across three local authority areas chosen to be illustrative, as far as possible, of national conditions.", "Two local authority areas were situated in the central eastern region of mainland China and consisted of a mixture of large urban/city areas, together with small town and rural areas.", "The western region was markedly different with mostly poor rural and small town areas, and included a significant minority of non-Han residents in the local population.", "Individual schools were identified, based on a willingness to take part in the study, with six catering for students from 16 to 19 years, and two for students between the ages of 13 and 19 years.", "All included boarding facilities, which for some accounted for up to 82% of their intake.", "Within the regions, the project schools were located in a variety of contexts: large urban districts, residential suburbs, and poor rural areas.", "Within each of the three local authority areas, there was at least one rural and one urban project school.", "Individual interviews were conducted with the headteacher and/or deputy headteacher in each of the eight schools.", "Focus groups in each school were also conducted with a group of three to five senior high school teachers and three to seven senior high school students, all aged 18 years or above, within each school.", "In addition, four national policy makers, nine local policy makers (three from each region), and two policy makers from other local authorities voluntarily took part in the interviews.", "All participants were assured of confidentiality in the reporting of their contributions, and have been identified in code only according to the local authority where they were located, the institution in which they worked and the type of stakeholder they were: policymaker, headteacher, teacher or student.", "The individual interviews and focus groups, which involved over 90 stakeholders, generally lasted 1 h, were tape recorded, conducted in Chinese by native Chinese speakers, and carried out over a two-month period in 2008.", "The interview data were initially transcribed into Chinese and summarised into 13 reports of emerging themes related to the study research questions: including one for each study school; one for each local authority; one for the additional local authorities; and one at national level.", "These theme reports were then translated into English and further analysed drawing on the developing themes of stakeholder beliefs and attitudes towards quality education; classroom practice and professional activities; and classroom and school environment.", "Issues of curriculum development; equity; and further professional development were further themes that had particular relevance both for the current context in China and for the sample of respondents who took part in the study.", "However, given the relative size of China, it is recognised that only a relatively small sample of schools and stakeholder interviewees were possible, and so these findings are intended to be exploratory and illustrative, rather than definitive, and a more detailed discussion of the limitations of the work is included in the concluding section of this paper.", "There was a clear consensus from stakeholders that the nature of quality education should be centred around appropriate outcomes for students.", "It was important that the students received an education which contributed to their all-round development, and that they should be able to contribute to the economic prosperity of the nation.", "This view was in line with government policy and identified teachers’ work as extending beyond the classroom to include the cultivation of a certain optimistic attitude to life and a resilience that would support students in later life.", "As one teacher put it:", "pp. 21–32 once entering society it is impossible for a person to always live smoothly and in good times throughout their whole life.", "Frustration, failure and rough times are unavoidable.", "Therefore, to have an optimistic, positive and high-spirited attitude and the philosophy of life is very important ….", "I think education is to foster students as being a useful person but without forgetting to cultivate them as being a happy person.", "(Lea1Sch1TA, pp. 21–32)", "This was echoed by a student:", "From the nation's point of view, on one hand, various kinds of talents are cultivated via educational institutions in order to serve our country and society.", "On the other hand, education is to promote overall quality of citizens … from the view of individuals, I think, we’re now receiving education to serve our future life and job.", "(Lea1Sch2SC, pp. 45–54)", "Such sentiments led to the view that being a teacher in China has been traditionally regarded in very broad terms, having many responsibilities in relation to their students.", "As one deputy headteacher in a rural school put it:", "Schools are a place for incubation and development of talents.", "Therefore, ‘people’ are important.", "There are two aspects that should be reflected on.", "First, schools have to be student-centered and this should drive how schools arrange all their work including resource allocation … the second is about the quality of people who deliver teaching and the spirit of the team as these would influence students … the quality of people delivering teaching includes their personality, knowledge, approaches, spirit, and etcetera.", "All these are also one feature of an effective school.", "(Lea3Sch1HTC, pp. 387–394)", "Such professional characteristics also included commitment and a moral and ethical dimension for, as a headteacher in an urban context commented, quality education included, a teaching workforce with commitment which reflects the overall academic atmosphere, work ethic and responsibilities, team morale, and the ability of distinguishing right from wrong (Lea2Sch2HT, pp. 462–487).", "This theme was taken up by a student from the same school who said, I think a good teacher not only has a high teaching level but also has ethics which I think is particularly important.", "Being a teacher, he or she has to set an example by personally taking part (Lea2Sch2SA, pp. 402–409).", "This requirement for teachers to act as moral agents and role models reflects the influence of Confucian civilisation and long-established culture where teachers have, traditionally, been respected members of society, having a moral responsibility to attend to their students’ needs.", "A headteacher in an urban school put it this way:", "Since ancient times, we are influenced by East Asian Confucian civilization.", "There is a proverb saying that it is a teacher's laziness or irresponsibility if he or she does not strictly supervise his or her students and teach them with diligence… therefore, it should be said that, in Chinese schools, teaching [in basic education] is rather rigorous.", "Knowledge structure and courses on offer are fairly comprehensive.", "(Lea2Sch2HT, pp. 57–59)", "Chinese culture also puts great emphasis on collective responsibility, collaboration and success of the group.", "It is not enough to be successful in your own subject area, there is a responsibility to contribute actively to the success of the group.", "This is illustrated in the following quote by a headteacher in a rural school:", "If a class is very effective and energetic, it could be concluded that, firstly the teaching team … I think the team, should be very, very communicative and cooperative … the head teachers of a class should be able to unify all subject teachers of the class in studying their students on a regular basis … to learn what problems exist in students’ thinking and learning.", "If a student is performing poorly in Chinese, then his/her Chinese teacher can deliver specific, individual lessons.", "If it's in maths, then his/her maths teacher will look into where the problem is.", "Or if it's about a student's puzzled thinking and emotion, then the teachers who have a good relationship, rather than those who have a bad relationship, with the student can handle the issue.", "We have been employing a similar approach by asking individual teachers to take a few students to provide guidance in aspects of affective learning methods.", "That's it!", "Teachers should act in coordination with each other.", "Everyone has to clench their fist into one to work together.", "We can’t just, like you do your own job and I do mine without communication, is that right?", "The reason for many students who failed to get into a college is that [for example] they did well in mathematics but not in Chinese.", "Chinese dragged them down.", "(Lea3Sch1HTA, pp. 352–366)", "However, it was also clear from the responses that, in common with much research around the world (Skilbeck and Connell, 2004; Hextall et al., 2007), society in mainland China was changing.", "This had brought new pressures and challenges for teachers in terms of how they conceived of their role.", "One significant change was the complex nature of student experience, which required a broader understanding of the lives of students so as to address students’ social and emotional needs.", "This was particularly relevant in the more disadvantaged Western region.", "As one local policy maker put it:", "To be honest with you, I was a teacher for 20 years.", "But how can I put it?", "This is really different.", "When I was a teacher, I felt there were fewer disturbances but just … teaching.", "Now, there are issues including safety, moral and mental education of students facing teachers.", "In the past, we didn’t think that students would have such things as mental problems.", "They, even those from poor families, were all normal.", "We were in that kind of environment.", "It seemed that there were no contradictions.", "As for now, quite a few students whose families are worse off or poorer compared with others, for example, in terms of clothing, have a sense of inferiority.", "Some even just left a letter and ran away from the school because of this.", "So, students’ minds tend to be more complicated, and their endurance has become weaker.", "Being a teacher, you have to teach as well as be worried about all sorts of other issues.", "The pressure is greater [than before].", "It is no easy job to be a teacher.", "(Lea1LPM6, pp. 488–495)", "One of the reasons for this societal change was the effect of mainland China's one child policy which has meant that extended families, grandparents as well as parents, invest heavily in promoting academic success for their children.", "In a society which values hard work and commitment, and sees educational success as a way to improve the life chances of not only individuals but also their extended families, this could bring particular pressures for teachers.", "As one headteacher commented:", "During all these years, society has changed a lot.", "From a family perspective, nowadays, children who are the only child are blessed and cared for by their grandparents, maternal grandparents, in addition to their parents.", "They are a special generation, having more care, but also hoping to gain more.", "I have been a teacher for over 20 years since 1987.", "I feel that there are differences in the approaches and methods used to educate them as compared to those of previous generations.", "(Lea3Sch4HTB, pp. 215–218)", "In addition, there were new demands on teachers, in both urban and rural situations, to enable students to go beyond just obtaining knowledge and required that, in order to maximise their potential after graduation, they were prepared for a new and changing society.", "One student from an urban school expected teachers and the school, ‘to help students establish correct outlook on life and value.", "This includes being able to deal with problems independently after entering the society, being innovative at work …but more, it can nurture my thinking consciousness and me being in the habit of thinking’ (Lea2Sch2SB, pp. 17–25).", "Similarly, one student from a rural school required teachers to, ‘discover the strengths of each student and help them to inspire their interests and hobbies in order for them to make best of their strengths.", "After all, this is a diverse society that needs all aspects of talents’ (Lea3Sch1SD, pp. 318–321).", "However, such views were also tempered with an emphasis from all stakeholders on the importance of exams and competition in the employment market.", "This appeared particularly important for those in rural areas.", "As one headteacher explained:", "It is related to families’ own interests … within the current Chinese context, because of the high population, there is a particularly large pressure of employment.", "Now, above all, is the entrance exam for senior high school, or for college, including master and doctoral degrees, there is a basic rule that applies to all these situations.", "That is how to achieve higher levels of educational qualification in order to have a wider selection of jobs.", "(Lea3Sch1HTB, pp. 46–57)", "Similarly, a teacher from another rural school, while recognising their role as moral agent, also emphasises the need to enable students to gain good qualifications because it is unlikely that:", "… an industry would employ someone simply because he or she is morally good but has no educational qualification.", "No qualification probably means being jobless.", "The moral quality of a person does not matter as long as you have a qualification … (exam) scores are the very life of students.", "Students pursue scores.", "Teachers pursue scores.", "Parents pursue scores.", "This is the current situation.", "As a result we, as teachers, teach students how to achieve high scores in exams.", "(Lea2Sch1TC, pp. 129–137)", "However, such an approach is counter-balanced by concerns which reflect the recent curriculum reforms aimed at engaging students in a broader curriculum in order to develop the knowledge, skills and understanding necessary for a changing national and global context (Guan and Meng, 2007).", "These changes have sought to improve teaching resources and develop more generic skills, beyond focusing solely on examination success.", "As one local policy maker explained:", "There is this situation that our students have high examination score, but they are still lacking in comprehensive ability in terms of practical ability and creativity.", "We are doing that now actually.", "Since 2000, our nation has begun the 8th curriculum reform.", "The reform is trying to influence the situation via changing teaching materials … in order to improve students’ thinking ability, creative ability, studying and problem-solving ability as well as practical ability … gradually the situation of high scores and low abilities will change for the better as long as we persist in working hard (Lea1LPM5, pp. 182–194).", "This broad view of the role of the teacher and an emphasis on the support of individuals was also apparent when stakeholders talked about versatility and flexibility within classroom practice.", "Teachers talked of it being necessary to impress students with expertise beyond their particular subject specialism and offer the students more than the knowledge contained in the curriculum.", "Additionally, good teaching required an element of enthusiasm and charisma.", "One teacher spoke of using teaching to:", "… conquer your students.", "Second, you’d better master something else other than teaching as much as possible.", "Additionally, you should impress your students and make them admire you from their heart.", "Then I guarantee you a comfortable class atmosphere (Lea2Sch1TB, pp. 510–512).", "Another teacher, from the same school, gave the following explanation:", "If you are a maths teacher, you are also familiar with physics and you apply the knowledge of it in your maths classes; or you even know something about arts, then your lectures will excite your students … schools should encourage teachers to develop their multi-talents ….", "Of course, the prerequisite is that teachers must do their own job well.", "(Lea2Sch1C, pp. 516–522)", "One student in an urban school regarded the quality of their teachers as high and linked this to their ability to go beyond the curriculum and existing resources:", "As to the quality, I think it's very good in our school.", "At least the level of those who teach me is very high.", "Especially in class, they will supplement things that we are not able to get from textbooks, will provide new ways of thinking that are not confined to textbooks.", "(Lea2Sch2SC, pp. 50–52)", "There was also a concern with the need for teachers to ensure that they employed different teaching approaches in order to take account of differences in students’ aptitudes.", "A policy maker in the Western region drew attention to the ability to differentiate, and characterised good quality teaching as being able to:", "… recognise the differences between students and to be concerned in developing different levels of students … if the school is an effective one, it can suit the remedy to the case.", "That is to say, teaching students in accordance with their aptitude.", "(Lea1LPM6, pp. 383–384)", "This sentiment was supported by a student who saw clearly the need for good teachers to address the needs of individuals while, at the same time, recognising the pressure that this would put on teachers and the need for appropriate resourcing:", "If teachers can enable each student to improve, it must be that they seek different methods aimed at individual strengths and weaknesses … to some extent the cost of this practice can be very high, including the input of teachers’ energy … and organising this is pretty difficult.", "In practical terms, this probably involves teacher allocation in order to provide each student with teachers that are suitable for them.", "(Lea2Sch2SB, pp. 394–395, 395–398)", "This recognition of the value and importance of appropriate teaching strategies and teaching materials was also expressed in relation to the use of feedback data at the local level to aid in identifying good practice.", "As one local policy maker explained:", "… this year, for example, we (the LEA) will choose two subjects … according to the exam results of a few randomly chosen schools.", "We will find out which subject has reached the expected goal.", "This is not intended to assess a school or teachers, but to determine what efforts have been made to improve the teaching of the subjects.", "(Lea1LPM4, pp. 89–96)", "There was also evidence of the development of more systematic educational evaluation at the local authority level to inform practice.", "One policy maker supported the recent introduction of value added approaches by saying:", "As the headteacher, if you use this method, the result will be very obvious and quantitative, and the performance of all the teachers will be put onto paper – and it is very convincing.", "(Lea1LPM10, pp. 189–194)", "This view was endorsed by a headteacher who said:", "[LEA district] has recently been planning to implement a so-called relative evaluation [similar to value added evaluation] into the current school evaluation system so that the evaluation would become more scientific … I think it is very good, very good, and is of real education science.", "I have benefited from learning about this innovation in school evaluation even though I have been a practitioner for many years.", "(Lea1HTA, pp. 18–20)", "Though these new techniques were often focused on a narrow range of academic student outcomes, it should be recognised that, as highlighted in the previous section, Chinese teachers also saw all round development as a high priority and recognised the need for assessment and evaluation improvement across a broad range of educational outcomes.", "In line with the broader view of teaching quality that has been expressed in recent literature within China, the dual importance of a supportive school culture and appropriate classroom climate was also referred to by most interviewees.", "One urban headteacher drew attention to the contextual nature of this culture and the need to ensure that it was continually reviewed to take account of changing circumstances:", "To judge whether or not a primary or secondary school is good, first we observe its campus culture to see if it is a good place for studying, a good environment for children's physical and mental development … a good school should be the one that implements the ideas about what is good according to its own understanding.", "It is fine even if you revise your ideas in the process of practice.", "(Lea2Sch2HT, pp. 341–363)", "In particular, there was a view that when students understood, and were in agreement with, the overall culture of a school, learning could be enhanced.", "One teacher used the term ‘cultural penetration’ to address the importance of a student's recognition of, and alignment with, a school's cultural identity:", "The crucial point is cultural penetration.", "That is, what is the culture of the school.", "In this school, it is about indomitable spirit, in other words, being able to strive, being cautious, being diligent, and being innovative at work.", "If a student has characteristics similar to these school cultural identities … then obviously, he or she can live and learn very, very well in this school.", "(Lea3Sch3TC, pp. 640–643)", "Such a clear vision and statement of aims could be considered important in terms of motivating individual students to do well.", "As one student said, ‘schools need to make students understand themselves and find their own direction for development.", "Moreover, schools need to encourage students to have motives to improve and move forward’ (Lea1Sch1SC, pp. 162–164).", "The importance of a supportive and positive school culture was also seen as important in terms of enabling students to further their education at a higher level.", "One rural headteacher attributed their yearly increase in college entrance rates over the past 10 years, to a good teaching and learning ethos which had been developed within the school.", "He recognised the support that this gave to teachers by saying that:", "Our teachers have a positive teaching manner because they feel, “I am proud of working for this school and being able to cultivate more and more good students.", "It's an honour mission to me”.", "Then our students have very affirmative learning attitudes.", "They know why to learn, how to learn and learn the good things.", "(Lea3Sch2HT, pp. 407–409)", "A positive school environment went hand in hand with the atmosphere within the classroom, which, for some, included an equal relationship between teachers and students, so that students did not feel undermined but part of a supportive learning environment which enabled them to focus on their work.", "As one teacher pointed out, ‘because students have a sense of identity and belonging, they recognise the class, the collective and the environment, and of course, they also recognise their study’ (Lea3Sch3TC, pp. 647–648).", "As one student put it, ‘a class will gain progress as a whole if it has a good atmosphere, which definitely leads to a good learning environment’ (Lea2Sch1SC, pp. 293–294).", "However, this could be disrupted by student action.", "One student considered that, at his school, learning time was wasted because he understood that students at another school used break time to continue their studies.", "This was not the case in his school where he considered that it was more common for students to play around.", "A student from a third school had similar issues and was worried about the situation where, ‘if our classmates one by one are following someone else to just play around, our mood for learning would be soon disappearing, because our focus would be turning to play and totally ignoring the learning aspect’ (Lea2Sch1SB, pp. 306–309).", "Such peer influence could particularly disadvantage rural students who, even if they wanted to learn, could be disrupted because, as one student put it:", "Other classmates are interested in something else, which is nothing to do with learning.", "Sometimes, if people have low-self control, it's difficult to concentrate on learning … they will be affected by the surrounding environment.", "That is, the influence from classmates.", "(Lea1Sch2SA, pp. 306–309)", "There was evidence to suggest that some urban students were regarded as more difficult to control because of differing socio-economic circumstances.", "For example, some urban parents might have enough social and cultural power to intervene with regard to how a school was dealing with their child's misbehaviour.", "This was rationalised by recognising that it might be less critical for students from wealthy backgrounds to achieve academically because an influential family background would help their future prospects.", "As a student from another school said, ‘urban students come from families with money and power.", "No matter how they disturb [the flow of schooling] the school has no way of controlling them because of a conflict of interests’ (Lea3Sch4SC, pp. 339–340).", "Another issue that emerged from the student interviews was whether or not competition within the classroom had positive or negative effects on student learning.", "Students’ views differed and the two quotes below represent, to some extent, the differing views of rural and urban students.", "One rural school student emphasised the need to encourage competitive collaboration in learning:", "An effective school should know how to teach students to compete.", "Because competitiveness in the future and in the past is very important, a school should have sufficient competition.", "I have a feeling, in a class, the effect that comes out of one student working on questions is not the same as the effect that comes from two students competing with each other when working on questions.", "It involves improvement no matter the speed or the accuracy [of answering questions] … if everyone knows [the essence of] competition, then all students will compete [with each other], and then all will be full of energy, and then all will make rapid progress.", "If a school can fully mobilise their students’ sense of competition, then the effectiveness of the school would be, should be, high.", "(Lea3Sch2SC, pp. 285–290)", "By contrast, a student from an urban school in the same region disagreed and argued that competition could be counterproductive, ‘the school should not create such a competitive environment.", "I think you should compare the progress you’ve made yourself and find your value.", "It is not necessary that you must exceed other students and have to compare yourself against them’ (Lea3Sch4SD, pp. 547–549).", "The concept of collective collaboration is a significant feature in a society which emphasises the collective over the individual (Hofstede, 1991; Cheng and Wong, 1996).", "One teacher gave an example of how evidence of cooperation and collaboration had, she felt, been helpful to one student who gained a place at an overseas university, ‘in the interview, the student didn’t tell the university [about his involvement in a school choir] from the point of view of him leading the choir, but from the angle of sharing experiences with members in success and joys’ (Lea3Sch3TD, pp. 549–551).", "Despite some clear views on what it means to be an effective teacher, it was also evident from the interview responses that there were barriers to ensuring quality and the future development of teaching.", "This largely centred around issues of continuous professional development and disparities between wealthier urban and more disadvantaged rural districts, both of which are currently the focus of government policy.", "As a deputy headteacher said, there was a need to:", "… promote not only student development but also teacher development.", "During the process, teacher development could have a positive and assuring impact on the school's overall development in the future.", "Therefore, it should also be our priority.", "(Lea3Sch4HTC, pp. 57–58)", "A deputy headteacher in the same school gave examples of what was currently available to teachers, ‘the professional development of teachers includes opportunity for postgraduate study … professional training … [and] relevant subject activities organised by the Provincial Educational Research Institute every year’ (Lea3Sch4HTB, pp. 358–363).", "Respondents reported that teachers had various opportunities to further their studies, including some courses that were award bearing.", "Training programmes were organised at school, county/city, provincial and national levels.", "For example, one Western region local policy maker talked of an 8-day training programme in his area which involved 14 expert teachers, across 10 subjects, who visited from another province to focus on a new curriculum for all senior high school teachers in the county, in order to improve teacher capabilities.", "There was also evidence of school-based initiatives where there were high numbers of new teachers.", "Such initiatives could involve mentoring, observation of teaching and visits to and from other schools, and were considered to be thorough and were received favourably:", "Currently, we have more school-based training, mainly focusing on teacher improvement … for the past few years, the school has been recruiting a relatively higher number of new teachers.", "In order for these teachers to adapt to teaching, the school has been organising different approaches to support them.", "For example, new teachers are paired with senior teachers.", "Besides that, we also invite experts, famous teachers from other schools, higher education institutions, as well as within the school, to comment on new teachers’ teaching.", "Moreover, we send teachers out of the school to observe, study and learn from other teachers’ teaching in order to improve teachers’ professional level … personally speaking, training, when organised by schools, is able to look into in every aspect.", "This type of training is more effective.", "(Lea1Sch2T, pp. 389–393, 466–467)", "However, financial limitations and difficulties with workload and cover for absence due to training attendance were seen as restrictions.", "This tension is illustrated by the following two comments from a teacher and headteacher from the same school:", "If there were more funds then we could have more opportunities to learn from outside school.", "This is a concern also for the development of the school.", "(Lea3Sch4TA, pp. 304–305)", "For example, if a teacher leaves for one-month's training, during this month, all his or her class lessons have to be shared by other teachers who would be overloaded … teachers benefit from this training but this creates additional difficulties in school management.", "Even so, we want them to receive training.", "(Lea3Sch4HTA, pp. 373–377).", "Again, these pressures can be especially acute for staff in rural schools.", "As one rural headteacher explained:", "Nowadays there are higher expectations for teachers than before, and many young teachers cannot adapt to the expectations.", "The training for professional development is urgently needed.", "However, the training system fails to develop owing to lack of personnel, even though there are various kinds of training within the school.", "But I think it is still unsatisfactory.", "(Lea1Sch2HT, pp. 303–308)", "This situation, in terms of training and development, could be exacerbated by a shortage of teaching staff.", "Several interviewees drew attention to a serious structural problem in rural areas where there was a mismatch between supply and demand for some subject teachers.", "The lack of sufficient non-core subject teachers in rural high schools could have serious repercussions for the breadth of the curriculum.", "As one national policy maker explained:", "For example, the ratio of teachers to students is set at 1:20.", "If you reach 1:22 or 1:23, it should be fine.", "But most of the teaching staff are language and maths teachers.", "There are not enough music, sports and art teachers.", "The problem of structural vacancies is very serious.", "(NPM3, pp. 313–315)", "This was further explained by another national policy maker who said:", "Since funding is not sufficient, headteachers have no other way but to use the limited funds they have for core subject teachers.", "To the extent that some schools cannot even afford to buy a basketball.", "How can you run a PE lesson without basketballs?", "Consequently, the overall development of children is influenced.", "(NPM1, pp. 420–422)", "A student reported that in her rural school there was a problem with English specialists saying that:", "Teachers in the school when I studied there were not formal teachers.", "I think it is a drawback too.", "That is, even now our rural school has no English teachers … English as a curriculum subject has been provided for the students of the 3rd grade in primary schools in urban schools here, but our school still hasn’t got English teachers.", "(Lea1Sch2S, pp. 119–124)", "Such concerns can be particularly acute in remote rural areas.", "This reflects the fact that the population is more disperse, the terrain can be challenging and the distances travelled to school greater.", "This can lead to small schools with limited resources where professional qualifications and teaching quality are poor.", "Though the curriculum content is common across China, the level of implementation can differ from region to region.", "As one student said:", "The level of some teachers in small and remote schools is very limited.", "For example, some teachers who teach Chinese phonetics can’t even speak good Chinese.", "I feel the limitation of those teachers’ ability will influence students’ pronunciation in the future … the route to the school was a mountain road.", "It's tough.", "Because only more or less 10 students went to this school, there was only one teacher … practically the school was just a single room … I think it is a little bit unfair … for teachers recruited by such schools were generally not qualified … the government has to do something about it, to resolve the difficulty for remote rural children to access school.", "(Lea1Sch2SA, pp. 150–155, 109–113, 117–120, 146)", "The need to improve teacher training and supply, and disparities between rural and urban areas have been recognised by the government which has implemented various policies to ensure that teaching facilities and equipment are prioritised for poorer areas (National People's Congress, 2009; Chinese Ministry of Education, 2010).", "One student recalls how their junior high school benefited from such policies:", "In relation to the distribution of educational resources, I think things such as computers, TV sets, multi-media classrooms or audio classrooms, according to our policy, are given firstly to poorer areas.", "For example, the junior high school that I went to is a small rural school and has more scholarship places than any other city school; moreover, it was one of the schools to get access to the Li Jiacheng remote educational network in Hong Kong … all of these show that our government and educational administrative units, they are all making efforts to improve education as much as possible, with the hope of distributing the educational resources more equally to every student, especially to improve the reasonable distribution of funds to poor areas.", "(Lea1Sch2SC, pp. 204–223)", "There is also a nationwide policy to encourage new teaching graduates to work in rural schools and for highly performing schools to send their teachers for one or more years to support less developed rural schools.", "However, there are questions about the extent to which this is successful.", "As one headteacher said:", "Graduates want to go back to the city when they become mature after serving rural schools for a few years … for teachers like those in our school, who were sent to rural schools, what is in their mind is that, ‘I want to go back to my original school once I finish my task here’.", "The policy is just an expedient measure, which can’t resolve the fundamental problem.", "It's difficult to achieve the solution.", "China is geographically huge.", "The number of teachers we need is amazing.", "Therefore, the problem won’t be resolved itself within a short term period.", "From a long term perspective, the solution relies on the economy.", "As long as there is balanced economic development, remuneration and living standards are the same no matter where I work [then it will succeed].", "(Lea3Sch1HTA, pp. 142–153)", "Further, respondents pointed to a salary gap between teachers in rural and urban locations.", "Some saw differences even within areas.", "There was a general view that the salaries of rural teachers did not match their workload, despite the fact that they felt morally obliged to carry out their duties for the benefit of society.", "There was also recognition that a new generation of teachers may not feel the same way in terms of the balance between workload and salary, as one rural teacher said, ‘for new teachers, they consider that they should be paid for what they actually give, if it was not the case, they got a bad sense of balance’ (Lea3Sch1TB, pp. 573–574).", "So, it can been seen that, despite a strong government commitment to educational improvement and strategies to ameliorate the disparity between schools and regions, serious concerns remain over teacher training, supply and professional development especially in poorer rural areas.", "The implications of this will now be discussed in the following section.", "These illustrative findings reveal many similarities with countries around the world in terms of both stakeholder understandings of what constitutes quality education, and government concerns with the changing needs of society.", "However, they also reveal a slightly different emphasis in a country where central planning is the norm, and where large disparities in socio-economic conditions impact on the provision of schooling.", "Teacher development is regarded as central to the provision of high quality education within mainland China but it is clear that certain global and local pressures were making this difficult.", "Being a teacher within many societies around the world is not easy.", "The impact of globalisation and an information and communication revolution has contributed to a rapidly changing society which puts new demands on education systems.", "From our findings, it is clear that within mainland China the views and demands of students, as well as policy makers, were changing and becoming more diverse and sophisticated.", "This, together with a culturally determined, inclusive view of what a teacher should be could make their work exceptionally challenging.", "The requirements of a strict code of moral and ethical standards for Chinese teachers which, by law, requires them to be patriotic and law-abiding, dedicated to their work, to care for their students, to teach and educate them, to serve as role models, and to pursue lifelong learning (Chinese Ministry of Education, 2008) could be overwhelming, especially in poorly resourced and understaffed situations.", "This is supported by previous research (Hu, 1994; Yang and Guo, 2010) which draws attention to the expectations of an excellent teacher, which include addressing all aspects of their students’ development; possessing a rich subject knowledge; displaying such personality traits as happiness, kindness, confidence, diligence, intelligence and humour; to innovate and use modern teaching strategies and resources, and to frequently evaluate and update their teaching techniques; to carry out their own research; to network both socially and academically; and, above all, to achieve high student outcomes.", "Our findings illustrate how these demands were also set in a context of ongoing curriculum reform which brought its own pressures.", "Such reform was attempting to move Chinese society away from an overwhelming and deep seated emphasis on examination results towards the facilitation of all round development and lifelong learning, although so far with some evidence of limited success (Thomas et al., 2012).", "The impact of curriculum reform was not a central focus of the study but it was clear that some difficulties were being experienced.", "This supports previous research (Wang, 2010; Brown et al., 2011) which has drawn attention to concerns over teachers’ abilities to engage with new concepts, teaching materials and teaching strategies, new forms of student evaluation, new management systems and the need to teach optional subjects.", "This appeared especially acute for older, more experienced teachers and there was some evidence that the key discourse of student-centred learning has been especially challenging for teachers in rural schools with large class sizes (Wang, 2011).", "The findings of this study support previous research which draws attention to the tremendous stress that meeting the new curriculum requirements can bring for teachers (Sun and Li, 2005; Zhu and Si, 2006; Yazhuan et al., 2010).", "There was also evidence from the research that one approach to supporting teachers’ reflective practice is a renewed emphasis, at the local, regional and national levels, on educational evaluation and the introduction of more sophisticated and relevant methods of measuring student and school performance.", "One national policy maker reported on the recently established Centre for National Assessment for Education Quality (NAEQ), located in Beijing Normal University, which has been set up to monitor educational achievement in every year grade of basic education through national tests.", "However, such monitoring will go beyond the raw data of academic achievement to include measures for four other aspects of education quality: ideological, moral and civic literacy; physical and mental health; art literacy, practical skills and innovative awareness; and the influence of educational and social environments on students’ development (see NAEQ, 2013).", "It was also evident that stakeholders recognised the local need for a more nuanced, value-added approach to the assessment of school quality in order to enhance feedback to schools, improve teacher self-evaluation, and inform the development of teachers’ practice (Thomas et al., 2012).", "The findings also demonstrate that, similar to work in Western societies, a key dimension of the effective teacher was seen to be the ability to create a supportive teaching and learning environment.", "Interestingly, this was seen to be a collective and collaborative responsibility and not the individual teacher's alone.", "The school, the teacher and the students all had their part to play.", "On the one hand, it was considered important that teachers were able to balance creating new opportunities for collaborative work with the more traditional, familiar and widely entrenched approach of stimulating individual competition in the classroom in order to motivate students to fully engage with their learning.", "They were expected to maintain sound relationships with students while acting morally in line with their professional commitment and positive attitudes.", "However, there was also recognition by all stakeholders that students had a responsibility to actively contribute to this positive environment.", "This was demonstrated by the way in which some student respondents were keen to maximise their study time in school and resented disruption by other students.", "This resonates with previous research (Anderson, 2004) which drew attention to the importance of student involvement in their own learning.", "Finally, the importance of a supportive school ethos which could engender pride and allow teachers and students to identify with the school's aims and objectives was also seen as an important aspect of quality.", "Within a vast country like China which, while currently emerging as a major economy, still has areas of great disadvantage, it is important to consider the context when thinking about issues such as teacher supply and retention (Scheerens, 2000; UNICEF, 2000; UNESCO, 2004).", "A key aspect with regard to improving the education system nationwide is the need to improve teacher quality and support teacher professional development.", "However, our findings suggest that all stakeholders were aware of the inequalities with regard to the distribution of resources between schools, especially differences between the West and other areas of china, and regarded this as a key barrier to improving professional development and teacher quality.", "Such evidence supported previous research which has shown that, while various initiatives had been inspired by the introduction of Shulman (1986, 1987) ideas for strong pedagogical content knowledge in order to develop effective teaching, new or less experienced teachers—more likely found in rural western areas—were shown to be lacking in their ability to integrate knowledge around subject knowledge, teaching strategies and collaborative learning (An et al., 2004; Tang and Chen, 2005; Li et al., 2006; Xu and Gong, 2007; Sun, 2008; Liu and Zheng, 2010).", "Similarly, Brock (2009) discusses a participatory approach which was introduced by the well known Gansu Basic Education Project in western China to train teachers in new ways of teaching rather than relying on the inculcation of content knowledge alone.", "Evidence from the respondents in this study suggested that opportunities for further training, organised from national to school levels, were available but that the ability of individual schools and teachers to take advantage of these could vary.", "Insufficient resources to cover for absent colleagues, and an imbalance in terms of teacher qualifications and subject specialisms could limit the opportunities for some teachers.", "This situation was particularly acute for schools in rural, remote and western areas.", "In light of these limitations, attempts were being made to encourage school-based training which was likely to be more closely connected with local issues while, at the same time, providing more opportunities for more teachers.", "There was some evidence that current government priorities to increase resources in disadvantaged Western areas was having some effect in improving quality but there was scepticism about the long term effectiveness of relocating teachers from urban to rural schools.", "Concerns about current disparities between high expectations, excessive workloads, multiple roles and poor pay were evident in our data, and previous research (Liu, 2010) has drawn attention to the extent to which this might drive people away from teaching as a career.", "This is reflected in the conclusion of the 2011 International Summit on the Teaching Profession which concluded that there was a need to make teaching more attractive and to solve problems with recruitment and retention (Schleicher, 2011; Stewart, 2011).", "Johnson (2012) has pointed out that, given the current changes to teachers’ work; it may no longer be possible to count on new generations taking up teaching as a lifetime career.", "This can have serious consequences for the quality of education because, as Barber and Mourshed (2007) have pointed out in their survey of what makes effective schools effective, there is some evidence of a correspondence between high performing schools and the qualifications and training of the teachers.", "Though this is a complex area and no causal link can be proved, the findings of this study have shown that there are concerns about the level of initial and further training and development available to teachers, especially in rural areas.", "This paper has discussed some illustrative findings from individual interviews and focus groups conducted with a range of stakeholders (national and local policy makers, head teachers, teachers, students) across a sample of schools from three local authorities within mainland China.", "In a country like China with such vast geographical and social differences, encompassing as it does both extremely poor, disadvantaged communities together some of the world's most advanced high tech cities, it would be foolish to place too great an emphasis on conclusions drawn from such a small sample.", "It should be recognised that evidence from this study sought to broaden the usual metropolitan perspective by including schools in rural and disadvantaged areas, together with those in the Western region of the country where a lack of resources and issues of minority inequalities are most evident.", "Given the word limitations of this article, it has only been possible to represent a small number of voices in detail, though the intention of the authors has been to draw out common themes and link these to both the existing international literature and current Chinese policy context.", "The researchers recognise that, within the constraints of the study, it was not possible to examine either school or local authority level documentation, or observe in detail classroom practice which could have triangulated the data collected.", "The data can say nothing in detail about issues of school and class size, the background or previous experience of teachers or pupils, or the perceptions of Chinese parents.", "It should also be recognised that the participants in the study did so voluntarily and so were a self-selected group who may have demonstrated a particular view of educational development within China.", "Social, cultural and political pressure can constrain the ability of individuals to share their perceptions within the context of a focus group interview and, even within individual interviews, participants can be wary of sharing unpopular or exceptional views.", "The data were collected by Chinese speakers, from both within and outside the national system, and were then translated by the bi-lingual project members.", "Trouble was taken to represent both the original meaning and emphasis of the participants, and the analysis of the substantial interview data collected was conducted with care and rigour, including attention to the central concerns of cross-cultural researchers in establishing conceptual, rather than strict linguistic equivalence.", "So, while recognising the limitations in the data discussed, and despite the limited scope and exploratory nature of these findings, it is argued that this paper can still provide greater understanding of the current situation of teaching and teachers’ work within mainland China.", "The findings help to illustrate the ways in which the demands of ongoing educational reform, which include the active involvement of students and a focus on lifelong learning for both students and teachers, can motivate against quality of schooling.", "Tensions within a traditional structure of an exam-based environment can make it difficult for teachers to address the demands of sù zhì jiào yù while ensuring the support of parents and the reputation of their school.", "Structural and financial inequality was evident in the responses, and strategies for further professional development were being compromised in some areas.", "The findings also support the view from Reynolds et al. (2011) that teacher behaviour is influenced by their underlying attitudes and values, although more research is needed on the way in which the formation and expression of such values shapes, and is shaped by, the historical, social and political context, in which they work.", "It can be argued that this is especially needed in developing country contexts where little such research already exists.", "With these exploratory findings in mind, a key aim of the on-going research is to extend these findings to investigate the nature of teachers’ professional development and learning in mainland China.", "The relevance of such Western concepts as professional learning communities (PLCs) in a context where tradition and practice emphasise collectivism and the collaborative nature of the group, rather than the promotion of the individual, is also being investigated.", "Preliminary findings strongly suggest that such a concept already exists within teachers’ working practice within China, although the emphasis is somewhat different (Thomas et al., 2013).", "For example, in the Chinese context such PLCs tend to include only teachers, rather than all professional staff who have contact with the pupils.", "It is hoped that a combination of Western and Confucian heritage concepts, and the identification of current localised practice, will help to enhance future approaches to teacher professional learning." ]
[ "Education reform impacting adversely on teaching quality, especially in rural areas.", "Inequities in the distribution of resources, and some subject specific skills, especially in rural areas.", "Lack of opportunity for good quality professional development.", "Need for improved data collection and evaluation systems to inform practice.", "Need for more emphasis on lifelong learning and student involvement." ]
Rapid Encoding of New Memories by Individual Neurons in the Human Brain
The creation of memories about real-life episodes requires rapid neuronal changes that may appear after a single occurrence of an event. How is such demand met by neurons in the medial temporal lobe (MTL), which plays a fundamental role in episodic memory formation? We recorded the activity of MTL neurons in neurosurgical patients while they learned new associations. Pairs of unrelated pictures, one of a person and another of a place, were used to construct a meaningful association modeling the episodic memory of meeting a person in a particular place. We found that a large proportion of responsive MTL neurons expanded their selectivity to encode these specific associations within a few trials: cells initially responsive to one picture started firing to the associated one but not to others. Our results provide a plausible neural substrate for the inception of associations, which are crucial for the formation of episodic memories.
[ "NEUR" ]
[]
[ "2800" ]
[ "Neuroimaging investigations in humans and behavioral studies of neurological patients have substantiated the importance of the medial temporal lobe (MTL) for episodic memories (Davachi, 2006; Eichenbaum, 2004; Eichenbaum et al., 2007; Moscovitch, 1994; Squire et al., 2004; Tulving, 2002).", "Furthermore, neurophysiological and lesion studies in animals have shown that the MTL is involved in the encoding of associations (Bunsey and Eichenbaum, 1996; Day et al., 2003; Kahana et al., 2008; Sakai and Miyashita, 1991; Wirth et al., 2003), which is a key mechanism for episodic memory formation.", "In spite of the major significance of these works in advancing our understanding of episodic memory, their contribution has been limited.", "On the one hand, human studies have not addressed episodic memory formation at the single neuron level.", "Animal studies, on the other hand, have relied on extensive reward-driven training with numerous repetitions of non-natural stimuli, thus offering a limited account on how single exposures to natural stimuli can give rise to the rapid encoding of new episodic memories.", "Neurons in the human MTL have been found to respond to concepts that are related to each other (Quian Quiroga, 2012; Quian Quiroga et al., 2005), such as two co-stars in the same television series or a few researchers (previously unknown to the patients) involved in the experiments (Quian Quiroga et al., 2009; Viskontas et al., 2009).", "Here we designed a paradigm to study how fast these associations can be created and whether this speed is compatible with basic mechanisms of episodic memory creation.", "We postulate that associations can be formed by partially overlapping cell assemblies encoding related concepts (Quian Quiroga, 2012) and show experimental evidence of rapid changes of single-cell responses while contextual associations are learned.", "As detailed below, in order to gain such evidence, we combined the ability to analyze trial-by-trial changes in the robust firing of highly selective MTL neurons (Quian Quiroga et al., 2005, 2008, 2009), with the rapid facility that humans have for learning complex associations and consciously declare them.", "Patients first participated in a “screening session” (Quian Quiroga et al., 2005) in which a large number of images of people, animals, and places were presented to find out which (if any) of the recorded neurons responded to a picture.", "Data processing (spike detection, sorting, and identification of responsive cells) was done quickly (typically within 1 hr) and 3 to 8 (median 7) pairs of pictures were selected.", "Each pair consisted of a picture of a person (or animal) and a picture of a landmark, for which there was a neuron firing to one of them (the preferred “P” stimulus) and not to the other one (the non-preferred “NP” stimulus).", "For each pair, we created contextual “composite” images, in which each individual was digitally extracted from the original picture and placed in front of the landmark, mimicking a real photo of seeing the individual at that landmark (Figure 1).", "Using presentations of the single and composite images of each pair, we evaluated changes in neural activity while subjects performed five consecutive tasks (Figure 1).", "First, to get an estimation of the pre-learning firing to each picture, in Task 1, the screening was repeated showing each of the single pictures for 1 s 6 times in pseudorandom order, and patients were asked to indicate whether the picture contained a human face or not.", "Then, a block of “learning and evaluation trials” (median of 15 trials) comprising interleaved tasks 2 and 3 were shown.", "In Task 2, the composite images (each of them being a specific person in a specific place) were presented in pseudorandom order, which were then followed by the presentation of the single pictures, also in pseudorandom order.", "The instructions were the same as in Task 1 (i.e., indicate presence of a human face).", "After each run of Task 2, the learning of associations was tested in Task 3 (the patient was presented each face at a time and had to select the landmark corresponding to it).", "After Task 2 and Task 3, in Task 4 the patient was presented 6 times each landmark in pseudorandom order and had to name the person that was there.", "Finally, in Task 5 (“re-screening”) all single pictures were presented again in pseudorandom order, to compare with Task 1 (before learning).", "Typically, the entire experiment lasted between 25 to 30 min.", "In 14 patients, who participated in 25 experimental sessions (and only 22 for Task 5), we recorded the activity of multiple single neurons using electrodes implanted in the MTL for clinical reasons.", "Figure 2 shows a neuron in the hippocampus that responded strongly to the picture of a member of the patient’s family (with a mean firing rate of 13.1 spikes/s, SD = 3.9, median = 12.5) but not to the Eiffel tower (3.6 spikes/s on average, SD = 3.4, median = 3.3.", "The firing to the Eiffel tower during the response period did not differ significantly from the one during baseline (3.9 spikes/s on average, SD = 2.0, median = 4.2), according to a Wilcoxon rank-sum test (p = 0.84, W = 40.5, n1 = n2 = 6).", "With our experimental design, we aimed to establish whether MTL neurons will widen their tuning to encode the formed association by selectively increasing their firing to the associated stimulus.", "After a single exposure of the composite picture, the subject learned the association (i.e., family member at the Eiffel tower) and the firing rate in response to the Eiffel tower increased to 7.6 spikes/s on average (SD = 5.1, median = 8.3), a 230% increase compared to the presentations of the Eiffel Tower before learning took place (Task 1).", "This difference was significant (p = 0.002, W = 563, n1 = n2 = 27, Wilcoxon rank-sum test between baseline and response periods, see Experimental Procedures).", "In contrast, the response to the preferred stimulus (family member) did not change significantly after learning the association (9.4 spikes/s, SD = 4.5, median = 10.8) and it was similar to the response to the composite image of “family member at the Eiffel tower” (7.8 spikes/s, median = 8.3; p = 0.96, W = 325, n1 = 27, n2 = 15, Wilcoxon rank-sum between the response to the Eiffel tower and the composite image).", "In order to verify that the increase in firing after learning was specific to the associated stimulus pair (NP) and not common to other stimuli used in the experiment, for example, due to an increase in familiarity, we also examined the response to the other stimuli.", "For each neuron X with a preferred stimulus Px and a non-preferred stimulus NPx, we defined the non-associated (NA) stimuli for neuron X to be all the other pictures used in the association experiment corresponding to the same category of the NPx stimulus (person or landmark).", "The bottom-right plot of Figure 2 shows the average response to all the NA stimuli, which decreased from a mean of 5.3 spikes/s (SD = 5.6) to 3.8 spikes/s (SD = 4.9) after learning.", "For some other units, the association was established the other way around, i.e., a neuron initially responding to a landmark changed its firing to the associated person after learning.", "Figure 3 shows a multi-unit in the parahippocampal cortex that, in Task 1 (before learning), originally fired to an image of the White House (mean = 17.8 spikes/s, SD = 7.2, median = 15) and not to American beach volleyball player Kerri Walsh (mean = 5.0 spikes/s, SD = 3.6, median = 3.3).", "After the patient learned the association between these two concepts (trial 1 in Task 2, see Experimental Procedures for learning criterion), there was an increase in the firing of the neuron to the picture of Kerri Walsh (mean = 13.8 spikes/s, SD = 9.2, median = 14.2), which was statistically significant (p < 0.05, Wilcoxon rank-sum test between baseline and response periods).", "This increase in the neuron’s response to Kerry Walsh (NP stimulus) after learning was observed in all tasks: a mean of 12.9 spikes/s in Task 2 (post-learning trials only), 16.7 spikes/s in Task 3, and 9.4 spikes/s in Task 5.", "The response to the preferred stimulus (the White House) increased slightly after learning to 25.6 spikes/s (SD = 8.9), but this difference was not significant (Wilcoxon rank-sum test).", "Additional examples are shown in Figure S1 and Movie S1.", "We recorded from a total of 613 units (438 multi-units and 175 single units) from the hippocampus (138 units), entorhinal cortex (117 units), amygdala (194 units), and parahippocampal cortex (164 units).", "We first identified visually responsive units, defined as those that, before learning, showed a significant difference in the response to at least one stimulus using a Wilcoxon rank-sum test between baseline and response (see Experimental Procedures).", "Altogether, we found 51 visually responsive units (31 single units and 20 multi-units) that significantly increased their firing rate in response to the preferred stimulus (P), with P being one individual (27 units) or landmark (24 units).", "Figure 4 shows the population results for all visually responsive units.", "Figure 4A shows the increase in response strength (comparing before and after learning) for each of the 51 visually responsive units and for all stimuli.", "The population averages are shown at the bottom of Figure 4A for all types of stimuli, where we observe a larger increase in firing after learning for the NP compared to the other stimuli.", "The change in firing rate after learning (see “Visually Responsive Units” in Experimental Procedures) was significantly different for the different stimuli according to a one-way ANOVA F(11,492) = 3.15, MSE = 0.46, p = 0.0001 (n = 42 cells with at least 12 stimuli—9 units that corresponded to sessions where less than 12 stimuli were presented were excluded from this analysis to avoid unbalanced data).", "This significant difference was largely due to the change in the NP stimuli and not any other non-associated stimulus.", "In fact, the difference was still significant when excluding the P stimuli (p = 0.01) but not when also excluding the NP stimuli (p = 0.76).", "Moreover, the only two stimuli that showed a median significantly different from zero were the preferred stimulus (decrease, p = 0.001; see below for interpretation in terms of repetition suppression) and the NP stimulus (increase, p = 0.005).", "Furthermore, paired t tests showed that the increases in the NP responses were significantly larger than the ones to any other stimulus (all p values between 0.0008 and 0.03).", "To further validate these results, we performed a permutation test, adjusted for multiple comparisons, by shuffling the labels of the stimuli and taking as test statistic the smallest difference between the activity to the NP stimulus and the one to any other stimuli.", "We ran 5,000 permutations and found the p value of the NP stimulus to be statistically significant (p = 0.012, see Supplemental Experimental Procedures for details).", "Of all the 613 units that we recorded from, 51 were visually responsive and 562 were non-responsive (i.e., did not have a significant response compared to baseline before learning).", "Of the 562 non-responsive units, 12 (2.1%) exhibited a significant increase to at least one image (mean = 4.1 images, SD = 1.5) after learning took place, according to a Wilcoxon rank-sum test between the baseline and response periods.", "This number is within what could be expected by chance (n = 28) with a false positive rate of 0.05.", "Only three of the non-responsive units had a change in response to an association pair (P and NP) that was larger than the one to the other pictures (Wilcoxon rank-sum test, p < 0.05).", "To further quantify the responses of all visually responsive neurons (to all of the presented stimuli), we calculated a pair-coding index (PCI), a correlation coefficient for each neuron between the mean response to each stimulus and its paired associate (as defined in Higuchi and Miyashita, 1996).", "This statistic has been used to assess how neurons acquire stimulus selectivity through associative learning and is expected to approach zero for a large number of neurons firing with a pattern independent of the stimulus pairs (Naya et al., 2003).", "Across the population of visually responsive units, we found that the pair-coding indices after learning (median = 0.35) were significantly higher (median = −0.03, D = 0.36, n1 = n2 = 42, p = 0.007, Kolmogorov-Smirnov test, see Figure 4B), thus showing the formation of an association between the P and NP stimulus pairs.", "To assess the changes that occurred in different tasks, we calculated, for the whole population of visually responsive units, an average differential activity index DAI = (Pr − NPr / Pr + NPr), where Pr, NPr denote the mean activity in the response interval (see Experimental Procedures).", "The DAI is expected to be positive, since Pr > NPr, and it quantifies the difference in the response to the preferred and non-preferred stimuli.", "As expected, the largest DAI values were obtained for Task 1 (Figure 4C) before learning took place, indicating a large difference in the response to the P and the NP stimuli.", "For the following tasks, DAI values were significantly smaller (p < 0.001, see Experimental Procedures).", "To study the time course of the responses, we separated the normalized population response for all visually responsive neurons according to the type of stimuli (P, NP, and NA) and condition (before and after learning).", "After learning (Figure 4D), we found a 172% increase in the response strength to the NP stimuli compared to the pre-learning value.", "This increase was statistically significant (p = 0.05, n = 51, Wilcoxon rank-sum test between the mean response before versus after learning).", "In contrast, the mean response to the preferred stimuli decreased to 87% of its pre-learning value (p = 0.3, n = 51, Wilcoxon rank-sum test), while the mean response to the non-associated stimuli did almost not change (101% of the pre-learning value, p = 0.9, n = 51, Wilcoxon rank-sum test).", "Given these population results, we next evaluated how many of the visually responsive neurons encoded the enforced associations.", "For this, we defined “pair-coding neurons” as the ones that: (1) showed a significant response to the NP stimulus after learning, using a Wilcoxon rank-sum test comparing baseline and response periods (with p < 0.05), and (2) the distribution of increases of single-trial responses to the NP stimulus after learning was larger than the distribution of increases of single-trial responses to all the other pictures (excluding P) after learning (see “Pair-Coding Units” under Experimental Procedures).", "Of the 51 visually responsive units, 21 (41%) were “pair-coding neurons” and selectively increased their response to the NP stimuli after learning.", "As expected by construction—since based on the screening sessions we chose the NP stimuli to be one that the neuron originally did not fire to—these units showed no significant response to the NP stimuli before learning took place (Wilcoxon rank-sum test).", "The number of neurons encoding the association (pair-coding neurons, n = 21/51) far exceeded the number expected by chance (p < 10−13), according to a binomial test with a chance level of 0.05 (see Supplemental Experimental Procedures).", "We also verified that the observed distribution of p values was significantly lower than the one generated by neurons with a Poisson firing probability and the same mean firing rates as the responsive units (p < 0.004; see “Proportion of Pair-Coding Units” under Supplemental Experimental Procedures).", "In what follows, we concentrate on the 21 neurons that encoded the associations.", "Among the 21 pair-coding units, 14 (67%) originally fired to a person and started firing to the associated landmark after learning (like the one shown in Figure 2).", "In the remaining 7/21 cases, the association was established the other way around, i.e., the neuron originally responding to a landmark, changed its firing to the associated individual after learning (like the one shown in Figure 3).", "Across the population of pair-coding neurons, the responses to the non-preferred stimuli showed an average increase of 281% (from 1.44 ± 0.22 to 4.06 ± 0 0.38, mean ± SEM) after learning, which was statistically significant (p < 10−5; Wilcoxon rank-sum test between the mean response before versus after learning) (Figure 5B).", "Similar results were obtained when considering only single units (n = 11).", "In this case, there was a significant increase of 412% in the response to the NP stimuli after learning (p = 0.0001, Wilcoxon rank-sum test).", "In line with the results for all visually responsive units (Figure 4C), the responses to the P (Figure 5A) and NA (Figure 5C) stimuli did not change significantly after learning (88% of the pre-learning value, p = 0.65 for the P stimulus and 134% of the pre-learning value, p = 0.27 for the NA stimuli).", "In order to compare on a trial-by-trial basis the neural and behavioral changes, we calculated the neuronal learning curves by rescaling the activity across the population of neurons encoding the association for all trials in all tasks to the range 0–1 (see Experimental Procedures).", "A direct comparison between the behavioral and neural learning curves exhibited a significant positive correlation for the non-preferred stimulus (r = 0.25, p < 10−11, Pearson’s correlation coefficient r), due to the increase in firing after learning the associations.", "There was a non-significant correlation for the non-associated stimuli (r = 0.05, p = 0.2) and also a negative correlation for the preferred stimulus (r = −0.08, p = 0.03), consistent with the decrease in firing to the preferred stimulus reported in Figure 4A, which is likely due to repetition suppression in line to a previous work without an association paradigm (Pedreira et al., 2010).", "To further investigate whether this behavior is due to repetition suppression, or whether it also reflects the formation of associations, we compared the decreases found in pair-coding units with the ones found in the other visually responsive units.", "For this, for each visually responsive unit, we calculated the percentage change as 100∗(Ppost − Ppre)/Ppre, where Ppost and Ppre indicate the mean activity in the response window.", "Both populations of pair-coding and non-pair-coding units exhibited similar trends, with a mean percentage decrease of −7% (SD = 40, median = −15%) for the pair-coding units and −11% (SD = 33, median = −20%) for the non-pair-coding units.", "The median of both populations did not differ significantly (p = 0.64, Wilcoxon rank-sum test) and the median of both populations differed significantly from zero (p = 0.03 and p = 0.02 for pair-coding and non-pair-coding, respectively), thus the decreases for the P stimuli seem to reflect repetition suppression rather than an encoding of the association.", "Given the variability on the number of trials that subjects needed to learn each association pair, to further evaluate how tightly correlated were the observed firing changes to the actual learning of the associations, we realigned the response of each cell to the learning time and compared these neural responses to the ones obtained as a function of the actual trial number in the experimental session.", "For this, we fitted logistic functions to the average behavioral and neural learning curves to the non-preferred stimulus for both alignments (absolute trials and relative to learning time; see Experimental Procedures for details).", "In Figure 5D, we first observe that aligning to learning time gives a more accurate matching between the behavioral and the neural learning curves.", "In fact, for data aligned to learning there were no significant differences between behavioral and scaled neural data (Kolmogorov-Smirnov test, p = 0.59).", "Additionally, the fits were more accurate for data aligned to learning time, as quantified by the Akaike Information Criterion (AIC) (Akaike, 1974)—data relative to learning: AICBeh = 142; AICNeu = 298; data not aligned: AICBeh = 175; AICNeu = 325, where smaller values denote higher accuracy.", "The same logistic model did not fit the data for the preferred and non-associated stimuli as accurately as it did with the non-preferred stimuli (Figure S2).", "Moreover, both for the P and NA, Pearson’s correlation coefficient was larger for the unaligned (R2 = 0.73/0.04 for P/NA stimuli) than for the learning-aligned data (R2 = 0.46/0.03).", "When the NP data were aligned to learning time, there was a large increase in the slope of the behavior curve: βBeh = 4.6 after re-alignment, compared to βBeh = 4.2 for the unaligned data.", "But the interesting fact was that this change in the behavioral learning curve was accompanied by an abrupt increase in firing to the non-preferred stimuli, when re-aligning the neural data to the learning time: βNeu = 3.1 aligned to learning, compared to βNeu = 1.6 without alignment.", "The slope difference (with and without alignment to learning) was significant, according to a non-parametric bootstrap test (p < 0.05; see Figure S3 and Experimental Procedures for details).", "Next, to rule out that changes in neuronal responses were just driven by one of the specific tasks we used (as each task varied in complexity and attentional demand) and not by the formation of new associations, we used the differential activity index DAI introduced before for the population of visually responsive units.", "Altogether, after learning there was a decrease of DAI values by a factor of 5.5 on average (range: 4.2–6.7).", "Moreover, differences between the tasks after learning were not significant, thus suggesting that these neuronal changes were not task dependent.", "Supporting this view, a direct comparison of the response to the non-preferred stimuli in the two identical tasks (Task 1, pre-screening and Task 5, re-screening) showed significant differences (p = 0.001, Figure 5E), which can be attributed to the learning of the particular association.", "There was also an increase in the response during Task 5 when considering all visually responsive units but in this case the difference was not significant (p = 0.14).", "From a readout viewpoint, the learning of the associations should be accompanied by a decrease in the discriminability between the NP and the P stimulus, given that the neuron originally firing only to the P stimulus starts also firing to the NP after learning.", "This selective increase in firing to the NP stimuli should also lead to more discriminability between the NP and NA stimuli after learning.", "This is indeed what we observed using a linear classifier to decode the identity of the stimuli before and after learning (see Supplemental Experimental Procedures).", "When considering the whole population of visually responsive units, the discrimination between P and NP stimuli went down from a 74% average performance before learning to 68% after learning.", "The decrease was significant according to a paired t test, t(100) = 1.95, p = 0.03.", "For pair-coding units, the discrimination between P and NP stimuli went down from a 72% average performance before learning to 56% after learning.", "Altogether, the decoding performance was significantly larger than chance with p < 0.05 (see Supplemental Experimental Procedures) for 11 of the 21 responses (52%) before learning and for 6 of the 21 responses after learning (38%).", "Two possible mechanisms can in principle account for the increased response to the NP stimuli after learning.", "On the one hand, neurons can rapidly change their tuning and start firing to the NP stimuli directly—that means, a neuron originally encoding the P stimulus starts encoding the NP stimulus after learning—in which case, the time courses of both P and NP signals are expected to be similar.", "On the other hand, the NP stimuli can act as a cue to evoke the representation of (and in turn the neuron’s firing to) the P stimuli.", "Following previous works (Naya et al., 2001, 2003), we distinguished between these two putative mechanisms—namely between Type 1 and Type 2 neurons—by analyzing the differences in the latency response onsets between the NP and P stimuli.", "In the first case (Type 1), we expect similar latency onsets for the P and NP stimuli, whereas in the other case (Type 2), we expect a larger latency onset for the NP stimuli.", "We used Poisson spike train analysis (see Experimental Procedures) to estimate the onset latency for all presentations and performed a Wilcoxon rank-sum test to compare the latency values for the P and NP stimuli.", "Of the 21 pair-coding units that selectively increased their firing to the NP stimuli after learning, 13 were “Type 1,” as in the example shown in Figure 2, and the remaining 8 were “Type 2,” as in the example shown in Figure 3.", "The scatter plot of the response onset latency values with the classification details is shown in Figure S4.", "Interestingly, both Type 1 and Type 2 units exhibited a significant positive correlation between behavioral performance and neural activity for the NP stimulus (Pearson’s r = 0.24, p = 10−7 and r = 0.28, p = 4∗10−6 for Type 1 and Type 2, respectively).", "Altogether, we identified 51 visually responsive units across different regions within the MTL: 10 in hippocampus, 7 in the entorhinal cortex, 29 in the parahippocampal cortex, and 5 in the amygdala.", "We observed pair-coding units throughout the MTL: (6 out of 10 [60%] visually responsive units in the hippocampus), 4 out of 7 (57%) in the entorhinal cortex, 11 out of 29 (38%) in the parahippocampal cortex, and 1 out of 5 (40%) in the amygdala.", "We consistently found both Type 1 and Type 2 neurons in these regions: 4 out of 8 pair-coding units in H/EC were of Type 1, where we have grouped responses in hippocampus and entorhinal cortex that were previously shown to exhibit similar properties (Mormann et al., 2008; Quian Quiroga et al., 2009).", "In PHC, 7 out of 11 pair-coding units were of Type 1.", "Pair-coding cells in H/EC were more prominently firing to pictures of persons instead of landmarks (6 out of 8 pair-coding units) compared to cells in PHC (n = 7 out of 11) but the difference was not significant (χ2 = 0.28, p = 0.60).", "Despite the small sample size of the recorded neurons, we found that the time courses of the responses in PHC were qualitatively similar to the ones in H/EC (Figure S5).", "As a cautionary note, we wish to point out that a larger number of recorded neurons is necessary to address the issue of regional differences (and similarities) more conclusively.", "Episodic memory—the ability to consciously recall personal experienced events and situations (Moscovitch, 1994; Tulving, 2002)—relies on the very rapid and effortless formation of new associations (Bunsey and Eichenbaum, 1996; Quian Quiroga, 2012; Wirth et al., 2003; Kahana et al., 2008).", "Animal studies have previously shown that single neurons can change their selectivity after learning in associative tasks (Erickson and Desimone, 1999; Gochin et al., 1994; Messinger et al., 2001; Sakai and Miyashita, 1991; Wirth et al., 2003).", "In particular, Miyashita and colleagues trained macaque monkeys to associate pairs of fractal patterns and found picture-selective neurons in IT cortex (areas TE and perirhinal cortex) that showed significantly correlated responses to the paired associates (Sakai and Miyashita, 1991).", "This coding was later hypothesized to emerge from separate TE neurons coding perceptual information about the individual paired associates that would converge onto the same neurons in the perirhinal cortex (the selective-convergence model) (Higuchi and Miyashita, 1996; Naya et al., 2001, 2003).", "But the learning of paired associates in animals is a demanding task that requires extensive reward-driven training, typically taking place before recordings begin (Erickson and Desimone, 1999; Higuchi and Miyashita, 1996; Sakai and Miyashita, 1991).", "Moreover, these recordings were performed in extra-hippocampal regions, which show distributed representations and are not thought to support fast learning according to modeling studies (McClelland et al., 1995).", "One notable exception was reported by Wirth and colleagues (Wirth et al., 2003; Yanike et al., 2004), who demonstrated a significant correlation between behavioral performance and neuronal hippocampal activity during the acquisition of associations between background scenes and specific actions (a saccade toward one of four cardinal locations).", "However, in this case the task also involved explicit reward-driven training, and learning occurred in two-thirds of the cases only after 14–17 trials (Wirth et al., 2009).", "These timescales are longer than the ones concomitant with episodic memory, which is seemingly effortless and often triggered by single presentations.", "Besides the need of reward-driven training, a major caveat to develop animal models of episodic memory is the lack of verbal or complex feedback to assess conscious recollection.", "In an earlier study, we showed that neurons in the human MTL respond in a reliable and specific manner during viewing of video episodes such as a clip of The Simpsons and also during the free conscious recall of that same clip (Gelbard-Sagiv et al., 2008).", "Human MTL neurons have also been reported to act as novelty and familiarity detectors (Rutishauser et al., 2006).", "A recent work (Miller et al., 2013) has studied modulations in the firing of place-responsive neurons in the human MTL while subjects learned item-location associations during a virtual navigation task followed by free recall.", "The authors calculated a neural similarity index between the ensemble activity of these place cells during navigation and during item recall and found that such index was higher for the ensemble of place cells near the location of the item.", "Considering the previous finding that MTL neurons show an invariant representation of concepts (Quian Quiroga et al., 2005), our results of association formation in these neurons suggest conceptual associations.", "In particular, we show: (1) the encoding of associations at the single-cell level, (2) the learning of the associations on a trial-by-trial basis (showing the emergence of robust responses at the exact moment of learning), (3) the precise latency of the responses, distinguishing two type of neurons, (4) the neurons’ responses in different tasks, including free recall, also comparing the exact same task before and after learning (Task 1 versus Task 5), (5) that these changes were specific to the associated (compared to the other non-associated) stimuli, and (6) a decoding approach provided differences in discrimination performance after learning consistent with our other analyses.", "Overall, by showing that such associations can be created with arbitrary but conceptually coherent concepts (i.e., persons in particular scenes, in contrast to pair association tasks in which two arbitrary pictures are associated), our results provide strong evidence pointing toward a role of the MTL beyond a spatial representation of the environment.", "Moreover, the emergence of associations of concepts established after single trials linked to rapid neural activity changes is ideal for the creation of new episodic memories (Quian Quiroga, 2012).", "How different MTL regions contribute to episodic memory formation is still a subject of intense discussion (Diana et al., 2007; Eichenbaum et al., 2007).", "Neuroimaging works have advocated that episodic encoding is mediated by the hippocampus, which supports the relational binding of the individual elements to the context of an episode (see Davachi, 2006; Quamme et al., 2007), and the parahippocampal cortex, which is involved in item memory (Kirwan and Stark, 2004) and/or in relational memory (Diana et al., 2007).", "The PHC has been shown to be involved in both spatial (Buffalo et al., 2006) and nonspatial contextual associations (Aminoff et al., 2007; Law et al., 2005).", "Related lesion studies in animals have suggested that the hippocampus is important for item-item associations, while parahippocampal cortex is critical for recognition memory for object-place associations (Higuchi and Miyashita, 1996; Malkova and Mishkin, 2003).", "In line with these studies, we found pair-coding units not only in H/EC (8/21) but also in PHC (11/21).", "A long-lasting debate in the psychology literature (Roediger and Arnold, 2012), refers to whether the formation of associations occurs gradually (Hull, 1943) or all-or-none (Estes et al., 1960; Rock, 1957).", "In the first case, the strength of association between each pair develops gradually until the first item produces a recall of the second.", "This assumes that learning reflects a continuous buildup of the strength of memory traces.", "Alternatively, association pairs could be learned at once and repeated trials are just giving several opportunities for the formation of the association (Estes et al., 1960).", "In line with the latter view, the formation of associations to NP stimuli changed abruptly, with a large increase in the slope of both the behavioral and neural learning curves, thus supporting all-or-none learning.", "A majority of neurons that changed their tuning after learning had a similar response-onset latency for the P and NP stimuli (Type 1), thus suggesting the creation of associations by combining distinct concepts through partially overlapping representations—namely, some of the neurons initially encoding one concept started firing to the associated one after learning (Quian Quiroga, 2012).", "One could in principle relate gradual learning with Type 2 neurons—in other words, the later response onset latency of NP compared to P may imply a recall of P when showing NP.", "However, learning occurred in a median of 1 trial for Type 2 neurons (and a median of 2 trials for Type 1 neurons).", "So, rather than supporting gradual learning, Type 2 neurons could be showing an evoked representation based on associations created using partial overlapping representations by Type 1 neurons (and it is also plausible to expect that after more repetitions the latency difference between P and NP may disappear).", "Importantly, associations were encoded by widening the tuning of neurons previously encoding one of the concepts, rather than by recruiting new neurons encoding each association because less than 1% of the initially non-responsive units started firing to a pair of associated images, compared to a 41% of visually responsive neurons that expanded their tuning to encode the associations.", "Due to clinical constrains and day-to-day variability of the recordings, it is currently not possible to assess how the changes in neuronal tuning reported here may evolve at longer timescales (days, weeks, months, or years) to establish long-term memories and a robust encoding of related concepts.", "It seems, however, reasonable to postulate that this initial encoding of associations, established after single presentations, may be further consolidated with time in some cases but may also disappear in others, considering that a relatively large proportion of neurons encoded the associations.", "Although the inception of episodic memories—like remembering the context and sequence of salient events when meeting a friend at a particular café—goes beyond the formation of contextual associations, our study suggests a fundamental mechanism of neuronal plasticity that may support episodic memory formation.", "14 patients with pharmacologically intractable epilepsy (10 right handed, 6 male, 18 to 53 years old) participated in this study.", "Patients were implanted with chronic depth electrodes for 7–10 days to determine the seizure focus for possible surgical resection.", "The number and specific sites of electrode implantation were determined exclusively on clinical grounds and were verified by MRI or by computer tomography co-registered to preoperative MRI.", "Patients volunteered for the study and gave written informed consent.", "The study conformed to the guidelines of the Medical Institutional Review Board at UCLA.", "Each electrode contained nine platinum-iridium microwires at their end.", "Eight of the microwires acted as the active recording electrodes and the ninth microwire acted as a reference.", "The differential signal from the microwires was amplified and filtered between 1 and 9,000 Hz.", "Data from six patients were recorded with a 64-channel Neuralynx system with a sampling rate of 28 kHz.", "In the remaining eight patients, data were acquired at 30 kHz using a 128-channel acquisition system (Blackrock Microsystems).", "The extracellular signals were band-pass filtered (300 Hz to 3 kHz) and later analyzed offline.", "Spikes were detected and sorted using wave_clus (Quian Quiroga et al., 2004).", "Single- and multi-unit activity was classified by one of the authors (M.J.I.) based on spike shape, variance, and the presence of a refractory period for the single units (i.e., <1% spikes within <3 ms interspike interval distributions) (Quian Quiroga et al., 2005).", "Subjects sat in bed facing a laptop computer on which pictures were presented.", "In the screening sessions, they were instructed to respond whether the image showed a person or not with a button press.", "Approximately 105 pictures were displayed six times in pseudorandom order (Ison et al., 2011; Quian Quiroga et al., 2005).", "In each recording session, a median of 8 (range: 2–28) of the recorded neurons responded to one or more pictures.", "Each of these responsive neurons fired to a median of 2 pictures (range 1–18 stimuli), which gives an average selectivity of 2.6% (range: 0.8%–30%), in agreement with values reported in a previous study showing an invariant representation by these neurons (Quian Quiroga et al., 2005).", "After each screening session, we selected a subset of the stimuli (mean: 14, range: 6–16) to create the images to be shown in the “association sessions,” as depicted in Figure 1 and described in the main text.", "Further information about these sessions can be also found in the Supplemental Experimental Procedures.", "All the methods described below correspond to the analyses of the association sessions.", "For each image presentation, we considered two intervals based on the response latency of neurons recorded from the medial temporal lobe (Mormann et al., 2008): a baseline interval starting 500 ms before stimulus onset and ending 100 ms after stimulus onset and a response interval between 200 ms and 800 ms after stimulus onset.", "Responses were defined as the median firing rate in a segment (baseline/response) across trials (Quian Quiroga et al., 2005).", "We identified visually responsive units as those that significantly responded to at least one individual or landmark before learning took place.", "The criterion for significance of the response was based on a Wilcoxon rank-sum test (with p < 0.05) between the baseline and response periods and we additionally required a median firing rate of at least 2 Hz following stimulus onset.", "For each stimulus presented (P, NP, and others), we quantified the firing rate changes after learning for stimulus “i” as Delta_i = Resp_i(AL) − Resp_i(BL), using a Z score normalization for each unit and phase (BL/AL): Resp_i = (mean(FR_i) − mean(FR))/SD(FR).", "We evaluated the quality of the fits following an information theoretic approach by means of the Akaike Information Criterion (Akaike, 1974).", "The lower the value of AIC, the more accurate the fit.", "To test the significance of the difference in the parameters (slope, AIC) for the neural data with different alignments, we performed a non-parametric bootstrap procedure (Kingdom and Prins, 2010).", "We defined pair-coding units as the ones that selectively changed their response to the associated picture after learning (see below for the definition of learning time), fulfilling the following criteria: (1) they had a significant increase in the response to the NP stimulus (the paired associate of the preferred stimulus) with respect to baseline after learning (Wilcoxon rank-sum test), and a non-significant response to NP before learning (Wilcoxon rank-sum test), and (2) the distribution of single trial increases after learning (i.e., subtracting the mean number of spikes before learning in the response window) for the NP stimulus was significantly larger than the distribution of single trial increases after learning for all the other pictures (excluding P) according to a Wilcoxon rank-sum test across trials.", "In the examples shown in Figures 2 and 3 and Figure S1, we used the raw data (number of spikes in the response window) and Wilcoxon rank-sum tests to compare between different conditions.", "For comparing the population responses before and after learning, we used normalized data (see “Time Courses of Behavioral and Neural Data”) and Wilcoxon rank-sum tests between responses before and after learning.", "To quantify the difference in firing in the different tasks, we computed a differential activity index DAI = (Pr − NPr / Pr + NPr), considering the mean activity in the response interval of the normalized response (where Pr and NPr are the mean normalized responses to the preferred [P] and nonpreferred [NP] stimuli, respectively).", "We used z tests to assess the significance of the difference in the DAI across different tasks (Figure 5F).", "Onset latencies for responsive units were determined by Poisson spike train analysis (Hanes et al., 1995; Mormann et al., 2008).", "To compare the latency values for the P and NP stimuli, we estimated the onset latency for all presentations and then performed a Wilcoxon rank-sum test.", "This procedure allowed us to separate the neurons into Type 1 neurons, which fired to the P and NP stimuli with a latency that was not significantly different (Wilcoxon rank-sum test and interquartile range < 250 ms), and Type 2 neurons, which showed a significantly longer latency to the NP compared to the P stimulus.", "M.J.I., R.Q.Q., and I.F. designed the electrophysiology study; I.F. performed the surgeries; M.J.I. collected the electrophysiological data; M.J.I. analyzed the data; M.J.I., R.Q.Q., and I.F. wrote the paper.", "R.Q.Q. and I.F. contributed equally to the study.", "All authors discussed the results and implications and commented on the manuscript at all stages." ]
[ "Contextual associations were used to model the formation of new memories", "Human single neurons changed their firing patterns to encode new associations", "Changes occurred at the exact moment of learning, even after single presentations", "The rapid speed of neural changes is compatible with episodic memory formation" ]
A new method for selecting sites for soil sampling, coupling global weighted principal component analysis and a cost-constrained conditioned Latin hypercube algorithm
Analysing spatial patterns of soil properties in a landscape requires a sampling strategy that adequately covers soil toposequences. In this context, we developed a hybrid methodology that couples global weighted principal component analysis (GWPCA) and cost-constrained conditioned Latin hypercube algorithm (cLHC). This methodology produce an optimized sampling stratification by analysing the local variability of the soil property, and the influence of environmental factors. The methodology captures the maximum local variances in the global auxiliary dataset with the GWPCA, and optimizes the selection of representative sampling locations for sampling with the cLHC. The methodology also suppresses the subsampling of auxiliary datasets from areas that are less representative of the soil property of interest. Consequently, the method stratifies the geographical space of interest in order to adequately represent the soil property. We present results on the tested method (R 2 = 0.90 and RMSE = 0.18 m) from the Guinea savannah zone of Ghana. • It defines the local structure and accounts for localized spatial autocorrelation in explaining variability. • It suppresses the occurrence of model-selected sampling locations in areas that are less representative of the soil property of interest.
[ "BIOC", "HEAL" ]
[ "Auxiliary dataset", "GWPCA", "Localised spatial soil variability", "Optimised soil sampling design", "Sampling design to represent both the feature and the geographical space", "cLHC" ]
[ "1308", "3607" ]
[ "Sampling designs aim at representing either the investigated soil property or the geographical space of a study area, or both [1,2].", "However, there are still shortcomings in the geographical-space sampling designs [3].", "Minasny and McBratney [2] proposed the conditioned Latin hypercube (cLHC) method as a feature-sampling approach, to address the shortcomings of the geographical-space sampling design.", "To ensure that a sampling strategy adequately represents both the geographical and the feature space, Minasny and McBratney [2] recommended considering the whole range of auxiliary data available for a study area.", "Based on this suggestion, Levi and Rasmussen [4] proposed a hybrid approach, in which they coupled an iterative principal component analysis (iPCA) with the cLHC.", "However, the iPCA hardly accounted for local spatial effects in their auxiliary datasets.", "The iPCA algorithm generally obscured the localized spatial effects in the auxiliary datasets [5–7].", "Generally, the iPCA algorithm is unable to correlate the measured soil property and its local environment, which depicts the geographic variations in the soil and environmental characteristics across space.", "Hence, iPCA ignores spatial characteristics.", "Therefore, several authors suggested to correct this shortcoming by including a localized weighted spatial auto-correlated principal component analysis [5,6,8].", "However, even coupling iPCA with cLHC still does not account for geographical weightings that provide principal component scores and loadings at all data locations [7].", "Therefore, in this paper we propose a global weighted principal component analysis (GWPCA) as an alternative to the iPCA.", "The advantage of the GWPCA is that it is able to recover the known dimensional spatial structures.", "Hence, it accounts for localized spatial autocorrelations in the algorithm that can explain the variability of auxiliary datasets [9].", "Consequently, we propose a new method, in which we couple GWPCA and a cost-constrained cLHC, to optimize the representation of both the feature and the geographical space.", "Similar to the scorpan concept [10], auxiliary datasets, represented as indices, were used to explain the local spatial heterogeneities and the soil property of interest at the selected sampling locations.", "We evaluated all localized spatial effects, trends and variabilities in the auxiliary datasets by GWPCA, adopting an automatic bandwidth in the GWPCA calibration.", "Next, using the selected GWPCA principal components as model input parameters, we selected optimal sampling locations using the cLHC algorithm executing 1 * 104 — 5 * 104 iterations.", "We incorporated a cost layer in the cLHC algorithm to suppress subsampling from areas that had only minor influence on the soil variable.", "Finally, we evaluated the model selections using root mean square error and correlation coefficient between model selected and actual locations.", "We chose soil moisture (SM) to test the method.", "In all stages of the proposed hybrid approach, we used R software [11], specifically the R packages factoextra [12], rsaga [13], stats [11], psych [14] and clhs [15].", "The study area (Fig. 1) is a major agricultural area of Ghana.", "Agriculture is the main base of the economic livelihood of the local population.", "The area is semi-arid, characterized by a single rainfall season, peaking in June, and a mean annual rainfall of 1200 mm.", "The daily mean temperatures vary between ˜32 °C (in August) and ˜43 °C (in March), and mean daily relative humidity varies between 18% and 97%.", "Elevation ranges from ˜39 m to ˜255 masl.", "The study area is largely flat with gentle slopes of 0.5–5% inclination.", "The study area has seven benchmark soils, reported here according to the World Reference Base for Soil Resources [18].", "The soils developed mainly on sandstone, shale, mudstone and quartzite of the Voltain platform, and alluvial sediments [19].", "Three main topographical units can be distinguished in the study area, i.e. upper slopes, middle to lower slopes, and toe slopes.", "The upper slopes are widely covered by Eutric Plinthosols (Kpelesawgu series, in the local classification system).", "On the middle to lower slopes, Petric Plinthosols (Changnalili series), Chromic Lixisols (Kumayili series) and Gleyic Panosols (Lima series) occur.", "The soil association on the toe slopes includes Fluvic Gleysols (Volta series), Plinthic Lixisols (Siare series) and Gleyic Fluvisols (Dagare series) (Fig. 2).", "Details on these soils were reported by Adu [16] and Adu and Asiamah [17].", "The vegetation of the area is mainly grassland with interspersed shea trees (Vitellaria paradoxa), Borassus palm (Borassus aethiopum) and Senegal mahogany (Khaya senegalensis).", "We selected twenty auxiliary datasets, which we considered to represent factors that contribute to the local spatial variability of SM.", "We thus expected these datasets to be suitable to explain the SM dynamics at the model selected sampling locations.", "They were also included in the modelling process, in order to optimize the selection of sampling locations.", "Collinearity between the auxiliary datasets was accounted for with the GWPCA algorithm [5].", "Table 2 and Fig. 3 present summaries of the auxiliary datasets used in this study.", "Each auxiliary dataset was either sourced or resampled at 100 m resolution via the cubic spline model of the R-GDAL package [20].", "DEM-derived surfaces were obtained via the SAGA-ta_morphometry and SAGA-ta_hydrology functions [21].", "Selecting an optimal bandwidth was the key to achieve an optimized GWPCA algorithm.", "Following the approach of Harris et al. [6], we adopted an automatic bandwidth selection process.", "The stack of auxiliary datasets had dimensions of 315, 579 and 21 (number of rows, columns and layers respectively) and 182,385 pixels.", "It was thus impossible within a reasonable time-span to automatically select a bandwidth using the entire auxiliary datasets, which had GWPCA loadings at each 182,385 pixel sites.", "This challenge was related to the required computing power and processing time in the cross validation algorithm, because each observation omitted was reconstructed using the principal components (PC) derived from the observations of the entire stack of auxiliary datasets.", "Therefore, we randomly selected 10% of the auxiliary datasets for use in the automatic bandwidth selection process.", "The selection of 10% of the datasets was guided by a series of cross validation evaluations.", "We identified an adaptive bandwidth of 16.2 km at k = 10 (principal components) as the optimum minimized fit between the score and auxiliary data.", "In the cLHC annealing simulation process, model optimization was implemented by executing 1*104–5*104 iterations (increments of 1*104).", "Because the user can define the number of cLHC selection outputs, we defined 38 locations in this study, as this was the maximum number of access tubes we had for the SM measurements.", "Thus, depending on the objectives and resources available for a particular study, users of this methodology can assign any maximum number of outputs.", "Roudier et al. [15] used rough terrain, surface gradient and distance to road or trail network as criteria to design their cost constraint in simulating the annealing process.", "In this study, we used a similar approach but a different key criterion, namely the topographic wetness index (TWI), as a suitable constraint indicator directly affecting SM.", "The TWI is generated with an upslope contribution area [22] and accounts for water redistribution within crest, ridges and depressions in an area [23].", "The TWI gives an indication of the potential SM contribution areas within the top and bottom soil layers [24] and quantifies the spatial scale effects on hydrological processes [25–27].", "In the TWI, slope inclination is classified at very short ranges to account even for slight changes in topography and local slope.", "In this study, low values represented crests and ridges, whereas high values represented depressions.", "We assumed that rainfall, infiltration, percolation and flooding by rivers during the rainy season are the only means by which SM is replenished in the soil layers of the study area.", "This assumption is based on the fact that in the study area, there is an inherent strong plinthic to petroplinthic horizon at ˜60–100 cm depth, which largely hinders contribution of ground water to SM [16,17,28].", "Hence, we chose the TWI as cost-constraint criterion for assessing SM in the study area in the cLHC annealing simulation (Fig. 4).", "Using the rsaga package [13], we developed a TWI layer at a 100 m resolution.", "Only PCs that accounted for eigenvalues ≥ 1 were considered.", "Temperature showed the strongest positive correlation, followed by bulk density, silt and clay contents (Fig. 5).", "Increasingly negative correlations were found for water balance < precipitation < DEM < drainage < sand content < available water content and sentinel-1 A. Within the rotational matrix of the global weighted PCs, available water content always showed the largest contribution in the list of auxiliary datasets, whereas sand content contributed least.", "Thus, we conclude that most of the variation was explained by available water content.", "PC1 to PC6 together explained 91.5% of the observed variance (Supplementary material (SUPP) Table S1).", "Generally, the highest positive loading of 0.552 was recorded for PC5 and the lowest negative loading of -0.453 was recorded for PC6.", "Together, PC1 and PC2 accounted for 74.7% of the local variation in the list of auxiliary dataset used.", "These findings suggest that AWC, clay and silt content are the key factors that need to be considered to explain local variability of SM.", "By adding PC3, the explained variability reached 82.1%.", "PC4 explained an additional 3.5% of the observed local variation.", "In PC4, soil type showed the highest positive loading of 0.446, whereas river distance exhibited the lowest negative loading of -0.445.", "We thus conclude that the local soil types, affected by their distance to rivers, markedly influence the spatial variability of SM.", "Adding PC5 and PC6 resulted in a cumulative explained variability of 88.1% and 91.5%, respectively.", "River distance exhibited the lowest negative loadings in PC4, PC5 and PC6.", "AWC showed maximum positive loadings in the GWPCA two times.", "Thus, among the list of auxiliary datasets used in this study, AWC and river distance showed the strongest influence on the spatial variability of SM.", "The reason for the important role of distance to a river that we found in this case may be the dense river network and the generally low elevation within the area that leads to a far-reaching riparian influence.", "While the PCs showed the observed variances in the auxiliary datasets, it also indicated the collinearity between the auxiliary datasets (SUPP Table S1).", "The larger or smaller the first or last PC, respectively, the stronger the collinearity between the auxiliary datasets [5].", "Hence, local variables that caused the local collinearity in the auxiliary datasets was identified and eliminated via the multivariate glyph cartogram plot prior to its use in the cLHC [5].", "The local spatial variability was analysed in the GWPCA using a 16.2 km bandwidth window around each data point in the auxiliary datasets.", "This bandwidth is equivalent to half of the maximum distance from river networks (minimum = 0 km, maximum = 32.3 km).", "The selected automatic bandwidth interprets as ˜50% of the entire auxiliary datasets were retained each time in the cross validation algorithm to calibrate the GWPCA.", "Thus ˜50% of the auxiliary datasets were used each time to explain the localised spatial variabilities of the study area.", "In addition, the automatic bandwidth ensured a balance between the local variation and locations with less influence on the spatial variability, as reported by Kumar et al. [7].", "Our findings suggest that the use of GWPCA will account for the local influences and collinearity of each auxiliary datasets on the proposed SM measurements with regards to their locality.", "The final output of the cLHC ensures that the proposed sampling locations cover 91.5% of the locally occurring site conditions, represented by the auxiliary datasets.", "To implement the cLHC, the algorithm analyzed the selected PCs of the GWPCA to identify points in the landscape representing a Latin hypercube, similar to the approach of Minasny and McBratney [2].", "In the subsequent series of iterations of this spatial representation of site conditions, the level of optimization of the objective function of the cLHC was shown at each iteration.", "Perturbations were recorded in both the objective and the cost-function optimization processes at <5*103 iterations, similar to what Roudier et al. [15] reported.", "Contrary to the findings of Roudier et al [15], our results showed a clear full model optimization after 5*104 iterations, both in the evolution and in the cost functions of the cLHC algorithm (Fig. 6).", "Between 1*104 and 22*103 iterations of the objective function, the optimization steadily increased until 5*104 iterations were completed.", "A comparison of the subsamples drawn from the selected PCs of the GWPCA used in the cLHC simulating annealing process is presented in SUPP Figure S1.", "Generally, the subsamples were within the first and third quartiles of the selected PCs of the auxiliary datasets.", "Only very small subsamples were drawn at locations with low TWI (ridges and crests).", "At sites with high TWI (depressions), large subsamples were drawn for use in the cLHC simulating annealing process.", "The reason for this difference is that the cLHC simulating annealing process suppressed sampling of locations with little or no influence on spatial SM variability in the study area (in this case, from ridges and crests).", "Thus, the probability of model selected sampling locations on ridges and crests was low.", "A single realization of the cLHC output shows that the cost layer in the cLHC influenced the selection of sampling locations (Fig. 7).", "The cost-constrained cLHC stratified the selected sampling locations, based on the influence of TWI (similar to results reported by Levi and Rasmussen [4]).", "In our study, the selected sampling locations represented the soil property of interest and in the geographical space, as also reported by Hengl et al. [1].", "Locations selected by unconstrained cLHC algorithms were dispersed across the entire study area (Fig. 7).", "The reason is that the cLHC optimization process accounted for all key landscape heterogeneities that occurred within the study area.", "The wide spatial distribution of sampling locations indicates that the cLHC effectively selected sampling locations, in terms of both the soil property of interest and the geographic space (Fig. 7).", "However, some locations were selected similarly under both the unconstrained and the cost-constrained cLHC algorithms.", "This hybrid approach enables a scientist to assess the local variability of a soil property of interest and to derive adequate sampling designs for analysing that soil property across a defined study area.", "The highest (1) and lowest (0.3) average scores were achieved by 71% and 2.63%, respectively, of the model selected sampling locations.", "Test of the method showed a RMSE of 0.18 m with a correlation coefficient (R2) between actual and model selected location of 0.90.", "Our findings indicates that the model selected sampling locations were very close to the in-situ sampling locations.", "AT14 and AT21 were found not suitable probably due to the main input dataset in the GWPCA, which affected the output of the cLHC algorithm.", "Although 91.5% of the observed local variability in the auxiliary dataset was explained by the GWPCA, it must be emphasized that incorrect input parameters can limit the performance of the cLHC.", "In this paper, we have presented a new approach for selecting soil sampling locations that adequately represent both the soil property of interest and the geographical space across a study area.", "We implemented the proposed hybrid approach under a cost-constrained conditioned Latin hypercube algorithm, by using inputs from a global weighted principal component analysis.", "This method defines the local structure and accounts for localized spatial autocorrelation in explaining soil-landscape variability.", "The method also suppresses the occurrence of model-selected sampling locations in areas that are less representative of the soil property of interest.", "In addition, the method provide an appropriate base for selecting adequate sites for a given number of possible measurements.", "The proposed approach can guide the selection of adequate sites for soil measurements and installations of soil-monitoring stations, in the context of scientific studies and agricultural interventions.", "Authors declare no conflict of interest." ]
[]
Data on migration of the non-invasive breast cancer cell line, MCF-7 treated with Bevacizumab using Real Time Cell Analyzer (RTCA)
Bevacizumab or Avastin® (Av), the recombinant antibody targeting VEGF, improves progression-free but not overall survival of metastatic breast cancer patients due to development of Av resistance. We showed that Av-therapy-induced inflammatory microenvironment contributes to the refractoriness to Av treatment. Here we present data regarding the effect of Av treatment on migration of a non-invasive breast cancer cell line, MCF-7. The data presented hereis related to the research article “Bevacizumab induces inflammation in MDA-MB-231 breast cancer cell line and in a mouse model” (Hajjar et al., 2018).
[ "MULT" ]
[ "Bevacizumab", "MCF-7", "Migration", "RTCA" ]
[ "1000" ]
[ "MCF-7 cells were treated with 50 μg/ml of Av for 24 h. MCF-7 cells showed no change in their morphology (Fig. 1).", "RTCA was used to measure the proliferation and migration of treated MCF-7 cells.", "The interaction of cells with a gold electrode correlated with impedance, which was reported as the cell index.", "Cells were treated, trypsinized and seeded in RTCA E- and CIM- plates to assess proliferation and migration, respectively.", "An inhibition of cellular proliferation by 22% in MCF-7cells was observed.", "There was an increase in migration which reveals the motility behavior of Av-treated MCF-7 cells.", "This observation was accompanied by a significant increase in mRNA expression levels of epithelial to mesenchymal transition (EMT) markers (Twist and Snail) (Fig. 2).", "Quantitative analysis of the effect of Av treatment on the proliferation and migration of MCF-7 cells was performed as previously described [2] with slight modifications using RTCA(×CELLigence RTCA[A2]DP, Roche Applied Science, USA).", "Cells were grown in 6-well tissue culture plates at a density of 10,000 cells/cm2 and treated or not with 50 μg/ml Av for 24 h. For migration assays cells were harvested, counted, re-suspended in 120 μl of serum-free media and seeded at a density of 20,000 cells/well in the upper chamber of CIM-plates.", "For proliferation assays, cells were seeded similarly, but in an E-plate at a density of 7000 cells/well with an additional 120 μl of media containing 10% serum.", "Migration and proliferation were monitored every 15 min for a minimum of 18 h by recording the cell impedance produced as the cells attached and detached from the gold electrodes in the CIM and E-plates.", "The RTCA software generated a survival curve and estimated the cell survival or cell index (CI).", "CI correlates directly with cell number.", "Data were expressed as bar graphs of CI % of control.", "Total RNA was isolated from cells in culture using Nucleospin® RNA II Kit (Machery-Nagel, USA) according to the manufacturers’ instructions.", "1 μg of total RNA was first reverse transcribed to cDNA using RevertAid 1st strand cDNA synthesis kit (Thermo, USA) and then amplified by qPCR using iQ SYBR Green Supermix in a CFX96 system (Bio-Rad Laboratories, USA).", "Primers were designed against human genes (TIB MOL BIOL, Germany) with the following sequences:", "Twist: F: AGCTACGCCTTCTCGGTCT and R: CCTTCTCTGGAAACAATGACATC", "Snail: F: CTTCCAGCAGCCCTACGAC and R: CGGTGGGGTTGAGGATCT", "GAPDH: F: TGGTGCTCAGTGTAGCCCAG and R: GGACCTGACCTGCCGTCTAG", "ΔΔCq was used to calculate the relative fold change in gene expression after normalization to the housekeeping gene, GAPDH.", "Results are expressed as average ±SEM.", "Statistical comparisons were done using student׳s t-test in order to determine statistical significance.", "P value was determined and significance level was set at P < 0.05.", "Microsoft Excel was used to perform statistical analysis." ]
[]
Outcomes and comorbidities of SCN1A-related seizure disorders
Purpose: Differentiating between Dravet syndrome and non-Dravet SCN1A-related phenotypes is important for prognosis regarding epilepsy severity, cognitive development, and comorbidities. When a child is diagnosed with genetic epilepsy with febrile seizures plus (GEFS +) or febrile seizures (FS), accurate prognostic information is essential as well, but detailed information on seizure course, seizure freedom, medication use, and comorbidities is lacking for this milder patient group. In this cross-sectional study, we explore disease characteristics in milder SCN1A-related phenotypes and the nature, occurrence, and relationships of SCN1A-related comorbidities in both patients with Dravet and non-Dravet syndromes. Methods: A cohort of 164 Dutch participants with SCN1A-related seizures was evaluated, consisting of 116 patients with Dravet syndrome and 48 patients with either GEFS +, febrile seizures plus (FS +), or FS. Clinical data were collected from medical records, semi-structured telephone interviews, and three questionnaires: the Functional Mobility Scale (FMS), the Pediatric Quality of Life Inventory (PedsQL) Measurement Model, and the Child or Adult Behavior Checklists (CBCL/ABCL). Results: Walking disabilities and severe behavioral problems affect 71% and 43% of patients with Dravet syndrome respectively and are almost never present in patients with non-Dravet syndromes. These comorbidities are strongly correlated to lower quality-of-life (QoL) scores. Less severe comorbidities occur in patients with non-Dravet syndromes: learning problems and psychological/behavioral problems are reported for 27% and 38% respectively. The average QoL score of the non-Dravet group was comparable with that of the general population. The majority of patients with non-Dravet syndromes becomes seizure-free after 10 years of age (85%). Conclusions: Severe behavioral problems and walking disabilities are common in patients with Dravet syndrome and should receive specific attention during clinical management. Although the epilepsy course of patients with non-Dravet syndromes is much more favorable, milder comorbidities frequently occur in this group as well. Our results may be of great value for clinical care and informing newly diagnosed patients and their parents about prognosis.
[ "MEDI", "NEUR" ]
[ "Behavioral problems", "Comorbidities", "Dravet", "GEFS +", "SCN1A" ]
[ "2728", "2802", "2808" ]
[ "Pathogenic variants in SCN1A can cause several different epilepsy syndromes, with varying disease severities [1–5].", "The most common and severe associated condition is Dravet syndrome, which is characterized by intractable epileptic seizures and a slowing of the psychomotor development in the second year of life, which results in mild to severe intellectual disability (ID).", "Walking difficulties and behavioral problems are common comorbidities [6–9].", "Milder phenotypes include genetic epilepsy with febrile seizures plus (GEFS +) syndrome and febrile seizures (FS and febrile seizures plus (FS +)), in which usually no ID is present, and the epilepsy has a milder course [4,10].", "Although the majority of school-aged, adolescent, and adult patients with SCN1A-related disease can easily be classified as having Dravet syndrome or not, these different phenotypes may have a similar presentation at onset [11,12].", "SCN1A encodes for the α-subunit of a neuronal sodium channel, Nav1.1.", "Pathogenic variants cause a reduction in sodium currents in gamma-aminobutyric acid (GABA)-ergic inhibitory interneurons, which leads to hyperexcitability of neuronal networks and the occurrence of seizures [13,14].", "These reduced sodium currents, furthermore, impair Purkinje cells, causing motor disorders [15,10] and contribute to the development of behavioral problems and cognitive disabilities [16].", "The association of SCN1A pathogenic variants with multiple syndromes can be partly explained by the consequences of different mutation types: pathogenic variants that lead to a complete loss of function of the channel are virtually always associated with severe phenotypes whereas milder disturbances in channel function usually cause milder phenotypes [17].", "However, in clinical practice, it remains difficult to fully predict the effects of all variants on channel function.", "Differentiating between Dravet syndrome and non-Dravet SCN1A-related phenotypes is understandably of extreme importance for families and physicians.", "The presence or absence of ID and the frequency and severity of seizures both significantly alter the level of care an affected child requires.", "Furthermore, severe comorbidities, such as walking disabilities or behavioral problems, are frequently reported in Dravet syndrome [7–9,18–23] and may pose a heavy burden on affected families.", "Behavioral problems have been identified as the strongest independent predictor for lower quality-of-life (QoL) scores [24] and are reported to often be a cause of stress and concern for parents [23,25].", "Motor disorders have been shown to contribute significantly to lower health-related QoL (HRQoL) scores as well [24].", "However, also, when a child is diagnosed with a milder SCN1A-related disorder, such as GEFS + or FS, accurate information about the disease course and prognosis is essential.", "Many studies have reported on the clinical spectrum of different SCN1A-related syndromes [12,17,26], but detailed information on seizure course, seizure freedom, medication use, and comorbidities is lacking for the milder patient group.", "Although ID is thought to be exclusive to patients with Dravet syndromes, there are reports of patients with non-Dravet syndromes that show a mild cognitive impairment or neuropsychiatric symptoms [27–29].", "However, the incidence of these problems in patients with non-Dravet SCN1A-related epilepsy is unknown, as they are mostly described in case reports.", "We describe here the detailed clinical data of a large cohort of Dutch patients affected by SCN1A pathogenic variants (n = 164), consisting of patients with Dravet syndrome as well as GEFS +, FS +, or FS.", "We explore the nature, occurrence, and relationships of SCN1A-related comorbidities in both the Dravet and non-Dravet groups, to improve the counseling of patients and their parents.", "We furthermore give a detailed overview of the disease course of patients with non-Dravet syndromes to provide more insight in the clinical spectrum of the less severe SCN1A-related disorders.", "A previously described cohort [11,30] of 164 patients affected by SCN1A-related seizures was included in this study.", "Only symptomatic participants with heterozygous pathogenic or likely pathogenic variants (classes IV and V, according to the American College of Medical Genetics and Genomics criteria [31]) in SCN1A were included.", "All eligible individuals of at least 4 years of age known to the University Medical Center Utrecht were approached.", "Patients below the age of 4 years were excluded since syndrome classification and estimation of disease severity are less reliable for younger children.", "Informed consent was obtained from participants or their legal caretakers, according to the Declaration of Helsinki.", "The study was approved by the Ethical Committee of the University Medical Center Utrecht.", "Detailed clinical data were retrospectively collected from medical records of all participants, and a semi-structured telephone interview was conducted when possible at the time of inclusion (n = 155).", "Interviews were conducted with participants themselves if they were adults and mentally competent; in all other cases, interviews were conducted with parents of patients.", "Participants, or parents of participants, were asked the kind of education the participants were following or had followed and whether they had any learning problems or psychological problems.", "If parents of patients were interviewed and not the patients themselves, they were also asked whether their children showed any behavioral problems (for other patients reported as missing data).", "The same information was extracted from medical files.", "Furthermore, three questionnaires were completed by participants in specific age groups, or their parents, at the time of inclusion:", "The Dutch version of the Functional Mobility Scale (FMS), to classify the general functional mobility in six categories for children aged 4–18 years [32] (Hugh Williamson Gait Laboratory, The Royal Children's Hospital Melbourne, Australia, Part of the Gait CCRE, www.rch.org.au/gait, Graham 2004);", "The Dutch version of the PedsQL Measurement Model, to measure HRQoL on a 0–100 scale for participants aged 0–25 years [33];", "The Dutch parent report version of the Child Behavior Checklist 1.5–5 years (CBCL 1.5–5) or 6–18 years (CBCL), or the Dutch version of the Adult Behavior Checklist 18–59 years (ABCL) to evaluate behavioral and emotional problems [34,35].", "Behavioral problems in the clinical range on the “total problems” scale are reported (p-scores > 90, according to the CBCL manual).", "Cognitive functioning at the time of inclusion was classified in a consensus meeting by a child neurologist, neuropsychologist, and clinical geneticist and rated on a five-point scale based on available data on intelligence quotient (IQ) and developmental level, (1 = no ID (IQ or developmental quotient (DQ) > 85), 2 = borderline ID (IQ or DQ 70–85), 3 = mild ID (IQ or DQ 50–70), 4 = moderate ID (IQ or DQ 30–50), 5 = severe or profound ID (IQ or DQ < 30)).", "When no (recent) IQ or DQ was available, the assessment was made based on school functioning, communication, and/or adaptive behavior.", "All participants were categorized into two clinical subgroups: Dravet syndrome or non-Dravet syndrome.", "Dravet syndrome was diagnosed based on previously published criteria [36].", "The diagnoses were in line with recently published recommendations [37].", "The non-Dravet group consisted of patients with either GEFS + or FS.", "Seizure severity was classified based on seizure frequency for both minor seizures (defined as short absences, short focal seizures, or myoclonias) and major seizures (defined as all other seizure types with loss of consciousness or prolonged seizures) at the time of inclusion (score 4 = daily seizures, score 3 = weekly seizures, score 2 = monthly seizures, score 1 = yearly seizures, score 0 = seizure-free (> 1 year)).", "Data on major disease outcomes (cognitive functioning, seizure severity, walking difficulties, behavioral problems, and HRQoL) are reported as total counts, percentages, or mean/median scores for all patients, and additional detailed clinical information (on seizure frequency, antiepileptic drug (AED) use, learning problems, and psychological/behavioral problems) is reported per age group for patients with non-Dravet syndromes.", "No statistical testing was performed to formally assess differences in outcomes between patients with Dravet and non-Dravet syndromes, since these outcomes were used to classify each patient and therefore, differ per definition between the groups.", "Correlations between different outcomes were calculated with Spearman's rank-order correlation.", "Differences between groups were calculated with either Pearson's chi-square test or Fisher's exact test for binary and categorical variables or a Mann–Whitney U test for continuous and ordinal variables.", "Statistical analyses were performed using SPSS statistics software (IBM SPSS Statistics for Windows V21, Armonk, NY: IBM Corp.).", "All reported tests were performed 2-tailed with an alpha-level for significance of p < 0.05.", "Correlations for the non-Dravet group only were only investigated for the group of patients below 20 years old, because childhood learning and behavioral problems may not have been reported reliably for older patients because of recall biases.", "The characteristics of the study population are depicted in Table 1.", "The Dravet syndrome subgroup consisted of 116 patients belonging to 112 different families, and the non-Dravet group consisted of 48 patients belonging to 28 different families.", "Six families had members in both the Dravet and non-Dravet categories.", "The median age of patients with Dravet syndrome and patients with non-Dravet syndromes was 14 years and 22 years respectively.", "Important differences between both groups were seen for all five major outcomes.", "All patients could be assessed for cognitive outcome.", "Almost half of all patients with Dravet syndrome had a severe cognitive disability (score 5, 45%) whereas almost all patients with non-Dravet syndromes had normal cognitive capacities (score 1, 90%) except for five, who had a slight delay (score 2, 10%) (Table 1).", "Since cognitive functioning is a defining characteristic used to classify Dravet syndrome, this is an expected outcome.", "Cognitive disabilities worsened with age in patients with Dravet syndrome; over 60% of patients of 20 years and older had a score of 5, in contrast to only 25% of patients aged 7–8 years (Fig. S1).", "Interestingly, nine patients with non-Dravet syndromes experienced a slowing of development after the first year of life as well; however, in two cases, the delay was only temporary, and none of these patients developed ID.", "All patients could be assessed for seizure frequency.", "Many patients with Dravet syndrome experienced major seizures weekly (44%) and minor seizures daily (41%).", "In the non-Dravet group, both major and minor seizures occurred much less frequently, with most patients being seizure-free at the time of inclusion (73%) (Table 2).", "If seizures did still occur in the non-Dravet group, these were mostly yearly events (23%).", "The youngest patients with non-Dravet syndromes had most seizures: only 38% of 4- to 9-year olds were seizure-free whereas 82% of 10- to 19-year olds and 100% of patients older than 40 years were seizure-free.", "Although 71% of patients with non-Dravet syndromes had used maintenance treatment, 60% was medication-free at the time of inclusion.", "Larger percentages of older patients were medication-free than younger patients were medication-free than younger patients (87% of 40 +-year olds versus 38% of 4- to 9-year olds).", "Interestingly, one patient with non-Dravet syndrome experienced minor seizures (myoclonias) daily.", "This 16-year old girl used to have generalized tonic–clonic seizures but had been seizure- and medication-free between ages 4 and 10 years old, after which seizures reappeared.", "At the time of study, she had monthly focal seizures with impaired awareness and a few myoclonias per day while being treated with topiramate and levetiracetam.", "She was not diagnosed with Dravet syndrome because she had a normal intellect (IQ 91), and her seizure course is unusual for Dravet syndrome.", "Sixty-three patients with Dravet syndrome and 20 patients with non-Dravet syndromes could be assessed for functional mobility.", "Patients with Dravet syndrome showed either no walking disabilities (independent walking on all surfaces, score 6), minor walking disabilities (independent walking on level surfaces, score 5), or used a wheelchair (score 1) on a 500-meter range.", "Over 40% of patients with Dravet syndrome already used a wheelchair at age 4–8 years; this was 50% at 14–18 years (Fig. 1).", "Patients with non-Dravet syndromes did not show any walking disabilities on the FMS.", "Eighty patients with Dravet syndrome and 42 patients with non-Dravet syndromes could be assessed for behavioral problems by CBCL/ABCL questionnaires.", "Thirty-seven patients (30%) showed behavioral problems in the clinical range (Table 1); all but three were patients with Dravet syndrome (43% versus 7%).", "One of the patients with non-Dravet syndromes with behavioral problems in the clinical range is a 32-year-old father of a son with Dravet-syndrome, who was found to be mosaic for their pathogenic SCN1A variant in previous research [30,38].", "Since his epilepsy was well controlled and no ID was present, he was not diagnosed with Dravet syndrome; he, however, does have psychosocial problems, among which an autism spectrum disorder, an active substance addiction, and aggressivity.", "The second patient (14 years old) has an autism spectrum disorder and an IQ of 90; she has been seizure-free since 2.5 years but has previously experienced different seizure types, including multiple absences per day.", "The third patient is 28 years old and has a normal intellect but was schooled at a low level and now works in a sheltered environment because of his epilepsy and memory problems.", "He has around 10 generalised tonic-clonic seizures (GTCS) per year.", "Most behavioral problems in the clinical range were reported in younger patients with Dravet syndrome: 60% of 4- to 11-year olds score within the clinical range whereas less than 30% of 20 +-year-old patients do (Fig. 2A).", "One hundred six patients with Dravet syndrome and 27 patients with non-Dravet syndromes could be assessed for behavioral problems during the telephone interview.", "Parents of patients responded “yes” to the question “are there any behavioral problems?”", "during the telephone interview in 66% of the patients with Dravet syndrome while only 7% did in patients with non-Dravet syndromes (Table 1).", "The CBCL/ABCL data were missing for 36 patients; similar percentages of parents reported behavioral problems during the telephone interview for patients with and without CBCL/ABCL results.", "Most behavioral problems were reported during the telephone interview for patients with Dravet syndrome between 8 and 11 years old (> 80%, Fig. 2B).", "Seventy-one patients with Dravet syndrome and 22 patients with non-Dravet syndromes could be assessed for HRQoL.", "Patients with Dravet syndrome showed lower HRQoL scores than patients with non-Dravet syndromes (average 52.6 versus 88.5, Table 1, Fig. 3).", "Scores tended to worsen with age (Fig. S2).", "Although patients with non-Dravet syndromes were significantly less affected on all five major outcome scales than patients with Dravet syndrome, we did observe subtle problems in this group (Table 2).", "Twenty-seven percent had encountered some kind of learning problem for which extra educational assistance was necessary (see Supplementary data 1 for specific problems).", "None of the patients over 40 years old reported to have experienced problems while following primary or secondary education.", "Although patients with non-Dravet syndromes were significantly less affected on all five major outcome scales than patients with Dravet syndrome, we did observe subtle problems in this group (Table 2).", "Twenty-seven percent had encountered some kind of learning problem for which extra educational assistance was necessary (see Supplementary data 1 for specific problems).", "None of the patients over 40 years old reported to have experienced problems while following primary or secondary education.", "Behavioral problems, like attention-deficit, autistic features, or anxiety, were reported for 38% of patients with non-Dravet syndromes among all age groups, although their CBCL score was not in the clinical range except for three.", "Two of these 18 patients had not completed the CBCL questionnaires (see Supplementary data 1 for specific problems).", "Behavioral problems, like attention-deficit, autistic features, or anxiety, were reported for 38% of patients with non-Dravet syndromes among all age groups, although their CBCL score was not in the clinical range except for three.", "Two of these 18 patients had not completed the CBCL questionnaires (see Supplementary data 1 for specific problems).", "In the complete cohort, FMS scores were strongly related to cognitive outcomes, seizure severity, and QoL scores: patients with more severe walking disabilities had a more severe cognitive disability (rs = − 0.682, p < 0.0005, Fig. S3A), a higher seizure frequency (rs = − 0.537 for major seizures and rs = − 0.492 for minor seizures, both p < 0.0005, Fig. S3B), and a lower QoL score (rs = 0.668, p < 0.0005, Fig. S3C).", "Most behavioral problems were observed in patients with a mild cognitive disability (Fig. 4A, B); less behavioral problems were seen in patients with cognitive outcome scores on both ends of the spectrum.", "Behavioral problems in the clinical range of the ABCL/CBCL questionnaires were positively related to major seizure frequency (rs = 0.260, p = 0.004, Fig. S4A) in the complete cohort; however, in patients with Dravet syndrome, most behavioral problems were reported in patients that are seizure-free or experience daily seizures (Fig. S4B).", "Furthermore, patients with behavioral problems in the clinical range scored significantly lower on the QoL questionnaire (rs = − 0.523, p < 0.0005, Fig. S4C).", "In the complete cohort, cognitive outcome scores were significantly related to major seizures (rs = 0.693, p < 0.0005) and minor seizure frequencies (rs = 0.511, p < 0.0005) (Fig. S5A, B).", "In the non-Dravet group only, the presence of any learning problems was significantly associated with a lower QoL score (median score of 86.45 versus 92.95, p = 0.006 (Mann–Whitney U, U = 19, z = − 2.708), Fig. S6A).", "No statistically significant differences in QoL scores were found between patients with and without psychological/behavioral problems (p = 0.188, Fig. S6B).", "Patients with psychological or behavioral problems more often used AEDs than patients without these problems (p = 0.013, χ2-test).", "No differences in percentages of medication-free patients were observed between patients with and without learning problems (p = 0.408, χ2-test).", "Furthermore, no differences in percentages of seizure-free patients were observed between patients with and without learning problems (p = 0.421, χ2-test) or psychological or behavioral problems (0.421, Fisher's exact test).", "No universally used consensus guidelines for the diagnosis of Dravet syndrome exist, and many studies use different criteria [37,39–41].", "The main diagnostic criteria in most studies relate to the epilepsy phenotype and cognitive development.", "Walking disabilities and behavioral problems are usually not included, even though many studies have already acknowledged that they are common and important features of Dravet syndrome [6–9,18,19,37,42] while they are virtually never present in patients with non-Dravet syndromes with pathogenic SCN1A variants.", "Both walking disabilities and behavioral problems can emerge early in the disease course of Dravet syndrome, affecting a large percentage of patients before the age of 8 years; 79% of children with Dravet syndrome between 4 and 8 years in our cohort had a walking disability, which is in line with recent research [19].", "Gait disturbances have already been described in patients as young as 2 years of age [9,19].", "Our overall percentage of patients with Dravet syndrome with a walking disability (score 5 or lower, 54%) is between percentages previously reported: Brunklaus et al. [7] found a motor disorder in 36% of patients with Dravet syndrome whereas Lagae et al. [19] found walking disabilities in 79%.", "These differences are likely due to used definitions; factors such as skeletal malalignment, behavioral issues, epilepsy severity, and pain can affect functional mobility as well.", "Furthermore, the FMS assesses functional mobility on a distance of maximal 500 m while other studies incorporated no distances [19].", "Behavioral problems in the clinical range were most frequently seen between 4 and 11 years of age, when assessed by CBCL/ABCL questionnaires.", "This is in contrast to previous studies that have found an increase of behavioral problems with age (although these problems had been observed in children < 2 years of age already) [7,19].", "Our different results might be due to differences in measurement tools and data collection.", "Interestingly, parents of patients with Dravet syndrome reported behavioral problems more frequently in the interview (66%) than patients scored in the clinical range of the CBCL/ABCL questionnaires (43%); this might indicate that scores in the subclinical range are perceived to be very burdensome as well.", "This high percentage underlines the impact of these issues on families and the importance of accurate management.", "Behavioral problems were much more common among patients with Dravet syndrome than in patients with non-Dravet syndromes (43 versus 7%), and the three patients with non-Dravet syndromes that scored in the clinical range of the ABCL/CBCL questionnaires had a relatively severe non-Dravet SCN1A phenotype.", "Our data suggest that walking difficulties and behavioral problems, based on official assessments, could be useful when counseling patients with SCN1A pathogenic variants; they are strong indicators for a more severe disorder when observed at a young age.", "Both walking disabilities and behavioral problems showed a strong correlation with cognitive outcomes, seizure frequencies, and QoL scores.", "Similar findings have been reported previously, and behavioral problems have been identified as the most important predictors of a worse HRQoL score [7,19].", "Interestingly, the relationship between cognitive disability and frequency of behavioral problems was not linear but hyperbolic: most problems were seen in patients with a mild ID.", "A reason for this could be that more severely disabled patients are less capable of aberrant behavior; however, it might also be due to the fact that CBCL questionnaires have been proven to be less reliable in the assessment of children with moderate, severe, or profound ID [43].", "Total scores cannot be calculated when many questions are not applicable because of cognitive impairment, which occurs more frequently in patients with a worse cognitive outcome.", "Nevertheless, when parents were asked if they thought their child showed any behavioral problems, a similar pattern was observed.", "A hyperbolic relationship was also found between seizure frequency and behavioral problems in Dravet syndrome, although inversed: most behavioral problems were seen in patients that were seizure-free or experienced daily seizures.", "This nonlinear relationship indicates that behavioral problems are not completely subject to an epileptic encephalopathy disease model, as previously suggested [19,44].", "Recent research has shown that SCN1A variants could lead to changes in the dopamine system that may contribute to behavioral problems, irrespective of seizure activity [45].", "This, together with the large impact of behavioral problems on HRQoL scores and the high number of families affected by it, calls for more attention regarding recognition of behavioral problems.", "Handling these issues and developing coping strategies for parents require more emphasis during treatment, which should not only focus on suppressing seizures.", "Patients with non-Dravet syndromes seem to have a good prognosis: the majority of our patients was reported to be seizure-free after 10 years of age, and seizure freedom was reported for all patients over 40 years of age, in most cases, without maintenance treatment.", "However, some of the older participants represented parents of children with SCN1A-related seizures that were tested subsequently after a diagnosis was made in their children.", "They may have gone undiagnosed if their children had not been assessed for mutations, and our results may therefore not be fully applicable to young probands with SCN1A-related epilepsy.", "Seven of the 15 patients in the 40 + age group were parents of affected children and had never used antiepileptic medication themselves, so it is unlikely that they would have been diagnosed individually.", "It is, furthermore, worth noting that pathogenic SCN1A variants do not have a 100% penetrance: although our study only describes symptomatic patients, we are aware of four mutation-carrying GEFS + family members that never experienced any seizures (not included in this study).", "However, although patients with non-Dravet syndromes showed better outcomes on the used questionnaires, subtle comorbidities were still observed in this group.", "It is known that neuropsychological and cognitive problems are common in epilepsy in general [46–50], and it is, therefore, not surprising that similar problems are observed in the non-Dravet group.", "Although the use of different methods makes comparing the exact results difficult, and we investigated a small cohort (only 24 patients with non-Dravet syndromes < 20 years of age), we, nonetheless, observe relatively similar percentages of problems in our patient group and cohorts consisting of patients only diagnosed with epilepsy: 26% of patients between 7 and 16 years old in our cohort had repeated a grade, and 60% required special educational assistance compared with 26% and 51% respectively in an epilepsy cohort [48].", "Psychological/behavioral problems were observed in 40% of patients between 5 and 17 years old and in 37% of patients over 16 years old in our cohort, compared with 31.4% in children with childhood seizures [49] and versus 30.6% in patients with epilepsy [47] in the same age groups.", "Furthermore, similar percentages of patients with symptoms of attention deficit hyperactivity disorder (ADHD) (10% vs 14.3%), depression (5% vs 4%), and anxiety (15% vs 12.3%) were seen in patients between 5 and 17 years old in our cohort and in patients with idiopathic epilepsy respectively [46].", "Although according to the International League Against Epilepsy (ILAE), criteria [51], usually, no developmental impairments are expected in non-Dravet SCN1A phenotypes, we report five patients with non-Dravet syndromes (10%) with a cognitive outcome score of 2 (borderline ID).", "Their disorder could not be classified as Dravet syndrome, since they were seizure-free with minimal or no medication at the time of inclusion, and three had never developed any secondary seizure types.", "Furthermore, a borderline ID (IQ 70–85) represents the IQ score range between − 1 and − 2 standard deviation (SD) from the mean (100) and is thus observed in 13.6% of the general population.", "It can consequently be expected to also occur in a small part of the non-Dravet group, as described by several authors [27–29].", "In addition, borderline ID might be a family characteristic in these patients.", "We therefore argue that a slight cognitive impairment does not necessarily indicate a diagnosis of Dravet syndrome.", "Our study has several limitations.", "In the non-Dravet group, all of the more subtle problems (not captured by the standardized questionnaires) were reported by patients or their parents during telephone interviews or mentioned by clinicians in medical files, and no official assessments were used to evaluate the problems.", "Our results therefore reflect issues that they regarded most important, rather than structured measurements and may be subject to recall and response biases.", "To minimize this effect, we limited our analyses in this group to patients younger than 20 years.", "In these patients, the occurrence of problems at school was independent of seizure and/or medication freedom at the moment of inclusion.", "These problems may however, already have occurred while medication was still being used or when seizures were still active, so we cannot exclude a causal relationship.", "Interestingly, although psychological/behavioral problems occurred independently of seizure freedom, they occurred more often in patients that still used medication.", "This implies that psychological/behavioral problems might at least be partly due to medication side effects.", "In other studies however, no such relation was found [46,48].", "It is worth noting that although significantly lower QoL scores were found in patients with problems at school, the average QoL scores of the non-Dravet group is comparable with that of the general population [33].", "By analyzing multiple outcomes in patients with Dravet syndrome together with patients showing milder SCN1A-related phenotypes, we are able to assess different disease burdens over a large part of the SCN1A-spectrum.", "Both groups show distributions at different ends of the spectrum, and in general, a clear distinction between these syndromes can therefore be made, not only based on regularly used clinical criteria such as cognitive impairment and seizure severity but also on other comorbidities: severe behavioral problems and walking disabilities, which can already occur at a young age in patients with Dravet syndrome and are almost never seen in patients with non-Dravet syndromes.", "These issues should receive specific attention during clinical management of the disease.", "However, as in patients with other epilepsies, comorbidities occur in a substantial part of patients with non-Dravet syndromes as well.", "Although these problems are less severe than in patients with Dravet syndrome, they can still have a large impact on patients and their families.", "Our study provides valuable information on the disease course and comorbidities in these patients, which can be of great value when counseling newly diagnosed patients and their parents." ]
[ "Walking disabilities and severe behavioral problems are specific for Dravet syndrome and can already occur between 4-7 years of age.", "These comorbidities should receive specific attention during clinical management.", "A substantial part of non-Dravet syndromes patients experiences learning and behavioral/psychological problems.", "The majority of non-Dravet syndromes patients over 10 years of age were seizure free." ]
High-dose testosterone treatment increases serotonin transporter binding in transgender people
Background Women are two times more likely to be diagnosed with depression than men. Sex hormones modulating serotonergic transmission are proposed to partly underlie these epidemiologic findings. Here, we used the cross-sex steroid hormone treatment of transsexuals seeking sex reassignment as a model to investigate acute and chronic effects of testosterone and estradiol on serotonin reuptake transporter (SERT) binding in female-to-male and male-to-female transsexuals. Methods Thirty-three transsexuals underwent [<sup>11</sup>C]DASB positron emission tomography before start of treatment, a subset of which underwent a second scan 4 weeks and a third scan 4 months after treatment start. SERT nondisplaceable binding potential was quantified in 12 regions of interest. Treatment effects were analyzed using linear mixed models. Changes of hormone plasma levels were correlated with changes in regional SERT nondisplaceable binding potential. Results One and 4 months of androgen treatment in female-to-male transsexuals increased SERT binding in amygdala, caudate, putamen, and median raphe nucleus. SERT binding increases correlated with treatment-induced increases in testosterone levels, suggesting that testosterone increases SERT expression on the cell surface. Conversely, 4 months of antiandrogen and estrogen treatment in male-to-female transsexuals led to decreases in SERT binding in insula, anterior, and mid-cingulate cortex. Increases in estradiol levels correlated negatively with decreases in regional SERT binding<inf>,</inf> indicating a protective effect of estradiol against SERT loss. Conclusions Given the central role of the SERT in the treatment of depression and anxiety disorders, these findings may lead to new treatment modalities and expand our understanding of the mechanism of action of antidepressant treatment properties.
[ "NEUR" ]
[ "Estradiol", "Hormone treatment", "Positron emission tomography", "Serotonin transporter", "Testosterone", "Transsexual" ]
[ "2803" ]
[ "The study sample consisted of 33 transsexual subjects and 35 control subjects: 14 female-to-male (FtM) and 19 male-to-female (MtF) transsexuals, 11 female control subjects (FC), and 24 male control subjects (MC).", "Transsexuals were recruited from the Transgender outpatient unit of the Department of Obstetrics and Gynecology, Medical University of Vienna.", "They were naive to steroid hormone treatment and were seeking sex reassignment.", "Transsexuals reported gender dysphoria since a relatively early age (before or at puberty).", "Mean age was numerically lower in FtM (28.21 ± 6.81, mean ± SD) compared with MtF (31.79 ± 9.21), FC (30.43 ± 10.07) and MC (34.14 ± 11.00), although this difference was not significant (p = .38, analysis of variance).", "To rule out internal medicine and neurological disorders, subjects underwent a standard medical examination including electrocardiogram, routine laboratory tests, and the Structural Clinical Interview for DSM-IV Disorders.", "Further exclusion criteria included intake of psychotropic medication within 6 months before inclusion, past or current substance abuse, pregnancy, and hormonal treatment before study participation.", "Diagnostic assessment of transsexualism followed DSM-IV-TR and ICD-10 and was performed after several semi-structured, sociodemographic, clinical, and psychiatric interviews based on legal requirements for cross-sex hormonal treatment in Austria.", "Psychotherapy before and during hormone treatment was also recommended.", "After at least 1 year of hormone therapy, subjects could decide to undergo sex reassignment surgery (36).", "The recommendations in Austria are largely based on the Standards of Care for the Health of Transsexual, Transgender, and Gender Nonconforming People, The World Professional Association for Transgender Health, 7th Version, Atlanta, Georgia, 2011.", "Therefore, none of our study participants had sex reassignment surgery before or during study participation.", "However, two FtM and one MtF already had their official names changed at screening visit.", "Written informed consent was obtained after thorough explanation of the study to the subjects.", "Data from a subsample of subjects (14 MtF, 9 FC, and 13 MC) have been published in a previous study (24).", "The study was approved by the Ethics Committee of the Medical University of Vienna.", "The study was designed as a longitudinal monocenter study.", "Fourteen FtM and 19 MtF transsexuals underwent a baseline scan before start of hormone treatment (PET 1).", "Scans of 11 FC and 24 MC served as control measurements, while 8 of the 24 MC were measured twice to determine the test-retest reliability of [11C]DASB (time interval: 10.13 ± 9.66 days).", "Baseline scans in FtM and those in FC were performed irrespective of their menstrual cycle phase.", "A subset of transsexuals underwent a second scan (PET 2; 9 FtM, 16 MtF) 4 weeks after treatment start, and a third scan (PET 3; 11 FtM, 13 MtF) 4 months after treatment start.", "Hormone treatment followed protocols routinely implemented at the Department of Obstetrics and Gynecology, Unit for Gender Identity Disorder, at the Medical University of Vienna.", "FtM received 1000 mg testosterone undecanoate every 12 weeks (Nebido 250 mg/mL, 4 mL vial, intramuscular; Bayer, Vienna, Austria).", "Two cases further received 10 mg to 15 mg lynestrenol (Orgametril 5 mg, oral; Organon, Oss, The Netherlands) daily.", "MtF received either daily 50 mg cyproterone acetate (Androcur 50 mg tablet, oral; Bayer; 14 subjects) or triptorelin acetate 4.12 mg/month (Decapeptyl 172 mg powder for suspension for injection subcutaneous or intramuscular; Ferring Arzneimittel, Vienna, Austria; 4 subjects).", "Additionally, MtF over 40 years of age received daily doses of 100 μg estradiol (transdermal therapeutic system applied twice a week; Estradot, Novartis, Vienna, Austria/Estramon, Hexal, Vienna, Austria; 3 subjects), while those less than 40 years of age received 4 mg/day estradiol hemihydrate (Estrofem 2 mg, oral; Novo Nordisk, Vienna, Austria; 9 subjects).", "Alternatively, six subjects received estradiol hemihydrate .75 mg/day to 1.5 mg/day (Estro-Gel .75 mg/1.25 g/day, transdermal; Meda, Vienna, Austria).", "Because of extensive hair loss, six MtF further received 2.5 mg/day of the 5-alpha reductase inhibitor finasteride (5 mg, oral; Ratiopharm, Vienna, Austria).", "One MtF terminated the study prematurely, moved out of town after the baseline PET, and received no medication at our hospital.", "Blood samples were collected before PET scanning for transsexuals at each visit.", "The analysis of plasma levels of estradiol, testosterone, and progesterone was done by the Department of Laboratory Medicine, Medical University of Vienna, Austria (http://www.kimcl.at).", "All PET scans were performed in a GE Advance full-ring scanner (General Electric Medical Systems, Milwaukee, Wisconsin) in three-dimensional mode at the Department of Biomedical Imaging and Image-Guided Therapy, Division of Nuclear Medicine, Medical University of Vienna.", "A 5-minute transmission scan was done using retractable 68Ge rod sources for tissue attenuation correction (24,37).", "Data acquisition started simultaneously with a bolus injection of [11C]DASB measuring brain radioactivity in a series of 50 consecutive time frames.", "Mean injected dose and specific activity were not significantly different between groups at any time point (t test, p > .05; Table 1).", "Total acquisition time was 90 minutes.", "Collected data were reconstructed in volumes consisting of 35 transaxial sections (128 × 128 matrix) using a fourier rebinning iterative filtered back-projection algorithm with a spatial resolution of 4.36 mm full-width at half maximum 1 cm next to the center of the field of view; for radiotracer preparation and radiochemical variables, see Lanzenberger et al. (38) and Haeusler et al. (39).", "Following between-frame motion correction, individual summed PET images were spatially normalized to a PET template in stereotactic Montreal Neurological Institute space using SPM8 (Wellcome Trust Centre for Neuroimaging, London, United Kingdom; http://www.fil.ion.ucl.ac.uk/spm/).", "The SERT nondisplaceable binding potential (BPND) (40) was quantified using the multilinear reference tissue model (41).", "Cerebellar gray matter (excluding vermis and venous sinus) was used as reference region, as postmortem and in vivo SERT quantification identified the cerebellar gray matter as optimal reference region for [11C]DASB (42,43).", "All modeling calculations were performed using PMOD image analysis software, version 3.3 (PMOD Technologies Ltd, Zurich, Switzerland; www.pmod.com).", "SERT BPND was computed in a region of interest (ROI) based approach.", "Twelve ROIs were selected including the insula; anterior, middle, and posterior cingulate cortex; hippocampus; amygdala; hypothalamus; caudate; putamen; thalamus; and dorsal and median raphe nucleus.", "ROIs were based on the automated anatomical labeling brain atlas (44), except for dorsal and median raphe nuclei, which were defined manually according to Kranz et al. (45).", "ROIs were selected based on their moderate to high concentration of SERT BPND (46,47), the overlap between sex steroid receptor distribution and adequate SERT BPND (48,49), and because of gender-specific SERT BPND abnormalities in mood disorders (13,15).", "Treatment-induced hormone level changes were assessed using linear mixed models analysis with group (FtM, MtF) and time (PET 1–3) as fixed factors and subjects as the random factor, followed by separate models for each group and post hoc pairwise comparisons, corrected for multiple comparisons using the Bonferroni procedure.", "Similarly, changes in regional SERT BPND over time were assessed using linear mixed models with group as the between-subjects factor, time and ROI as repeated factors adjusted for baseline values, and subjects as the random factor.", "Associations between treatment-induced hormonal changes and changes in regional SERT BPND were calculated using Pearson product-moment correlations.", "This was followed by exploratory partial correlations to correct for initial hormone level (e.g., testosterone) and for changes of other hormones (e.g., estradiol or progesterone).", "The significance level was set at 5% in all analyses.", "SPSS version 19.0 for Windows (SPSS Inc., Chicago, Illinois; www.spss.com) was used for statistical analysis.", "Estradiol, testosterone, and progesterone plasma levels at each time point and plasma level changes between time points were normally distributed for each group (assessed using Kolmogorov-Smirnov test and visual inspection).", "As expected, androgen treatment in FtM and antiandrogen and estrogen treatment in MtF had profound effects on hormone plasma levels, revealed by a significant interaction group × time (testosterone: F2,38.5 = 67.74, p < .001; estradiol: F2,53.6 = 5.92, p = .005; progesterone: F2,27.2 = 4.41, p = .022).", "Androgen treatment in FtM led to significant changes in testosterone (F2,14.1 = 27.94, p < .001) with post hoc pairwise comparisons revealing increases from PET 1 to PET 2 (p = .005), PET 1 to PET 3 (p < .001), and a trend for PET 2 to PET 3 (p = .086).", "Conversely, antiandrogen and estrogen treatment in MtF led to significant decreases of testosterone (F2,26 = 42.21, p < .001), when comparing PET 1 to PET 2 (p < .001) and PET 1 to PET 3 (p < .001) but not when comparing PET 2 to PET 3 (p > .1).", "Estradiol plasma levels did not change over the course of treatment in FtM (F2,12.81 = .64, p = .541), but they profoundly changed in MtF (F2,17.89 = 16.90, p < .001), showing significant increases from PET 1 to PET 2 (p = .001) and PET 1 to PET 3 (p = .011) but not from PET 2 to PET 3 (p > .1, all p corrected).", "Progesterone plasma levels showed a trend toward decreased values over time in FtM (F2,10.3 = 3.16, p = .085), whereas reductions were significant in MtF (F2,28.4 = 3.95, p = .031) with post hoc tests revealing a significant decrease from PET 1 to PET 3 (p = .034, corrected; Table 1).", "SERT BPND at each time point and BPND changes between time points were normally distributed for each region and group.", "The comparison between transsexuals and healthy control subjects revealed significantly higher SERT BPND in MC compared with FtM, whereas no other group comparisons showed significant differences (for further information, see Supplement 1).", "Testing the effect of treatment in transsexuals revealed a significant main effect of time (F2,872.3 = 12.14, p < .001), group (F1,335.6 = 4.13, p = .043), and ROI (F11,854.4 = 7.09, p < .001) as well as significant interactions for time × goup (F2,872.3 = 19.13, p < .001), time × ROI (F22,858.8 = 3.23, p < .001), and time × group × ROI (F22,858.8 = 2.03, p = .004).", "To interpret the three-way interaction with respect to potential group differences, post hoc two-sample t tests for group comparisons were made for each time point and region.", "This revealed that most regions in MtF had numerically higher BPND values compared with FtM at all three time points.", "However, only in the amygdala at baseline, the higher BPND in MtF was significant at an uncorrected level of p = .037.", "To interpret the three-way interaction with respect to treatment effects, separate models were calculated for each group and region, followed by post hoc pairwise comparisons of the three time points.", "These analyses revealed a significant increase in SERT BPND over time in FtM in the amygdala (p = .002), caudate (p = .002), putamen (p = .009), and median raphe nucleus (p = .045) and a trend for hippocampus (p = .080), thalamus (p = .053), and dorsal raphe nucleus (p = .053) (Figure 1).", "However, when correcting for the number of performed tests, only changes in amygdala and caudate remained significant.", "In contrast to FtM transsexuals, MtF transsexuals exhibited a decrease of SERT BPND over the course of 4 months in the insula (p = .023) and anterior and mid-cingulate cortex (p = .002 and p = .015, respectively) and a trend for putamen (p = .068), with changes only in anterior cingulate cortex surviving correction for multiple testing (see Table 2 for estimated means ± SE and post hoc pairwise comparisons).", "Controlling for individualized treatment regimens did not change the main findings (further information can be found in Supplement 1).", "In the subsample of eight MC, no significant differences were present between the two measurements and test-retest reliability was high (intraclass correlation coefficient > .8; Supplement 1).", "The significant treatment-induced increase in testosterone plasma levels in FtM showed strong positive correlations with changes in regional SERT BPND after 4 weeks of treatment (PET 1 vs. PET 2; Figure 2A).", "That is, increases in testosterone plasma levels correlated with increases in SERT BPND in anterior cingulate cortex (p = .027), amygdala (p = .010), caudate (p = .001), putamen (p = .001), and thalamus (p = .015) and a trend for median raphe nucleus (p = .053).", "No significant correlations were observed in other regions, indicating a regional effect.", "Controlling for baseline testosterone levels and changes in estradiol plasma levels in separate partial correlation analyses reduced the p value to a nonsignificant trend for the anterior cingulate cortex (p = .075 and p = .076, respectively) but did not change the significance of results in other regions.", "Likewise, controlling for changes in progesterone plasma levels had no significant effect.", "However, correlations between testosterone and SERT BPND became nonsignificant when correlating hormonal changes with changes in SERT BPND after 4 months of treatment (PET 1 vs. PET 3).", "In contrast to the results in FtM, treatment-induced decreases in testosterone plasma levels and increases in estradiol plasma levels in MtF after 4 weeks of treatment (PET 1 vs. PET 2) were not correlated with changes in SERT BPND in any of the investigated brain regions.", "Partial correlation analyses did not change the results.", "However, correlating significant estradiol changes after 4 months of treatment (PET 1 vs. PET 3) with changes in regional SERT BPND (PET 1 vs. PET 3) showed positive associations in the insula (p = .006), mid-cingulate cortex (p = .032), hippocampus (p = .048), and putamen (p = .046) (Figure 2B).", "When controlling for progesterone plasma levels, significances increased for insula (p = .004) and mid-cingulate (p = .026) but decreased to a trend for hippocampus (p = .079) and putamen (p = .063).", "Significant correlations were also observed when comparing values after 4 weeks with those after 4 months of treatment (PET 2 vs. PET 3) in insula (p = .027) and mid-cingulate cortex (p = .020) in regions that also showed a significant decrease in SERT BPND (Table 2).", "Finally, Pearson product-moment correlations revealed no significant associations between depressive symptoms and other clinical characteristics and regional SERT BPND at any time point (for clinical characteristics, see text and Table S1 in Supplement 1).", "In this study, we found that testosterone treatment in FtM transsexuals significantly increased SERT BPND after 1 month as well as after 4 months of treatment in regions with adequate signal-to-noise ratio, i.e., moderate to high SERT density (Figure 1, Table 2).", "Early animal studies demonstrated that, in contrast to 5α-dihydrotestosterone, a nonaromatizable androgen, testosterone increased SERT density and SERT messenger RNA in rats (50).", "While 5α-dihydrotestosterone cannot be converted to estrogen by aromatase, testosterone action is suggested to depend in part on its conversion to estradiol.", "Furthermore, brain masculinization is believed to depend on organizational effects of estrogen, triggered by aromatized testosterone, to which the initially undifferentiated brain is exposed (51).", "We therefore suggest that testosterone treatment in FtM affected SERT binding via aromatization to estradiol and activation of estrogen receptors.", "Plasma testosterone levels rose progressively over the course of treatment in FtM.", "Correspondingly, our data show a progressive increase in SERT BPND from subchronic testosterone administration (4 weeks) to long-term effects (4 months).", "Furthermore, acute increases in SERT BPND (after 4 weeks) in anterior cingulate cortex, amygdala, caudate, putamen, and thalamus were found to be positively correlated with changes in plasma testosterone levels.", "However, no correlations were observed after 4 months of treatment (PET 1 vs. PET 3).", "If one assumes a causal relationship, this apparent loss of SERT dependency on testosterone levels after 4 months of treatment therefore points to ceiling effects of the influence of testosterone levels.", "In contrast to SERT BPND increases in FtM after testosterone treatment, regional SERT BPND in MtF remained constant after the first 4 weeks of estrogen and antiandrogen treatment.", "However, values significantly decreased after 4 months of treatment in anterior and mid-cingulate cortex and insula, whereas hormonal levels significantly changed only within the first 4 weeks after treatment start.", "Assuming that the effects of testosterone on SERT density depend on its conversion to estradiol, SERT BPND should remain constant when reducing testosterone levels while increasing those of estradiol, which is in accordance with our data.", "The significant decrease of regional SERT BPND after 4 months of treatment, on the other hand, warrants a different explanation that apparently exceeds estradiol effects on SERT BPND.", "Still, negative associations between estradiol increase and regional SERT BPND downregulation after 4 months of treatment showed that regional SERT BPND decreased to a lesser extent when estradiol level increases were larger.", "In other words, estradiol increases seemed to have a protective effect against SERT BPND loss.", "Our results indicate increases in SERT BPND when testosterone (and aromatized estradiol) increases.", "According to a simple mechanistic view on SERT function, elevated SERT availability increases serotonergic uptake, which leads to reduced extracellular serotonin.", "Furthermore, according to the serotonin hypothesis of depression, serotonin deficiency is considered a hallmark underlying depressive symptoms.", "Thus, SERT BPND may be expected to be high in depressed subjects (16,52,53), which is, however, in contrast to our meta-analysis showing SERT reductions in several regions (12).", "Furthermore, studies indicate that testosterone supplementation improves depressive symptoms in hypogonadal men as well as in surgically menopausal women (54,55), and preliminary data indicate that short-term estradiol treatment may qualify as an effective therapy for perimenopausal major depression (56).", "Recent animal research indicates that androgen therapy elevates serotonin levels and that this is dependent on aromatase activity (57).", "Accordingly, exogenous androgen increases aromatization to estradiol, which leads to increases in serotonin synthesis and availability via estrogen receptors (58).", "Furthermore, according to the use it or lose it hypothesis formulated by Ramamoorthy et al. (59), SERT cell surface expression is increased in response to increased synaptic serotonin.", "Conversely, SERT proteins are downregulated when 5-HT levels are low.", "Taken together, our data indicate that testosterone treatment in FtM increased serotonin levels, which thereby increased SERT expression.", "With reference to depression, our interpretations are still very speculative since no associations were found between depressive symptoms and regional SERT BPND.", "Future research should investigate testosterone-induced changes of serotonergic neurotransmission and the potential benefits of testosterone as add-on therapy to selective serotonin reuptake inhibitor treatment in major depression.", "Our study includes limitations that compromise the interpretation of its results.", "First, our results in transsexual subjects cannot easily be generalized to other human studies.", "According to Swaab and Garcia-Falgueras (60), transsexuality may derive from a mismatch between sex differentiation of the brain and of the body.", "Transsexual subjects exhibit features in brain structure and function that reflect their gender identity rather than their genetic sex (24,61–63).", "However, they may also exhibit features that are specific to their condition, which can include the neural underpinnings of well-being, self-esteem, and psychological strain (64–66).", "We cannot exclude that these characteristics include alterations within the serotonergic system.", "However, when comparing transsexuals with their sex-matched control subjects (e.g., FtM with FC and MtF with MC), there was no significant difference in regional SERT BPND.", "This is in accordance with our previous publication (24), a finding which can be explained by the overlap in study participants between the current and the previous study.", "On the other hand, in the current study, MC had significantly higher SERT BPND than FtM in several regions.", "This is in accordance with Jovanovic et al. (19), showing decreased BPND in women compared with men, but in disagreement with Erritzoe et al. (20), showing the opposite pattern in the midbrain.", "Second, reductions of testosterone in most MtF are achieved by inhibition of the negative diencephalic pituitary testicular feedback system by the antiandrogen cyproterone acetate.", "This drug exhibits a close structural relationship with progestogens; therefore, we cannot exclude a more direct effect that goes beyond the modulation of testosterone levels.", "Indeed, progesterone levels decreased over time in MtF.", "Still, when correlating changes in testosterone plasma levels with those in SERT BPND, correction for progesterone changes did not influence the results.", "However, correction affected the relation between estradiol and SERT BPND changes.", "Third, no arterial blood samples were available to quantify SERT BPND independent of a reference region.", "However, previous studies investigating SERT occupancy using [11C]DASB identified the cerebellar gray matter as optimal reference (42,52).", "Even a 50% difference in cerebellar specific binding between subjects would lead to only 3.5% bias for group comparisons in target regions (52).", "Finally, since psychiatric disorders other than transsexuality were no exclusion criteria, this could have affected SERT BPND.", "Indeed, 12 of the 33 transsexuals (4 of 14 FtM and 8 of 19 MtF) were diagnosed with a previous mood or anxiety disorder (i.e., dysthymic disorder, major depressive disorder in full or partial remission, or social phobia) according to the Structural Clinical Interview for DSM-IV Disorders.", "However, comparing SERT BPND in subjects with and without a previous diagnosis, groups did not significantly differ.", "In conclusion, our data provide evidence that long-term high-dosage cross-sex steroid hormone treatment in transsexual subjects affects SERT binding in a hormone-specific manner.", "Testosterone treatment increased SERT binding, whereas chronic antiandrogen treatment decreased it.", "Given the central role of the SERT in the treatment of depression and anxiety disorders, our findings may lead to new treatment modalities and understanding of the mechanism of action of antidepressant treatment properties." ]
[]
A two scale mixed lubrication wearing-in model, applied to hydraulic motors
Wearing-in of a machine component can increase the conformity between contacting pairs and smoothen the surface topography. A two scale model, combining the wearing-in effects, resulting in changes in the surface topography, with the wear that occurs on the component, is presented. The geometry of the components is represented with measured coordinates. Wear leads to changes of the geometry, which has an effect on several tribological conditions, such as contact forces, relative velocities and conformity. Due to the wear on the topography scale, the load sharing is also affected. The model is applied to orbital hydraulic motors. The wear depth predicted with the model is qualitatively in good agreement with the wear depth recorded in experiments.
[ "ENGI", "MATE", "PHYS" ]
[ "Mixed lubrication", "Orbital motor", "Running-in", "Wearing-in" ]
[ "2210", "2211", "2508", "3110" ]
[ "The early life of a machine component is characterized by high wear rates.", "Under desirable circumstances, this wear rate will drop gradually and a steady state of low wear rate will occur.", "The process is called running-in.", "Running-in is caused by a number of factors.", "Changes in microstructure, activation of lubricant additives and changes in surface topography are examples of changes that can help us to protect the surface from further damage.", "Wearing-in is when wear during the early life of a machine component affects the wear rate through geometrical changes [6].", "Wearing-in can be seen as a two-scale process, which from a global scale perspective increases the conformity between the surfaces and on the local scale smoothens the topography.", "Smoothening of the topography, in turn, influences the load sharing between asperities and hydrodynamic lift, caused by pressure build up in the lubricant.", "Wear which leads to a smoothening of the surfaces, often with a low wear rate, is called mild wear.", "Wear causing an increased surface roughness, usually under high wear rates, is called severe wear.", "Favourable running-in is commonly initiated by severe wear, which is followed by a state of mild wear [7].", "Akbarzadeh and Khonsari [1] used a model to vary loads, initial roughness and sliding speed to investigate the influence of these parameters on wearing-in.", "The model illustrates mild wear wearing-in.", "The model and experimental investigations have shown that running-in under mild wear conditions can be favoured by high loads.", "This is only the case up to a limit, where a transition to severe wear occurs, as noted by Jamari [21].", "Experimental investigations of surface roughness by Jahanmihir and Suh [20] indicate that high loads will quickly cause a roughening of a surface, and that wear under low loads can be reduced by low initial surface roughness.", "Other investigations show both increases [13] and decreases [4] in surface roughness due to wear.", "The transition to severe wear can be explained by e.g. breakdown of oxide films, and depend on the conditions of the tribological system [25].", "A model investigating the details of the transition to severe wear has been published by Bosman and Schipper [8].", "The study included a transition from mild to severe wear due to local surface temperatures.", "The running-in is further complicated, and improved, by including lubricant additives in the tribological system.", "Changes in surface roughness due to the influence of lubricant additives have been investigated experimentally [16,35] and numerically [3].", "Full scale component wear modelling can be used to estimate the wear behaviour of machines and machine components.", "Such models allow new designs to be tested numerically.", "Contact conditions have been numerically evaluated using the finite element method [27,29], the boundary element method [33,37,2] and the elastic foundation material model [15,34].", "Numerical procedures to efficiently evaluate the wear problem have been elaborated on by e.g. Põdra and Andersson [28] and Lengiewicz and Suptkiewicz [24].", "Mattei et al. [26] compared a number of running-in models which assume changes in wear rate with time for the case of hip joints.", "Zhu et al. [41] treated the lubrication as a separate model component, solving the lubrication problem including a wear effect.", "Although wear models keep improving, many of them do not consider roughness, running-in and lubrication in a satisfying way.", "Treating the roughness deterministically is not always possible as time and geometry scales for a component grow large.", "Therefore, simplified running-in models are needed for full scale component wear modelling.", "Orbital hydraulic motors are suitable for delivering high torque at a low speed [19].", "In the centre of the orbital hydraulic motor there is a gear set, illustrated in Fig. 1.", "The gear set consists of an inner gear which is moving in an epicyclic motion inside an outer gear.", "The inner gear consists of m−1 teeth.", "As illustrated in Fig. 1, geometric positions on the teeth can be related with an angle θ.", "The inner gear orbits around the centre of the outer gear in a circle, which is called the excentre.", "The inner gear also rotates around its own centre.", "The time it takes for the inner gear to complete a rotation around its own centre is m−1 times the time it takes for the inner gear to orbit one lap on the excentre.", "Contact between the inner and outer gear causes wear, which in the worst case, can lead to seizure.", "Mixed lubrication effects and wear in orbital hydraulic motors were investigated by the present authors in a previous publication [18].", "Results from the study indicated that improvements of the mixed lubrication model would be needed in order to explain the deviance between simulation and experiments.", "In this study a two scale wearing-in model is presented.", "Global wear is modelled through material removal from the geometry of the components.", "Topography scale wear is modelled by changes in the mixed lubrication behaviour of the individual contact points between the surfaces.", "Measured surface topographies are used to tune the topography scale wear model.", "The two scales are illustrated in Fig. 2.", "The model is applied to carry out numerical simulations of wearing-in behaviour of gear sets in an orbital type hydraulic motor.", "The model is applied to unique, measured gear set geometries.", "The numerical simulation results include changes in the forces between inner and outer gear, as well as the changes in the load sharing between lubricant and asperities.", "Finally, the component scale wear recorded in the experiments is compared with the wear depths predicted by numerical simulations.", "A numerical model, developed by the authors of this work, which was presented in [17], is used to estimate tribological conditions in a number of orbital type hydraulic motor gear sets.", "The hydraulic pressure in the motor is balanced by contact forces Ftot, at each contact point.", "The contact forces together with speeds, geometry and material data constitute known tribological conditions.", "The conditions, in combination with measured surface roughness, are used to estimate the extent of mixed lubrication.", "The load which is carried by direct asperity contact, Fa, is then used to calculate a pressure distribution.", "The process is repeated for a number of time increments, as the inner gear moves all the way around the excentre cycle.", "Once the whole rotation is complete, a theoretical model is used to estimate the wear on two scales.", "On both scales material is removed based on an Archard type of wear model.", "The numerical simulation results are compared with experimental results, in terms of wear, for the gear set geometry presented in Fig. 1.", "The numerical model includes calculations of forces, contact points and velocities, based on what is observed on the global scale.", "These tribological conditions are calculated from measured surface profiles, as presented in [18].", "On the local scale, a relation between applied force and resulting average surface separation between two rough surfaces is calculated.", "This local scale is defined here as the topography scale.", "The curve depicting the relation between contact force and separation is referred to as the contact stiffness or contact stiffness curve.", "The two scales of the model, the topography scale and the tribological conditions from the global scale, are considered in the mixed lubrication model.", "Wear is included in the model both on the topography scale and on the global scale.", "The gears of the gear set, shown in Fig. 1, are symmetric around each inner and outer gear tooth.", "This means that the tribological conditions are cyclic and by using the symmetry, the calculation of tribological conditions can be done (m−1)m times more efficiently.", "For a more detailed explanation, the reader is referred to [18].", "The model presented in this paper does, however, not rely on this symmetry.", "That is because when measured profiles are used as input, each inner and outer gear tooth has unique features, and every time an inner gear moves through its contact with an outer gear, a unique set of contact forces (and other tribological conditions) will be the result.", "In order to account for the full range of contact forces, the whole movement of the inner gear is considered in this paper.", "In what follows, a description of the mixed lubrication model is first given in Section 2.1.1.", "Thereafter, in Section 2.1.2, a detailed presentation of the extension of the model that considers the topography scale wear is presented.", "In order to determine the severity of contact, a model for mixed lubrication is necessary.", "As the contacts between inner and outer gears are highly loaded and, for a large part of the time, non-conformal, the lubrication regime is assumed to be partial or full film elastohydrodynamic lubrication (EHL).", "The mixed lubrication model is crucial in this work, as it is where tribological considerations from the global scale are combined with the topography scale behaviour.", "Wear alters the surface roughness of machine components.", "Detailed changes in roughness are, however, hard to predict.", "Experimental investigations show both increase and decrease of roughness, depending on wear severity.", "Considering changes in roughness due to wear is therefore a complicated task.", "The model presented here is in part based on models found in existing literature and in part on experience from research and development of orbital type motors.", "For severe wear, a rough surface will result.", "Due to the upper limit on Hi, the roughest surface is the original surface.", "For simplicity, the model assumes that severe wear on a surface will set the value of Hi to Hi=0.5, for the severely worn contact points.", "Using pin on disk data from published work, their own experiments and their own calculations, Lim et al. identified maps of mild and severe wear [25].", "A strictly pressure dependent transition from mild to severe wear occurs due to increased pressure, under low temperature conditions.", "The reason for this transition was argued to be breakdown of oxide layers.", "In the case of orbital motor gear sets, where there are no steady state conditions and lubricant additives help protect the mating surfaces from severe wear, the wear maps for pin on disk contacts do not translate directly.", "However, it seems that a pressure dependent wear transition has been found for other EHL systems during running-in.", "According to Jamari, increasing the contact pressure will improve the quality of running-in up to a limit value [21].", "Cavatorta and Cusano [9,10] showed that the quality of running-in is increased with increased pressure, up to a pressure limit.", "The improved running-in effect is already captured by the relation in Eq. (10).", "The upper pressure limit, however, need to be determined.", "Simulation results of the tribological conditions in the motor have previously been compared with observations from experiments [18].", "Severe wear was observed on measured gear sets at some θ angles.", "These θ angles coincide with high local pressure, far exceeding the calculated pressures in regions of mild wear.", "The exact value for the transition to severe wear is not clear but based on experience, a limit value on the pressure was chosen at 2.5 GPa.", "The formulation is particularly useful in the case when there is a vast span of tribological conditions between the components.", "Different roughness values can be specified for any location on either surface.", "Another advantage with this formulation is that wear artificially affects the top of the asperities most, then gradually has a smaller effect with decreased separation.", "When the load sharing has been established, the asperity force is used to remove material on the global scale.", "The equation which is used to remove material globally is the same as the equation used locally; Archards׳ wear equation.", "In the equation, a wear coefficient is needed.", "In this work, the wear coefficient has been chosen to be the same on the local and global scales, with the exception that when pressures exceed 2.5 GPa, a transition to severe wear occurs on the topography scale.", "In this case, the value of Hi is directly set to 0.5, for the pressurized nodes of both surfaces.", "The wear coefficient of Archards׳ wear equation is an extensively studied topic.", "Derived by Archards as an equation for adhesion [5], the equation can also been derived for abrasive wear [31].", "The interpretation of the k-value for the two cases is different.", "In the case of adhesive wear, the coefficient represents a probability that two asperities adhere and originate mass transfer.", "In the case of abrasive wear, a simple derivation by Rabinovisch [31] shows that the k-value is a function of the asperity roughness angle, in the case of triangular asperities.", "In other words, the abrasive k depends on the shape while the adhesive k depends on the adhesive forces.", "As long as the wear mechanisms do not change, Archards wear equation is valid down to the nanometric scales [12].", "On the molecular scale, however, the continuum assumption loses validity, and along with it models for plastic deformation and crack propagation.", "At this scale, the wear occurs through dragging of atoms, according to predictions made by molecular dynamics simulations [23,38].", "But even on the molecular scale the wear rate has been found to be proportional to the contact pressure [38], with a many times lower wear rate.", "The wear model used in this work involves two scales.", "The global scale, in which wear occurs in the order of micrometers, and the topography scale, where wear depths are in the range of several hundred nanometres.", "The small scale is considered large enough to still use the continuum model and thus, the same wear coefficient on the two scales is assumed.", "Tests were performed as described in [18].", "The samples and their running conditions are displayed in Table 1.", "Roughness was measured using an optical profilometer after wear testing.", "Roughness was measured in regions of high wear, low wear and regions where no wear was observed, for samples B and J.", "The surface measurements were filtered, removing tilt and by removing shape in form of a second order polynomial.", "The filtered surfaces were mirrored around the origin in order to create a periodic surface.", "64 by 64 data points in the centre of the periodic surface were used to calculate contact stiffness.", "The contact stiffness was calculated by putting each surface pair in contact in every possible way, through shifting of the surfaces.", "Three regions each, of surfaces which had been mildly worn of sample B and unworn surfaces of sample J, was used as input to calculate contact stiffness.", "The surface topographies in mildly worn, severely worn and unworn regions on inner gears were measured and filtered.", "Table 2 shows Ra values for three different spots of the inner gears of samples B and J.", "The spots were chosen to represent unworn (at θ=30°), mildly worn (at θ=0) and severely worn (at θ=−20°) regions of the inner gears.", "The Ra values are only used to compare the surfaces, and are otherwise unrelated to the running-in model.", "The trend seems to be that surface roughness was higher than or equal to the unworn areas in regions of severe wear, while areas of mild wear seem to have a decreased surface roughness.", "All surface roughness measurements were performed on different spots, after testing.", "Parts of the actual surface measurements are shown in Fig. 4.", "The images of the surface profiles indicate that the choice of mildly and severely worn regions along the inner gear geometry was reasonable, as the surface is smoother in the areas selected as mildly worn, compared with those selected as severely worn.", "In terms of the presented wearing-in model, surface topography measurements are the basis for the model.", "The assumption that the roughest achievable surface in the model was equal to the original roughness is based on the data in Table 2.", "In the unworn regions we can see roughness shaped by the grinding tool used to produce the pieces.", "The unworn roughness was higher on sample J compared to sample B.", "In the mildly worn regions we can see a significant decrease in surface roughness for sample B. For sample J, there was an overall decrease in Ra, but the magnitude of the decrease varied depending on the measured location.", "In the severely worn regions, the surface roughness varied most dramatically.", "In some measured areas, the surface roughness was increased compared to the unworn case, and in some areas the surface roughness was decreased.", "The validation of the wearing-in model in terms of contact stiffness changes is first presented.", "After this the applied model is analysed.", "The force distribution between lubricant and asperity carried forces is analysed in depth, for the unworn original and for the worn final surface.", "The change in wear rate during running-in is also taken under discussion.", "Wear depths from the simulations are compared with wear depths from experiments.", "The model for topography scale wear as presented need to be validated.", "To calculate the unworn contact stiffness to be used in the model, unworn measured surfaces should be used.", "As depicted in Fig. 5, the contact stiffness calculated from contact between unworn surfaces of sample J is similar.", "The arithmetic average of the contact stiffness calculated from contact between unworn surfaces measured form sample J is used as the initial contact stiffness, fo, in the model.", "This contact stiffness is shown together with other contact stiffness curves, calculated from measured surfaces, in Fig. 5.", "The smoothest possible contact achievable in the model is when Hi=0.15 (and thus H=0.3).", "The contact stiffness fw, with H=0.3, is similar to the contact stiffness calculated from contact between the smoothest of the measured mildly worn surfaces.", "A difference between the contact stiffness curves calculated from contact between measured, mildly worn, areas and the smoothest curve in the model can be observed.", "Especially at the point of initial contact, there is a difference between the contact stiffness curves calculated from measurements.", "In the model this represents different values of H in the range between H=1 and H=0.3.", "We will focus on two different, characteristic, gear tooth contact events.", "The average force Ftot during the gear tooth contact events of gear sets A–D is shown in Fig. 6.", "The average computed forces Ftot of the gear tooth contact events of samples A and C can be compared with those of samples B and D.", "The latter reaches values above 400 kN/m, while the former does not.", "Also, the forces peak at different times during the excentre cycle.", "We call the trend of the average forces observed on samples A and C favourable, and the ones observed in samples B and D unfavourable.", "It should be noted again that the forces in Fig. 6 only depict the average values for the gear tooth contact events.", "In fact, some gear tooth contact events that lead to unfavourable conditions are present in all gear sets, they were just more frequent for samples B and D.", "Fig. 6 shows the contact forces, for gear tooth contact events before and after wear.", "The change in force Ftot is very small on average, leading us to conclude that the radius of curvature and contact point location remain approximately constant during the wear process according to the numerical model.", "While the contact force Ftot seems to be almost unaffected by the wear process, the changes in contact stiffness in the model may still cause a reduction in the asperity force Fa.", "It is therefore of interest to look at the change in Fa before and after wear.", "It is also of interest to compare the difference in Fa between the favourable and unfavourable gear tooth contact events.", "The forces Fa of samples A–D are shown in Fig. 7.", "The average asperity force Fa differs even more clearly between the favourable and the unfavourable gear tooth contact events.", "The Fa values in the unfavourable gear tooth contact events force curve are higher, and a large extra peak can be observed.", "The force Fa is reduced by about 21% for sample B, 19% for sample D and about 15% for samples A and C.", "The wear depth of all gear sets is shown in Fig. 8.", "Results from both experiments and simulation are shown.", "There is a large difference in terms of wear depth, between the samples A and C compared with samples B and D.", "The measured wear depths in samples B and D are larger than those of A and C.", "In the experiment the difference is larger than the difference in the simulation.", "This indicates that a transition to more severe wear occurs during unfavourable gear tooth contact events, which occur more often in samples B and D.", "In the experiment, the location of highest wear depth occurs in Zone I, marked in Fig. 8.", "This is also the zone where the highest contact forces are acting on the inner gear for unfavourable gear tooth contact events.", "The simulation underestimates the wear depths in Zone I.", "The large wear depths which are not proportional to the calculated pressure are most likely due to a transition to severe wear.", "This transition may be induced by pressure, or possibly high temperature which may occur at the later stage of the gear tooth contact event, coinciding with a low value on θ.", "SEM investigations in a previous publication [18] showed that the mechanism of wear in this region was abrasive wear.", "In Zone II in Fig. 8, the agreement between simulation and experiment was very good, in particular for sample A.", "This suggests that the model accounts well for the wear mechanisms in this zone.", "Zone III, marked in Fig. 8, is a zone of wear which is completely missed by the numerical simulation.", "As no contact pressure is expected in this region, and no third body abrasive wear has been seen from SEM investigations, we do not know exactly why there is wear in Zone III.", "The wear mechanisms which have been identified were 2 body abrasive and adhesive wear [18].", "Zone IV, also marked in Fig. 8, is captured by the simulation in gear sets A and C.", "The experiments also show wear in this region for samples D and B. SEM analysis indicates that wear in this region was partially due to third bodies [18], which could be an explanation to why the wear is not fully accounted for in the simulation results for sample B and D.", "After considering the force curves and the wear data, we can conclude that unfavourable force conditions during a gear tooth contact event lead to severe wear.", "Since the running-in model did not discover this transition, the transition pressure of 2.5 GPa, used in the model, could have been set too high.", "Furthermore, severe wear should lead to a higher k-value in terms of global wear, an effect currently not considered in the model.", "A numerical, two-scale, mixed lubrication, wearing-in model was developed.", "Numerical simulations were run on measured profiles from a number of hydraulic motor gears, which were also tested experimentally.", "The gear sets which showed the higher asperity carried contact forces in the numerical simulation also showed the highest wear depths in experiments.", "The wearing-in model includes a criterion for severe wear on the topography scale but not on the component scale.", "The wear coefficient is assumed to have a constant value on the component scale and the same value on the small scale.", "The criterion for, as well as variations in wear coefficient because of, a transition to severe wear is one possible explanation for differences between experimental results and simulation results.", "The suggested wearing-in model illustrates an efficient way to incorporate running-in effects in mixed lubrication wear simulations." ]
[ "A mixed lubrication running-in model is presented.", "Changes in the surface topography due to wear affect the load sharing.", "Running-in is observed in the model due to the wear on two length scales.", "Measured surface profiles are used as input to the model.", "Simulation results correlate well qualitatively with experiment." ]
[18F]fallypride characterization of striatal and extrastriatal D2/3 receptors in Parkinson's disease
Parkinson's disease (PD) is characterized by widespread degeneration of monoaminergic (especially dopaminergic) networks, manifesting with a number of both motor and non-motor symptoms. Regional alterations to dopamine D2/3 receptors in PD patients are documented in striatal and some extrastriatal areas, and medications that target D2/3 receptors can improve motor and non-motor symptoms. However, data regarding the combined pattern of D2/3 receptor binding in both striatal and extrastriatal regions in PD are limited. We studied 35 PD patients off-medication and 31 age- and sex-matched healthy controls (HCs) using PET imaging with [18F]fallypride, a high affinity D2/3 receptor ligand, to measure striatal and extrastriatal D2/3 nondisplaceable binding potential (BPND). PD patients completed PET imaging in the off medication state, and motor severity was concurrently assessed. Voxel-wise evaluation between groups revealed significant BPND reductions in PD patients in striatal and several extrastriatal regions, including the locus coeruleus and mesotemporal cortex. A region-of-interest (ROI) based approach quantified differences in dopamine D2/3 receptors, where reduced BPND was noted in the globus pallidus, caudate, amygdala, hippocampus, ventral midbrain, and thalamus of PD patients relative to HC subjects. Motor severity positively correlated with D2/3 receptor density in the putamen and globus pallidus. These findings support the hypothesis that abnormal D2/3 expression occurs in regions related to both the motor and non-motor symptoms of PD, including areas richly invested with noradrenergic neurons.
[ "MEDI", "NEUR" ]
[ "Dopamine", "Fallypride", "Neurodegeneration", "Parkinson's disease", "Positron emission tomography (PET)" ]
[ "2728", "2741", "2805", "2808" ]
[ "D2 and D3 receptors are expressed in high abundance in the striatum and ventral midbrain, and in lower levels in certain limbic and cortical regions (Gurevich and Joyce, 1999).", "Dopamine agonists that preferentially target these D2/3 receptors improve motor symptoms in Parkinson's disease (PD) (Shannon et al., 1997), and have been suggested to also reduce certain non-motor symptoms, such as depression (Barone et al., 2006).", "Early in the course of PD, striatal D2/3 binding potential (BPND) increases (Rinne et al., 1993; Rinne et al., 1995), potentially due to reduced receptor occupancy by endogenous dopamine, or post-synaptic sensitization induced increases in receptor expression (Knudsen et al., 2004).", "This upregulation of D2/3 receptors is more extensive in the putamen than the caudate nucleus, consistent with the earlier and more prominent dopaminergic denervation of the putamen in PD (Gibb and Lees, 1991).", "With longer disease duration, D2/3 expression diminishes throughout the striatum (Antonini et al., 1997).", "Less attention has been devoted to the effects of PD on extrastriatal D2/3 expression.", "Some studies have reported a decrease in D2/3 BPND later in the course of PD, including in the medial thalamus as well as anterior cingulate, inferior temporal, and ventromedial/dorsolateral prefrontal cortices (Kaasinen et al., 2003; Kaasinen et al., 2000; Ko et al., 2013).", "Due to intrinsic differences in properties of SPECT and PET radioligands, few studies have been capable of concurrently examining striatal and extrastriatal D2/3 binding in PD patients.", "The fact that most radioligands are best suited towards quantitation of binding in striatal or extrastriatal sites, but not both at the same time, makes a comprehensive understanding of the relative magnitude of PD-induced D2/3 BPND changes across areas compared to healthy subjects difficult to capture. [", "11C]raclopride is effective at measuring striatal (and to an extent thalamic) D2/3 levels but cannot provide reliable estimates in most extrastriatal areas, while [11C]FLB-457 can assess extrastriatal regions but is not able to quantify binding in the striatum (as it does not reach equilibrium in a reasonable timeframe) (Farde et al., 1997; Hall et al., 1989).", "The D3 preferring ligand [11C]-(+)-PHNO has infrequently been used to estimate dopamine receptor binding in PD (Boileau et al., 2009; Payer et al., 2015), but cannot provide full characterization of limbic and cortical regions (Egerton et al., 2010).", "[18F]fallypride ((S)-N-[(1 allyl-2-pyrrolidinyl)methyl]-5-(3[18F]fluoropropyl)-2,3-dimethoxybenzamide) is a high-affinity D2/3 radioligand that can provide accurate estimates of binding in both striatal and extrastriatal regions, allowing for concurrent estimation of dopamine D2/3 receptor levels (i.e. nondisplaceable binding potential, BPND) throughout the brain (Kessler et al., 2000; Mukherjee et al., 2002).", "Furthermore, [18F]fallypride has been successfully applied in a PD cohort (Deutschlander et al., 2016).", "We examined regional [18F]fallypride binding in a large cohort of PD patients and age-matched healthy control (HC) subjects in order to simultaneously determine differences in striatal and extrastriatal D2/3 BPND, with the goal of providing cortical and subcortical binding potentials that can be directly compared.", "As a secondary objective, we assessed if D2/3 BPND reflected motor severity in PD patients.", "PD participants were recruited from the Movement Disorders Clinic at Vanderbilt University Medical Center.", "All met UK Brain Bank criteria for a diagnosis of PD and were prescribed levodopa and DA agonist medications (including pramipexole, ropinirole, and rotigotine) for relief of motor symptoms.", "Daily doses of dopamine replacement therapy were converted to levodopa equivalent dose (Tomlinson et al., 2010).", "Patients were excluded if they had an implanted deep brain stimulator, received antipsychotic treatments, suffered from comorbid neuropsychiatric, cerebrovascular, or cardiovascular disease, could not tolerate a brain MRI/PET study, or dopaminergic medication withdrawal.", "HC subjects (Dang et al., 2017; Dang et al., 2016) did not have a history of psychiatric illness, head trauma, substance abuse, diabetes, or medical condition that precluded MRI collection, nor could they use tobacco.", "No participants took psychostimulant or psychotropic medications (with an exception for occasional use of benzodiazepines as sleep medication) over the preceding 6 months, and did not consume excessive alcohol.", "Urine drug tests were administered to all participants to ensure the absence of amphetamine, barbiturates, cocaine, marijuana, or opiates.", "A neurologic exam was performed on all participants, in order to exclude parkinsonism in HC subjects.", "PD patients completed part II of the Movement Disorders Society-United Parkinson's disease Rating Scale (MDS-UPDRS) (a self-reported assessment of the impact of PD on activities of daily living), and part III (an assessment of motor function in PD) in the Off-medication condition (Goetz et al., 2008; Weintraub et al., 2012).", "Dopamine medications were withheld for >40 h prior to PET imaging for DA agonists and >16 h for levodopa prior to PET imaging (the half-life of levodopa, ropinirole, and pramipexole are approximately 1.5, 6, and 8–12 h respectively (Bennett Jr and Piercey, 1999; Fabbrini et al., 1987; Tompson and Oliver-Willwong, 2009; Wright et al., 1997).", "Cognitive screening was performed using the Montreal Cognitive Assessment (MoCA) to rule out patients with frank dementia (Nasreddine et al., 2005), requiring a score of at least 22.", "In PD patients, depression was screened using the Center for Epidemiologic Studies Depression Scale Revised (CESD-R) (Radloff, 1977).", "The presence of medication-induced impulsive compulsive behaviors (ICBs) as a potential confounding factor was also assessed using a semi-structured interview with patient and partner.", "Demographic and clinical features for PD patients (n = 35), as well as HC subjects (n = 31) are presented in Table 1.", "Both groups had a similar average age and sex distribution.", "The side of symptom severity (both onset and based on motor testing) was more prominent in the left hemi-body of PD patients, who expressed moderate PD progression with an average disease duration of 5.9 ± 3.9 years.", "Of this cohort, 17 had symptoms of Impulsive Compulsive Behaviors.", "Written informed consent was obtained from all subjects, and the study was performed in accordance with the Institutional Review Board at Vanderbilt University, adhering to the ethical standards stipulated by the Declaration of Helsinki and its amendments.", "MRI scans were completed prior to PET scans in order to provide high-resolution structural delineation.", "Both PD and HC subjects were scanned at 3.0T (Philips, Best, The Netherlands) using body coil transmission and 8-channel SENSE reception.", "All underwent a T1-weighted high-resolution anatomical scan (MPRAGE; spatial resolution = 1 × 1 × 1 mm3; TR/TE = 8.9/4.6 ms).", "[18F]fallypride was synthesized in the radiochemistry laboratory consistent with the synthesis and quality control procedures outlined by US Food and Drug Administration INDs 47,245 and 120,035.", "Data were collected on a GE Discovery STE PET/CT scanner.", "Serial scan acquisition began simultaneously with a 5.0 mCi slow bolus injection of [18F]fallypride (specific activity >3000 Ci/mmol).", "CT scans were collected prior to each of the three emissions scans for the purpose of attenuation correction.", "Together, the scans lasted approximately 3.5 h with two breaks of 15–20 min (beginning approximately 70 min and 135 min after the beginning of the scan, respectively) included for patient comfort.", "During breaks, patients remained at rest but were permitted to stretch.", "Data for PD and HC subjects were acquired using identical MRI and PET technical parameters, with the single exception of a slightly different PET acquisition time protocol for the second and third dynamic runs, although total scan duration was similar (Supplementary Table 1; see Dang et al. (2017); Dang et al. (2016)).", "In past PET studies, differences in acquisition time protocol were not found to be a significant confound (Buckholtz et al., 2010; Smith et al., 2016).", "Following attenuation correction and decay correction, serial PET scans were co-registered using Statistical Parametric Mapping software (SPM8, Wellcome Trust Centre for Neuroimaging, London, UK, http://www.fil.ion.ucl.ac.uk/spm/software/) to correct for motion across scanning periods with the last dynamic image of the first series serving as the reference image.", "The mean PET image produced by realignment was then co-registered to the subject's corresponding high-resolution T1 MRI image using FSL's FLIRT with 6 degrees of freedom (FSL v5.0.2.1, FMRIB, Oxford, UK).", "D2/3 receptor levels were estimated using the simplified reference tissue model (SRTM) (Lammertsma et al., 1996), performed in PMOD software (PMOD Technologies, Zurich Switzerland) to measure [18F]fallypride binding potential(BPND; the ratio of specifically bound [18F]fallypride to its nondisplaceable concentration as defined under equilibrium conditions).", "Voxel-wise estimates were generated using a published basis function fitting approach (Gunn et al., 1997) conducted in the PXMOD module of PMOD.", "The rate constants were specified as k2a minimum = 0.006 min−1 and k2a maximum = 0.6 min−1.", "Due to the very limited expression of D2/3 receptors in the cerebellum (Camps et al., 1989), it was selected as the reference region (Kessler et al., 2000; Kessler et al., 2005).", "Subject-space analyses were conducted by warping baseline BPND images to T1 space with the saved FSL FLIRT transform matrices.", "For voxel-wise analyses, subject-space BPND images were registered to Montreal Neurological Institute (MNI) space using FSL's FNIRT (FSL v5.0.2.1, FMRIB, Oxford, UK).", "Voxel-wise analysis was first applied to assess putative subcortical and cortical differences between groups, with specific attention towards potential sub-regional distinctions in the broader mesiotemporal and prefrontal cortices.", "Next, subcortical regions of interest (ROIs), including the caudate (head), putamen (whole body), globus pallidus, ventral striatum, amygdala, hippocampus, ventral midbrain, thalamus, and cerebellum, were manually segmented on the T1-weighted MRI scans by a neuroradiologist (RMK) and neurologist (DOC) experienced in PET and MRI data analysis, and transferred to the co-registered PET studies through the FLIRT FSL transformation matrix.", "Manual segmentation methods followed established anatomical criteria, capturing the central portion of the selected region to gather the most representative sample.", "This method was selected in order to best avoid partial volume effects in densely arranged subcortical nuclei, and were applied so as to avoid the potential confound of inter-subject structural variability (Kessler et al., 2009).", "The caudate, putamen, and globus pallidus were manually drawn on axial slices approximately 2–12 mm above the ACPC line.", "The ventral striatum was segmented on coronal slices with the criteria of Mawlawi et al. (2001).", "The amygdala can be identified on axial slices 6–20 mm below the ACPC line, 12–28 mm lateral to the midline, and 2–12 mm behind the plane of the anterior commissure (Schaltenbrand and Wahren, 1998).", "To minimize partial voluming of the striatum, amygdala ROIs were defined 10–16 mm beneath the ACPC plane.", "The ventral midbrain was drawn on axial slices in 9–14 mm below the ACPC line, and the thalamus was segmented 2–12 mm above the ACPC line (Schaltenbrand and Wahren, 1998).", "The cerebellar ROI was drawn centrally within the structure to avoid partial voluming of ventral midbrain or cortical signal, and contained an approximately equal distribution of grey and white matter.", "Due to difficulty capturing the extent of the hippocampus using manual methods, it was defined using a previously published automatic subject space segmentation method (Asman et al., 2015).", "All ROIs were defined bilaterally.", "In order to account for potentially divergent structure size between groups, ROI volumes were collected and preserved for statistical analysis.", "Group differences in demographic and clinical parameters, as well as ROI volume, were evaluated using Mann Whitney U tests.", "Sex differences were evaluated with a chi-square test.", "A voxel-wise analysis investigated PD-related group differences in D2/3 BPND across cortical and subcortical regions.", "Age and sex were included as covariates, due to previous evidence that these factors influence D2/3 receptor status (Mukherjee et al., 2002; Pohjalainen et al., 1998), and significance criteria consisted of an uncorrected p < 0.005.", "Multiple comparisons correction was accomplished by controlling cluster-level FDR at 0.05.", "This analysis was completed using SPM8.", "In a secondary, exploratory voxel-wise analysis utilizing identical statistical parameters, BPND maps for subjects expressing more severe PD symptoms on the right side of the body were flipped across the rostral-caudal axis of the axial plane.", "This step was completed in order to ensure that the more degenerated side of the dopaminergic system (contralateral to more severe motor deficits) was aligned for all subjects (Antonini et al., 1997).", "To test the hypothesis that PD patients have different dopamine D2/3 receptor expression in specific striatal and subcortical extrastriatal areas, mean group regional [18F]fallypride BPND was analyzed via a general linear regression model (GLM), where within-ROI BPND was the dependent variable and PD status was the primary independent variable.", "Age and sex were included as covariates.", "ROI volume was also specified as a covariate, to ensure that any differences were not due to overall structural atrophy.", "False discovery rate (FDR) was controlled at 0.1 to correct for multiple comparisons, in concordance with the threshold recommended in the first description of the method (Benjamini and Hochberg, 1995).", "All analyses were performed using SPSS Statistics 24 (IBM, Armonk, NY, USA) and R (R Foundation for Statistical Computing, Vienna, 2016).", "In addition to the ROI analysis, we performed a post-hoc evaluation to quantify the magnitude of BPND differences in regions where voxel-wise distinctions were present, but were not captured by the hand-drawn ROI analysis.", "Mean group BPND values were extracted from standard space (MNI) in areas where voxel-wise clusters were manifested using the Automated Anatomical Labeling (AAL) atlas or manual segmentation methods.", "This served as a post-hoc method of evaluating the extent of regional differences identified by the voxel-wise analysis.", "The relationship between regional [18F]fallypride BPND and PD severity was also examined using a partial Pearson's correlation covarying for age, sex, ROI volume, disease duration, and levodopa equivalent daily dose (LEDD).", "This process was repeated for both MDS-UPDRS Part II (a measure of activities of daily living) and MDS-UPDRS Part III-Off (a measure of motor symptom severity), and the results were controlled at FDR of 0.1 to correct for multiple comparisons.", "A secondary voxel-wise analysis was also completed for MDS-UPDRS Parts II and III using SPM8, specifying age, sex, disease duration, and LEDD as covariates, an uncorrected threshold of p < 0.005, and cluster-level FDR controlled at 0.05.", "This voxel-wise analysis was also repeated using images flipped across the y-axis of the axial plane to align the side of the brain contralateral to the side of greater PD motor symptom severity in all subjects.", "Using the voxel-wise method covarying for age and sex, significant reductions of [18F]fallypride BPND in PD patients localized to subcortical clusters in the amygdala and hippocampus, ventral midbrain, LC, caudate nucleus, and globus pallidus.", "Cortical differences localized to the anterior and medial temporal cortical regions (temporal pole, parahippocampal, and entorhinal cortices).", "In no region was D2/3 BPND increased in patients with PD compared to HC participants.", "Figs. 1 and 2 display a map of significant clusters overlaid on a standard-space brain, in an array of coronal and axial slices respectively.", "A similar cluster pattern was evident when images were aligned so that the right side of the brain was contralateral to the side of greater PD motor symptom severity in all subjects; although largely symmetrical, cluster size was slightly more extensive in the right-sided midbrain and left-sided temporal cortex (Supplementary Figs. 3 & 4).", "To quantitate group differences in mean subcortical regional [18F]fallypride BPND we applied a GLM analytic approach, including group as an independent variable, and age, sex, and volume as covariates.", "In order of the magnitude of percent difference, significant BPND reductions in PD patients were evident in the ventral midbrain (p < 0.001; 39% decrease), amygdala (p < 0.001; 33% decrease), thalamus (p < 0.001; 30% decrease), hippocampus (p < 0.001; 22% decrease), globus pallidus (p = 0.002; 16% decrease), and caudate (p = 0.005; 11% decrease).", "No significant differences were evident in the putamen (p = 0.56; 2% difference), or ventral striatum (p = 0.66; 2% difference).", "(See Supplementary Table 3 for full description of mean regional binding potentials).", "Fig. 3 presents the individual and group mean regional BPND values for the ROIs, alongside segmentation visualization.", "To quantify the extent of BPND differences in regions where voxel-wise distinctions were present, but that were not captured by the hand-drawn ROI analysis, we applied the AAL atlas to define the bilateral combined entorhinal and parahippocampal cortices, the inferior and middle temporal gyri, and the temporal pole.", "The locus coeruleus (LC) was defined by uniting six contiguous 2 mm boxes bilaterally, centered in standard space (MNI) according to the mean values on the x and y axis as discussed by Isaias et al. (2011) and Keren et al. (2009).", "This approach revealed PD-induced BPND decreases of a similar magnitude to those observed in other extrastriatal areas: LC (27% decrease), entorhinal and parahippocampal cortices (19% decrease), inferior (25% decrease) and middle (23% decrease) temporal gyri, and temporal pole (24% decrease).", "(See Supplementary Table 5 for full description of mean regional binding potentials, and Supplementary Figs. 1 and 2 for depiction of the segmentation protocol as well as group and individual regional means).", "The relationship of BPND to MDS-UPDRS Parts II and III-Off scores was assessed using a voxel-wise analysis while covarying for age.", "We observed a positive relationship between [18F]fallypride BPND and UPDRS Part III-Off in a right hemisphere cluster localized to the globus pallidus and anterior putamen, and a left hemisphere cluster localized to the posterior putamen.", "No significant clusters were observed when BPND was tested for association with UPDRS Part II scores.", "Supplemental Fig. 4 displays a map of significant clusters overlaid on a standard-space map.", "When images were aligned so that the right side of the brain was contralateral to the side of increased PD motor symptom severity in all subjects, a significant cluster was evident only in the left-sided putamen and globus pallidus (Supplementary Fig. 6).", "To further evaluate the association between D2/3 BPND and motor severity, we assessed the relationship between [18F]fallypride BPND and UPDRS Part III-Off scores in the hand-defined bilateral putamen and globus pallidus, while covarying for factors that may influence motor severity.", "This included age, sex, ROI volume, disease duration, and LEDD.", "We observed a significant positive correlation between BPND and scores on the MDS-UPDRS Part III in the putamen (r = 0.488, p = 0.006) and globus pallidus (r = 0.449, p = 0.013).", "No significant association was present between MDS-UPDRS Part III and BPND in any other ROI.", "Again, we did not see a significant relationship between [18F]fallypride BPND and MDS-UPDRS Part II.", "Fig. 4 displays the graphs of MDS-UPDRS Part III score with BPND in the putamen and globus pallidus.", "We observed widespread reductions in D2/3 receptor binding in PD patients, indicating changes to D2/3 receptor availability in regions that relate to both the motor and nonmotor aspects of PD.", "The sites in which D2/3 BPND decreases were observed include basal ganglia, limbic, thalamic, and cortical regions, as well as the LC.", "Our discussion focuses on these D2/3 BPND group differences in the basal ganglia (and relationships with the severity of off-motor symptoms in PD), norepinephrine-related extrastriatal areas, and limbic and thalamocortical regions.", "Broadly, the pattern of BPND reduction in the basal ganglia is of a lower magnitude, indicative of a complex relationship between presynaptic receptor loss and postsynaptic receptor upregulation, while extrastriatal areas show larger differences between the PD and HC groups.", "The interpretation of data presented in this report requires the consideration of several technical parameters relevant to determining the neurobiological origin of PD-related changes reflected in [18F]fallypride BPND values.", "D2/3 receptors are expressed on dopaminergic neurons, where they can operate presynaptically as autoreceptors (Khan et al., 1998; Mercuri et al., 1992), or as postsynaptic receptors present on striatal medium spiny neurons and cortical pyramidal cells (Albin et al., 1989; Gaspar et al., 1995).", "Thus, a decrease in [18F]fallypride BPND values could potentially convey a loss of autoreceptors secondary to degeneration of mesotelencephalic dopaminergic neurons, or a decrease in postsynaptic D2/3 expression.", "However, the latter possibility appears unlikely to be a primary source of decline in receptor availability, as nigrostriatal dopaminergic denervation elicits a compensatory upregulation of postsynaptic receptors early in the course of PD (Falardeau et al., 1988).", "Considering that our subjects were relatively early in the course of motor parkinsonism, it is likely that the present report of decreased [18F]fallypride BPND is a measure of presynaptic D2/3 autoreceptor density, influenced by ongoing degeneration of dopaminergic neurons.", "Finally, interpretation of [18F]fallypride BPND must also account for the combined influence of long-term receptor expression and the level of dopamine in the synaptic cleft which competes with [18F]fallypride binding leading to an underestimation of the long-term D2/3 expression level.", "Reduced D2/3 levels were observed in the ventral midbrain, caudate nucleus, and globus pallidus of PD patients.", "The initial and greatest loss of midbrain dopaminergic neurons in PD occurs in the ventrolateral substantia nigra (Gibb and Lees, 1991).", "It is therefore likely that the ventral midbrain reduction in BPND reflects nigral dopaminergic cell loss (Gibb and Lees, 1991), and the consequent loss of somatodendritic D2/3 autoreceptors.", "In contrast, D2/3 BPND did not differ in the putamen and ventral striatum between PD and HC subjects.", "While the loss of presynaptic terminal autoreceptors due to dopaminergic cell death likely accounts for the decrease in BPND observed in several regions (caudate and globus pallidus), D2/3 BPND is unchanged in certain other areas (putamen and ventral striatum), although these regions also suffer dopaminergic denervation.", "This is particularly striking in the putamen where dopaminergic denervation is typically greatest.", "The lack of significant differences between subjects with PD and control subjects presumably reflects a combination of postsynaptic receptor upregulation (Falardeau et al., 1988), combined with a decrease in terminal autoreceptors and extracellular DA levels.", "Post-synaptically, long-term binding decrements over the course of PD likely involve the loss of D2/3 expressing dendritic spines (Stephens et al., 2005; Zaja-Milatovic et al., 2005) a concept further emphasized by past evidence of putaminal atrophy (Tanner et al., 2017).", "In our hands, the combined influence of these factors on D2/3 in the putamen sums to no significant change.", "This may explain equivalent putaminal BPND results between patients and controls, but divergent values in the caudate.", "Because ventral midbrain dopaminergic cell death occurs in the ventrolateral substantia nigra (containing cell bodies from which the dopaminergic projection to the more dorsolateral and posterior putamen emanates), the greatest DA reductions more heavily affect the putamen than the caudate; these losses likely exceed a critical threshold and produce notable receptor upregulation in the region (Gibb and Lees, 1991; Haber et al., 2000; Rinne et al., 1993; Rinne et al., 1995).", "By contrast, either this threshold is not reached in other brain regions, or those areas lack the same compensatory upregulation that occurs in the putamen.", "In addition to differences in long-term receptor expression, the lack of an observed BPND difference in the putamen could also be affected by acute reductions in synaptic dopamine, which may contribute to the positive correlation between motor symptoms and D2/3 binding in the region, which is likely weighted on the former factor.", "Patients with more severe motor symptoms after standardized dopamine withdrawal presumably have less endogenous synaptic dopamine levels after medication washout, resulting in less competition with [18F]fallypride binding.", "This concept could be further emphasized by the finding that this positive association was present only on the left hemisphere of the brain, following alignment of all BPND images so that more severe motor symptoms were expressed in the right hemi-body.", "The fact that the correlation is present on the side of the dopaminergic system less affected by denervation, where DA levels are likely more dynamic and sensitive to the presence of medication, could strengthen the association between observed striatal BPND and synaptic DA.", "Future studies employing pharmacological methods of manipulating dopamine levels may clarify this relationship between endogenous dopamine, regional differences in D2/3 receptor binding, and motor severity.", "Our findings of D2/3 reductions in the globus pallidus are similar to previous observations in a [11C]-(+)-PHNO study of PD reporting significantly reduced binding in comparison to control subjects (Boileau et al., 2009).", "However, this is the first study to indicate a relationship between motor symptoms and pallidal D2/3 expression after medication washout.", "This association is in agreement with a recent study linking external globus pallidus function to motor severity in dopaminergic denervation mouse models (Mastro et al., 2017).", "We also found D2/3 BPND changes in a number of regions that are classically known as noradrenergic sites, and receive less dense dopaminergic inputs.", "In PD patients, D2/3 BPND was decreased in the LC, a structure containing a large population of noradrenergic neurons that provide most norepinephrine in the forebrain.", "This effect was also present in the hippocampus, as well as several cortical sites in which norepinephrine concentrations are greater than those of dopamine (entorhinal/parahippocampal cortices, and temporal pole).", "While nigrostriatal denervation bears a clear link with the motor symptoms of PD, the association between the progressive loss of the noradrenergic system and many non-motor features has been amply described (Cumming and Borghammer, 2012).", "The degeneration of noradrenergic LC neurons in PD has long been known (German et al., 1992) and more recently the presence of α-synuclein inclusions in the LC has been reported (Braak et al., 2001).", "Moreover, noradrenergic loss in several cortical areas, including the prefrontal cortex, is comparable to or greater than the corresponding decrease in dopamine content in these structures in MPTP-induced parkinsonism (Elsworth et al., 1990) and in idiopathic PD (Scatton et al., 1983).", "D2 receptors are expressed on LC neurons (Mansour et al., 1990), suggesting that the degeneration of LC neurons may underlie the decrease in BPND due to postsynaptic D2/3 loss in subjects with PD.", "However, dopamine also positively regulates LC noradrenergic activity via reciprocal projections between ventral midbrain dopaminergic neurons and the LC (Deutch et al., 1986; Guiard et al., 2008).", "As such, the loss of presynaptic D2/3 expressed on dopaminergic midbrain projections to the LC may also contribute to the decrease in D2/3 binding in the region.", "In the hippocampus, the density of dopaminergic innervation from the midbrain is sparse (Otmakhova et al., 2013), meaning that decreased BPND there is less likely due to presynaptic D2/3.", "Recent evidence has indicated that noradrenergic projections to the hippocampus and associated cortical regions may account for a large proportion of dopamine release in these structures (Devoto et al., 2005; Smith and Greene, 2012).", "Combined with the finding that norepinephrine may act as a D2/3 receptor agonist (Sanchez-Soto et al., 2016), the D2/3 BPND reductions in the LC, hippocampus, and medial temporal cortex could instead indicate widespread dysfunction of the noradrenergic system and its terminal fields, a well-described.", "Degeneration of this system, centering on the LC, is suggested as a major contributor to cognitive and other behavioral non-motor symptoms of PD (Benarroch, 2017; Rommelfanger and Weinshenker, 2007; Weiss et al., 1986).", "The dopaminergic innervation of the amygdala arises from ventral midbrain neurons largely targeting the basolateral complex (Garcia-Amado and Prensa, 2013), where D2/3 receptors regulate reward learning processes (Berglind et al., 2006; Di Ciano and Everitt, 2004).", "The amygdala is known to be vulnerable to PD pathology, given the presence of Lewy body inclusions and susceptibility to atrophy (Braak et al., 1994; Junque et al., 2005).", "This pattern of degeneration is linked to the manifestation of depression in PD, where symptom severity correlates with reduced amygdalar volume (van Mierlo et al., 2015), and apparent hypofunction (Diederich et al., 2016; Sheng et al., 2014).", "Deficits in emotional processing constitute another non-motor symptom commonly attributed to dopaminergic innervation of the amygdala (Bowers et al., 2006), supported by the apparent influence of DA medication state on both behavioral and amygdalar hemodynamic response to emotional stimuli (Tessitore et al., 2002).", "D2/3 agonist medications show efficacy in ameliorating depression in PD (Barone et al., 2006), an effect that could potentially be induced by targeting D2/3 in the amygdala, where D2/3 agonists significantly displace receptor-bound [11C]FLB-457 (Ishibashi et al., 2011).", "Therefore, altered D2/3 BPND in the amygdala may account for some of the non-motor symptoms related to PD-depression.", "The extent of D2/3 receptor reductions to this region in our cohort of non-depressed patients suggests that this is an early area of vulnerability in PD which may contribute to subsequent depression risk.", "The thalamus expresses notable D2/3 populations in the midline intralaminar and mediodorsal nuclei (Garcia-Cabezas et al., 2007; Rieck et al., 2004).", "Due to the selection of an ROI in the middle of the superior-inferior thalamic axis, BPND in this ROI is heavily weighted towards D2/3 expression in the medial thalamus.", "Although the function of thalamic D2/3 receptors is largely unexplored, the role of altered thalamocortical networks in the manifestation of motor symptoms of PD is well established (Sarnthein and Jeanmonod, 2007).", "Anatomically, D2/3 populations in the thalamus integrate information from mesocortical and limbic regions and project to frontal cortical areas (Goldman-Rakic and Porrino, 1985).", "Previous [11C]FLB-457 PET studies that show reductions in thalamic D2/3 binding also restrict these regional reductions to advanced, but not early, PD populations (Kaasinen et al., 2003; Kaasinen et al., 2000; Ko et al., 2013).", "In contrast, however, the degeneration of neurons in the centromedian-parafascicular complex occurs in both Hoehn and Yahr stage I/II and stage III/IV (Henderson et al., 2000), suggesting that the loss of [11C]FLB-457 binding may preferentially reflect the loss of certain afferents to these intralaminar nuclei.", "In a similar manner to regions already described, losses of presynaptic autoreceptors could also play a role.", "We do not observe differences in frontal cortical areas.", "Previous [11C]FLB-457 studies have indicated reductions that localize to these regions (Kaasinen et al., 2003; Kaasinen et al., 2000; Ko et al., 2013); however, previous preclinical experiments utilizing MPTP models have not always proved consistent with this evidence (Gnanalingham et al., 1993).", "Our result could be a product of the pharmacodynamic distinctions between [11C]FLB-457 and [18F]fallypride (Narendran et al., 2009).", "However, this also may be due to our cohort of moderately affected patients, in whom previously described frontal cortical D2/3 reductions are not manifest until later in the course of the illness.", "We did, however, uncover differences in D2/3 receptors in the perirhinal and parahippocampal areas.", "These regions are critically involved in memory and learning processes (Zola-Morgan et al., 1989), which are often impaired in PD patients (Nagano-Saito et al., 2005).", "Future investigations on the clinical relevance of these findings may improve the characterization of cognitive changes early in PD.", "Although a decided advantage of our investigation was the number of patients with PD recruited to the study, our PD cohort included a larger proportion (~50%) of individuals with compulsive reward-based behaviors than would be expected in a typical PD population (Garcia-Ruiz et al., 2014).", "We noted differences between PD patients with and without compulsive behaviors only in the ventral striatum and putamen, which were not significantly different between the HC and overall PD group; the lack of significant differences in these areas were preserved even when the subgroups were compared with the HC group separately (see Supplementary Table 7).", "Thus, we believe this is not a significant limitation to the interpretation of changes in PD.", "Also, the generalizability of the sample is affected by the relatively specific subject selection criteria, including patients with mild to moderate PD, who could tolerate an extensive dopamine washout, and were without depression, dementia, or psychosis.", "In light of the close proximity of basal ganglia structures, there is potential for partial volume errors.", "We especially considered this given the significant association between D2/3 BPND and UPDRS Part III in the putamen and globus pallidus, two adjacent structures.", "However, the presence of a mean D2/3 BPND difference between groups in the globus pallidus but not in the putamen, in addition to the broad localization of the cluster observed in the voxel-wise correlation analysis, gives us confidence that our results in both regions are largely distinct.", "Additionally, all native subject space analyses included ROI volume as a covariate, which likely decreases the confounding effect of divergent patterns of atrophy in adjacent areas.", "As a cross-sectional study, the present work can only tentatively point to possible changes in D2/3 BPND changes that occur over the course of disease progression.", "Additionally, the use of D2/3 agonists by all those enrolled makes it difficult to disentangle the effects of chronic long-term medication exposure from those purely induced by PD.", "Although past studies have assessed changes to extrastriatal D2/3 binding and dopaminergic biomarkers over time (Kaasinen et al., 2003; Nandhagopal et al., 2009), future studies should longitudinally assess extrastriatal D2/3 expression in a de novo PD population.", "Relating non-motor symptoms (e.g. mood and cognitive impairment), to receptor-level changes in these regions will be necessary to define a causal relationship to non-motor symptoms in PD.", "Overall, our findings emphasize that PD is associated with widespread changes in the expression of D2/3 receptors, and point to increased scrutiny of the contribution of noradrenergic alterations as contributing to changes in dopaminergic systems in PD.", "These changes appear to affect regions implicated in the development of both motor and nonmotor features of the disease, where D2/3 losses proceed differently in the basal ganglia and extrastriatal areas.", "This was supported by the National Institutes of Health/National Institute of Neurological Disorders and Stroke (R01NS097783, K23NS080988) and National Institute of Aging (R01AG044838); and CTSA award No. UL1TR000445 from the National Center for Advancing Translational Sciences.", "The authors declare no conflict of interest." ]
[ "[18F]fallypride allows concurrent evaluation of striatal and extrastriatal D2/3.", "PD patients express widespread D2/3 reductions relative to healthy controls.", "Reductions are apparent in brain regions dominated by dopamine and norepinephrine.", "Percent reductions are larger in extrastriatal areas relative to the basal ganglia.", "D2/3 in the putamen and globus pallidus correlates with PD motor symptom severity." ]
Cotton certification in Sub-Saharan Africa: Promotion of environmental sustainability or greenwashing?
Environmental sustainability is a major challenge for cotton production in Sub-Saharan Africa (SSA). The creation of voluntary certification programs is an effort aimed at finding new ways to influence corporate conduct along supply chains. This article contributes novelty and insight on cotton certification in SSA by exploring the most relevant programs in detail with regard to the demands of international non-governmental organizations (NGOs). We selected the five most central NGOs dedicated to environmental sustainability in agriculture: Friends of the Earth (FoE), Greenpeace, Oxfam, Pesticide Action Network (PAN), and World Wildlife Fund (WWF). After identifying these NGOs’ demands regarding pesticides, fertilizers and genetically modified (GM)cotton, we analyzed the requirements of the four most dominant programs certifying SSA cotton cultivation: the Better Cotton Initiative (BCI), Cotton made in Africa (CmiA), the EU Organic Regulation and Fairtrade Labelling Organization (FLO). The results of our document analysis are bolstered by semi-structured interviews with stakeholders on NGOs’ stances and roles in standard setting and implementation. We find that the EU Organic Regulation scores best in terms of environmental sustainability. Those programs in which NGOs actively participate are less ambitious compared to the public standard. Therefore, we conclude that, if NGOs maintain their commitment to multi-stakeholder initiatives, they risk contributing to ‘greenwashing’ corporate conduct by mobilizing consumer support for labelling programs that actually fall behind existing public regulation on organic certification.
[ "ENVI", "SOCI" ]
[ "Certification schemes", "Cotton", "Organic standards", "Pesticides", "Pollution", "Supply chains" ]
[ "2303", "2306", "2308", "3305" ]
[ "Fifteen percent of global cotton lint exports originate in Sub-Saharan Africa (SSA), where it is an important cash crop expected to expand by 14% by 2025 (OECD and FAO, 2016, p. 83).", "An increasing share of these exports goes to China and Southeast Asia, before reaching consumer markets in North America and the European Union (EU) (OECD and FAO, 2016, p. 36).", "Intensified cotton production is accompanied by a number of ecological issues such as pollution and contamination from pesticide and chemical use (Sneyd, 2014).", "The creation of voluntary certification programs is an effort aimed at finding new ways to prevent such environmental harm along supply chains and to sustain or increase current cultivation levels in producing countries.", "Forty percent of cotton is already certified as being grown under less harmful conditions in SSA (CmiA, 2018a, p. 6).", "This article delivers fresh and novel insights on international certification by exploring the most relevant programs in detail with regard to the environmental demands of non-governmental organizations (NGOs).", "The Fairtrade Labelling Organization (FLO) started offering certification standards for cotton producers in SSA in 2005 (FLO, 2018a).", "In parallel, NGOs began to cooperate with conventional businesses in third-party multi-stakeholder schemes, such as the Better Cotton Initiative (BCI) and Cotton made in Africa (CmiA) (Sneyd, 2011, 2014).", "There are also certification standards defined by public authorities, such as the EU Organic Regulation (Pekdemir, 2018).1", "Scholars have highlighted the critical role NGOs have played in the emergence of environmental sustainability certification (Bartley, 2007; Bingen and Busch, 2006; Thorlakson et al., 2018).", "NGOs’ campaigns have raised a number of sustainability issues regarding cotton cultivation (Sneyd, 2014).", "Yet, while there is research on downstream textile certification, which involves the transport, distribution, sale and end-of-life of textile products (Bartley, 2007; Green, 2014; Koenig-Archibugi and Macdonald, 2013), scholars have widely neglected such governance efforts in the upstream portion of supply chains, where textile production starts with the cultivation and processing of raw materials, such as cotton (for an exception, see Sneyd, 2011, 2014).", "It is controversial whether NGOs’ participation in the diverse certification schemes is significant, or whether they only contribute to ‘greenwashing’ business as usual, i.e. helping companies to portray their products as environmentally friendly when they are not (Haufler, 2003).", "In this vein, this article examines whether cotton certification takes into account central NGO demands in regard to the use of pesticides, fertilizers and genetically modified organisms (GMOs) in cotton agriculture, and answers the following research question: Does certification provide an appealing prospect for international NGOs trying to ameliorate environmental issues within transnational supply chains?", "The following section provides background on voluntary certification as a form of governance of cotton production, with a particular focus on SSA.", "We selected the five most central international NGOs, in terms of membership, budget and public visibility, dedicated to environmental sustainability in agriculture: Friends of the Earth (FoE), Greenpeace, Oxfam, Pesticide Action Network (PAN) and World Wildlife Fund (WWF).", "There is a range of local NGOs involved in the implementation of certification standards on the ground.", "However, only international NGOs have formulated precise demands on pesticides, fertilizers and GMOs, and primarily these international NGOs actively participated in the formulation of cotton standards.", "In the third section of the article, we identify the selected NGOs’ stances on cotton cultivation.", "In the fourth section, we use these NGOs’ demands as a framework to analyze the four most dominant schemes certifying SSA cotton cultivation, based on the number of producers and volume of cotton certified, and presence in a plurality of countries across the continent.", "These are BCI, CmiA, the EU Organic Regulation (as farmers aim to export their ‘organic’ cotton to the EU, as well as other developed regions) and FLO (see Table 1).", "The fifth section discusses the results, comparing the empirical findings with literature on the role of NGOs in certification.", "This final section concludes that the multi-stakeholder certification schemes are generally inadequate in addressing environmental issues concerning pesticides, fertilizers and GMOs, and do not provide an appealing prospect for NGOs trying to ameliorate these issues within transnational supply chains.", "Instead, the EU Organic Regulation is most ambitious at tackling environmental sustainability problems in the SSA cotton sector.", "Hence, NGOs do best when promoting higher public standards in the case of SSA cotton, rather than participating in private schemes.", "Western consumers rely increasingly on voluntary certification initiatives to ensure that they are not unwillingly supporting detrimental practices, such as environmental degradation, in producing countries.", "In addition to product standards, certification initiatives also define an array of process standards related to the conditions under which items are produced or traded (Gupta, 2008).", "In essence, the idea is that if suppliers disclose information and certifiers guarantee compliance with specific standards, buyers are willing to pay a higher price for the products (Haufler, 2003; Koenig-Archibugi and Macdonald, 2013).", "In the cotton and textile sectors, the rise of certification initiatives has been fueled by increasingly globalized production and declining state regulation of environmental and social conditions, especially in international arenas (Brooks, 2015; Sneyd, 2014).", "The phase-out of the Multi-Fiber Arrangement (MFA) liberalized trade in apparel and, paired with China’s accession to the World Trade Organization (WTO) in 2001, shifted the geography of apparel and textile production (Quark, 2013, p. 26).", "Between 2000 and 2009, EU cotton and textile imports grew by 21.9% (European Commission, 2011), and in 2016, the EU imported one million tons of cotton and cotton products, while the United States imported 63.1 thousand tons (Chatham House, 2018).", "Activist movements and NGOs have played a significant role in exposing social and environmental problems associated with the globalization of the cotton and textile industry (Bartley, 2007; Sneyd, 2014).", "Due to the changes in trade, the environmental burdens associated with the cultivation of cotton have been disproportionately shifted to developing countries (Collins, 2003; Hoskins, 2014).", "Cotton is one of the most important cash crops in SSA (OECD and FAO, 2016, p. 83).", "Some countries, like those in the Sahel and West Africa, have focused on cotton exports for decades, whereas other countries, like Ethiopia, have recently intensified their focus on cotton production in response to the growing demand from the apparel industry and consumers in developed countries (USDA Foreign Agricultural Service, 2018).", "Like most other commodity crops, the intensification of cotton production is based on synthetic inputs, particularly pesticides and fertilizer, and a shift towards adopting GM technologies (FAO and ICAC, 2015).", "Most pesticides are toxic to humans and to the environment.", "Overuse and inappropriate application of pesticides increases the risks of unintended environmental consequences, in particular, soil and water degradation and contamination (FAO and ICAC, 2015; Jennings and Li, 2014).", "Despite being grown on only 2.3% of the planet’s arable land, cotton is estimated to use 6.2% of the total 4.6 million tons of chemical pesticides applied annually (ICAC, 2012; Zhang et al., 2011).", "Pesticides are applied to conventional cotton throughout its development in the form of fungicides, herbicides, nematicides and insecticides.", "Furthermore, cotton cultivation is particularly notorious for the use of highly hazardous pesticides (HHPs), ranging from Endosulfan to DDT, which are particularly harmful to producer health (Mengistie et al., 2016).", "A number of international agreements and guidelines have emerged to address these issues.", "In 2004, the Rotterdam Convention established a procedure and requirements for the trade and use of certain hazardous chemicals.", "Also in 2004, the Stockholm Convention entered into force, requiring parties to reduce or eliminate the release of listed persistent organic pollutants (POPs) into the natural environment (Galt, 2008).", "In addition, the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) provide toxicity classifications and define the specific characteristics of HHPs, which incorporates criteria from the WHO, the Globally Harmonized System on Classification and Labelling of Chemicals (GHS), the Rotterdam and Stockholm Conventions and the Montreal Protocol.", "In consequence, a number of chemicals are banned or restricted by the EU and the United States (FAO and WHO, 2016).", "Moreover, the FAO and the International Cotton Advisory Committee (ICAC) recommend that all HHPs should be banned in countries without advanced regulatory systems (FAO and ICAC, 2015).", "Despite maintaining membership in international agreements such as the Rotterdam and Stockholm Conventions, countries in SSA continue to experience issues with pesticides due to a lack of domestic regulatory capacity.", "According to Williamson (2011, p. 41), “African farmers are possibly the least equipped among the developing world to protect themselves and their community against the hazards of pesticide use” with “rural populations and the environment likely to suffer significant exposure”.", "Similarly, inappropriate fertilizer use is widespread among African farmers, the majority of whom are smallholders (Sheahan and Barrett, 2017).", "FAO predicts global fertilizer use to exceed 201 million tons by the end of 2020 (FAO, 2017, p. 3), and in SSA, increase in demand for fertilizer is expected grow 11.2% and 7.3% annually for nitrogen-based fertilizer and phosphate-based fertilizer, respectively, between 2018 and 2020 (FAO, 2017, p. 21).", "Along with the increase in fertilizer consumption comes a variety of environmental implications, particularly water pollution concerns from the two main ingredient nutrients: nitrogen and phosphorous (FAO, 2017).", "In addition to the use of pesticides and fertilizers, the use of GMOs is controversial in cotton agriculture (Klintman, 2002).", "Proponents of GM cotton cite its success in reducing the need for pesticides, its financial benefit to farmers and the resulting general increase in economic welfare and social development in areas where it is used (Adenle et al., 2013; Morse et al., 2005; Qiao et al., 2017).", "However, there are also concerns, such as threats to biodiversity, including non-pest organisms and soil biological processes; gene flow and genetic recombination; and development of herbicide resistance in target species (Tsatsakis et al., 2017).", "In SSA, Burkina Faso, Ethiopia, South Africa and Sudan have commercialized GM cotton crops, but Burkina Faso phased them out in 2015 after farmers did not receive expected crop quality, even though yields had increased.", "Use of the GM cotton varieties “ended up as a trade-off between quantity and quality” (Bavier, 2017, p. 1).", "Despite Burkina Faso’s phase-out, interest in the technology continues to grow in the form of research and development programs and trials (Braimah et al., 2017).", "There is a wide range of certification schemes addressing the use of pesticide, fertilizer and GM crops in cotton production (Sneyd, 2014; Sneyd, 2014).", "The agri-food governance and certification literature generally assumes that businesses participate in certification initiatives as a result of pressure from “the twin threats” (Haufler, 2003, p. 248): (1) environmental activism that targets the public reputation of specific firms and industry sectors as a whole, and (2) the ‘shadow of hierarchy’, i.e. continuous negotiations among governments on ‘command and control’ types of regulation.", "The general assumption is that it was only in response to these threats that multinational corporations started to express concern about environmental issues (Bartley, 2007; Green, 2014).", "Therefore, companies often ‘greenwash’—making misleading assertions about product benefits or environmental performance rather than pursuing a real sustainability transition (Lyon and Montgomery, 2015).", "However, as early as 1928, the Anthroposophical movement introduced the Demeter seal that labels foodstuff produced in accordance with Rudolf Steiner’s doctrine of organic agriculture (Demeter, 2017).", "Additionally, the concept of ‘fair trade’ dates back to the 1970s (Barratt Brown, 2007).", "Both early organic and fair trade movements formed in clear opposition to the conventional economic system.", "While organic movements used voluntary certification to resist the industrial expansion of agriculture (Pekdemir, 2018), fair trade movements sought to rectify trade injustice and aimed to overcome the colonial division of labor between producers in developing and consumers in developed countries (Barratt Brown, 2007).", "Since the 1990s, private regulation has won recognition and voluntary certification has become an increasingly integrated part of global markets (Brühl, 2001; Green, 2014).", "Not only businesses, but also NGOs, have dramatically increased their engagement in regulating through certification networks, which largely bypass existing state and intergovernmental structures (Bartley, 2007; Haufler, 2003).", "Raynolds et al. (2007) differentiate three types of certification:", "Third-party multi-stakeholder efforts are generally characterized by their participatory structures, clear standards and credible (third-party) verification system.", "In the cotton sector, BCI and CmiA are such examples of certification with NGO participation.", "In contrast to BCI, CmiA works exclusively with smallholders, who are the most relevant producers in SSA cotton cultivation.", "Both schemes have active NGO involvement and the support of WWF, one of the largest environmental advocacy NGOs in the world; furthermore, PAN and Oxfam were engaged during BCI’s establishment (BCI, 2018).", "With regard to our research question, scholars would likely hypothesize that BCI and CmiA, as multi-stakeholder initiatives, are the most ambitious certification programs in SSA (Dingwerth and Pattberg, 2009; Kemper and Partzsch, 2018).", "Fourth-party certification schemes are coordinated by government or multilateral agency bodies, but remain voluntary.", "The 2014 introduction of the EU Ecolabel for textile products is an illustrative case.", "It is based on the EU Organic Regulation, which defines how agricultural, food and textile products have to be grown and processed in order to use the EU Organic (‘green leaf’) or the Ecolabel for sales in the internal market.", "The United States Department of Agriculture (USDA) Organic label, developed by the National Organic Program (NOP), is the equivalent in the United States.", "Both EU and USDA Organic belong to the International Federation of Organic Agriculture Movements (IFOAM) Family of Standards.", "This Family also includes African public standards for organic labelling; for example, the East African Organic Product Standard in Burundi, Kenya, Rwanda, Tanzania and Uganda (IFOAM, 2018).", "However, if cotton is produced in these countries, producers need to comply with the standard of the final destination in order to label their products as ‘organic’ in those respective markets.", "This means that the EU Regulation’s scope includes textiles produced in non-EU member states, such as countries in SSA (Pekdemir, 2018).", "In order to be successful, public but voluntary certification schemes need the support of broad coalitions, and such coalitions must include corporations that often prefer weaker standards (Mutersbaugh, 2005).", "As it is administered by a government agency, the USDA Organic program is particularly susceptible to political interference by industry, which has resulted in a number of lawsuits and the subsequent dilution of the standard itself (Jaffee and Howard, 2010; DuPuis and Gillon, 2009).", "In the context of this study, it would hence not be surprising if the EU Organic Regulation is found to be less ambitious than third-party multi-stakeholder schemes like BCI and CmiA.", "First- and second-party ‘business only’ certifications represent either forms of internal self-regulation or certification involving industry associations.", "They typically have little credibility, given their self-interested nature (Raynolds et al., 2007).", "However, this third ‘business only’ category lumps together a broad range of differently-motivated businesses, including FLO, which is an umbrella organization comprised of 20 national initiatives that emerged from and are deeply rooted in fair trade movements (Barratt Brown, 2007).", "FLO is inherently based on ensuring a fair wage is paid to producers in developing countries.", "Like CmiA, the program exclusively works with smallholders.", "Many observers may consider FLO to be a NGO, as the organization emerged from the fair trade movement.", "However, the organization’s members are exclusively producers and it generates its income through the market: for example, through sales in world shops.", "Moreover, FLO does not use third-party verification (FLO, 2018a).", "Raynolds et al. (2007, p. 154) note that organic and fair trade certification are actually outstanding in terms of transparency and credibility.", "Since 65% of all FLO cotton is also certified organic (FLO, 2016), there is a clear connection between the ‘organic’ (EU Organic Regulation) and ‘fair trade’ (FLO) movements.", "With regard to our research question, we would not necessarily assume that FLO is less ambitious compared to BCI and CmiA.", "Seeking involvement in certification standards is appealing to NGOs as it can act as an alternative, although controversial, route to governing global supply chains (Bartley, 2007; Overdevest, 2009).", "Overall, these private or voluntary systems “offer the strongest regulation and potential to socially embed global markets” due to their ability to enforce standards based on compliance mechanisms (Bernstein and Cashore, 2007, p. 347).", "However, Sneyd (2014) argues to the contrary: although certification schemes may have more vigorous environmental or social standards with NGO involvement, they are still left at the mercy of large corporations.", "The “very real threat” associated with market approaches such as certification is that “lowest common denominator, lowest cost approaches will dominate” (Sneyd, 2015, p. 57).", "Some NGOs therefore refuse to participate in certification programs, and have generally lobbied against them (Partzsch, 2011).", "Some have warned that certification is a “privatization of world politics” (Brühl, 2001, author's translation) that is preventing stricter government rules (Green, 2014).", "Therefore, we study which type of certification is most ambitious regarding environmental sustainability in SSA and hence provides the most appealing prospect for NGOs’ commitment.", "Five central NGOs are advocating for greater environmental sustainability in agricultural production, which can be applied to cotton cultivation in SSA: FoE, Greenpeace, Oxfam, PAN and WWF.", "A detailed content analysis was conducted of the NGOs’ webpages and publications available on their websites in which they stated positions on agricultural production and explicit preferences or demands on pesticides, fertilizers and GMOs.", "These stances were grouped together to form the ‘NGO demand framework’ (see fourth and fifth column of Table 2).", "Additionally, a total of 23 semi-structured interviews were conducted between August and November 2017, with representatives of all selected certification schemes and NGOs.", "The interviews helped to clarify the five NGOs’ stances and roles in standard setting and implementation, and bolstered the results of our document analysis.", "For the content analysis of both the NGOs’ documents and the interviews, we used a manual coding process that included searching for key terms such as “pesticides”, “fertilizers” and “GMOs” within the texts and transcripts (see below and Annex 1 in Supplementary material).", "To account for overlap and to emphasize the common demands, the final framework combines the explicit preferences of the individual NGOs into nine pesticide demands, six fertilizer demands and one demand regarding GMOs.", "Each demand is elaborated below with reference to the particular document(s) that we used to identify the demand.", "The fourth and fifth column of Table 2 give an overview of the five NGOs’ demands.", "In order to address overuse and inappropriate application of pesticides in cotton cultivation, all five NGOs outlined specific demands pertaining to the types of pesticides allowed, as well as alternative methods to regulate pests in cotton cultivation (Castillo, 2014; Cook et al., 2016; FoE, 2018b; PAN, 2017, 2018b; Tirado, 2015; Watts and Williamson, 2015; WWF, 2013).", "Greenpeace and PAN had the most detailed and extensive demands with regards to pesticides.", "FoE, Oxfam, PAN and WWF all encouraged the avoidance of synthetic pesticides (Castillo, 2014; Cook et al., 2016; FoE, 2018b; PAN, 2018b; Watts and Williamson, 2015; WWF, 2013); however, Greenpeace was more stringent in calling for no chemical pesticides to be used in agricultural practices at all (Tirado, 2015).", "Only PAN explicitly demanded a complete ban on HHPs (PAN, 2018b), and PAN was also the only NGO to demand field sanitation and hygiene measures, such as removing old crop remains to reduce the spread of pests (PAN, 2017).", "In 1985, PAN also published a ‘dirty dozen’ list that includes mainly POPs: aldicarb, toxaphene, chlordane and heptachlor, chlordimeform, chlorobenzilate, DBCP, DDT, the ‘drins’ (aldrin, dieldrin and endrin), EDB, HCH and lindane, paraquat, parathion and methyl parathion, pentachlorophenol and 2,4,5-T (PAN, 2018a).", "The NGOs’ demands on pesticides and pest control can be categorized into nine core demands.", "The first four demands apply to the types of pesticides allowed in cotton cultivation: (1) avoidance of synthetic pesticides, (2) discontinued use of highly hazardous pesticides (HHPs), (3) elimination of pollinator-toxic pesticides and (4) avoidance of broad spectrum pesticides.", "The next five demands pertain to desired alternative practices to regulate pests beyond the use of conventional pesticides: (5) shift away from monoculture farming through means such as intercropping and crop rotation, (6) emphasis on biodiversity and enhancement of beneficial pest predators through the creation/preservation of habitat corridors, borders and/or insectaries, (7) utilization of biocontrol and other biopesticides (i.e. use of approved pesticides of biological/mineral/botanical origin, trapping methods/physical barriers, pheromone traps/mating disruption), (8) utilization of resistant cultivars and genetically diverse crops and (9) field sanitation and hygiene measures, such as removal of affected plants or plant parts and use of clean, healthy seeds.", "Regarding fertilizers, the selected NGOs explicitly recommended a number of agricultural strategies and methods to mitigate their impacts (Cook et al., 2016; Tirado, 2015; Watts and Williamson, 2015; WWF, 2013).", "All explicitly encouraged the reduced use of chemical and mineral fertilizers, and advocated for the use of cover crops, green manure, composting and crop rotation methods (Castillo, 2014; Cook et al., 2016; Tirado, 2015; Watts and Williamson, 2015; WWF, 2013).", "As with pesticides, Greenpeace and PAN again have the most extensive and detailed demands for fertilizers (Greenpeace, 2018; Watts and Williamson, 2015).", "FoE, Oxfam and PAN suggest reduced tillage of the soil (Castillo, 2014; Cook et al., 2016; Watts and Williamson, 2015), and only FoE, Greenpeace and PAN promote intercropping methods of agricultural production (Cook et al., 2016; Tirado, 2015; Watts and Williamson, 2015).", "The NGO demands for fertilizer use in cotton cultivation can be categorized into six core demands: (1) the avoidance of synthetic fertilizers, (2) utilization of cover crops/green manure, (3) application of manure, crop residues and food waste, (4) reduced/conservation tillage, (5) employment of crop rotation and (6) multiple cropping/intercropping.", "Regarding GMOs, the NGO demands are less nuanced.", "Three of the five selected NGOs—FoE, Greenpeace and PAN (FoE, 2018a; Greenpeace, 2018; PAN, 2007)—demand an outright ban on GMOs, while Oxfam and WWF remain neutral on the topic (Oxfam et al., 2010; WWF, 2015).", "Although Oxfam “has no policy position for or against GM technology”, the organization “does not support GMOs as the solution to hunger, poverty and development” (2010) and recognizes issues GM crops pose for yield improvement and the wellbeing of smallholder farmers (2018).", "This is in line with AGRA’s (Alliance for a Green Revolution in Africa) position on blending technologies “without ideological hang-ups” (ACB, 2012, p. 10).", "WWF’s position on this issue is based on a self-proclaimed precautionary approach: WWF acknowledges that GM technology “can help create benefits for the environment and society”, but advocates for “a strong precautionary approach to environmental impacts of GMOs” and utilizes a risk assessment framework informed by the Cartagena Protocol on Biosafety (WWF, 2015, p. 1).", "Thus, this demand in the final framework is not shared by all NGOs.", "Methodologically, based on the NGO demand framework, we assessed the principles and criteria of the four most relevant cotton certification schemes in SSA.", "Table 2 gives an overview of our results, and Annex 1 in Supplementary material outlines them in more detail.", "Below we make references to the documents that we used to identify the certification standards’ relevant requirements.", "Two of the selected certification schemes are multi-stakeholder schemes, i.e. with NGO participation: BCI and CmiA.", "The EU Organic Regulation was chosen as an example of a fourth-party certification scheme and FLO as an example of a second-party ‘business only’ certification scheme relevant to SSA, i.e. both without NGO participation.", "The BCI certification system is focused on six production principles and 24 criteria, which are relevant for all participating farms, regardless of size.", "The six principles are: (1) minimization of the harmful impacts of crop protection practices, (2) efficient water use, (3) care for soil health, (4) conservation of natural habitats, (5) preservation of fiber quality and (6) the promotion of decent work (BCI, 2013b, p. 10).", "The 24 criteria are the basis for two distinct levels within BCI’s performance scale: minimum requirements and improvement requirements.", "Minimum requirements are mandatory for certification and include production, management and reporting criteria.", "Most relevant to pollution concerns are the minimum production criteria that “immediately reduce stress on the environment in relation to pesticide toxicity level” (BCI, 2013b, p. 10).", "According to BCI, “minimum requirements are just the first stage.", "At the same time, farmers are encouraged to develop further through improvement requirements” (BCI, 2013b, p. 2), and “there are no requirements for farmers to step-up through the different levels and they can choose to stay at pass level [meeting only the minimum requirements] if they wish” (BCI, 2013b, p. 12).", "Therefore, we only considered the minimum requirements in the analysis below.", "Similar to BCI, CmiA also divides standards for production into minimum and improvement criteria.", "There are 17 so-called exclusion criteria, ranging from farm size (average 1–3 ha) to the prohibition of discrimination at the work place, and six improvement criteria at the cotton farm level, including maintenance of soil fertility and water protection, as well as transparency with regard to the price paid to farmers (CmiA, 2015).", "CmiA states that this separation of criteria serves as a way to motivate cotton farmers to “successively adapt their farming methods and the cotton companies their processing of the raw material to increase ecological, economic, and social sustainability” (CmiA, 2018b, p. 1).", "Farmers are required to have plans for progressively meeting the improvement criteria.", "The EU Organic Regulation defines criteria for cotton in textiles that carry the EU Ecolabel.", "According to Commission Decision 2014/350/EU, organic-labelled cotton products need to be produced in accordance with this EU Regulation (EC) no. 834/2007 (European Commission, 2014).", "The EU Organic Regulation contains specific minimum rules that must be complied with, ranging from soil treatment and preservation of fertility, a restriction on plant protection products and seed material, to cleaning products (European Union, 2007).", "FLO focuses its work on smallholder farmers, who are the most relevant producers in SSA cotton production.", "The FLO system has requirements on members and small producers, trade, traceability, sourcing, contracts, use of its fair trade label, business development and production.", "Stipulations on production include environmental issues and labor conditions (FLO, 2011).", "Again, these standards are organized into minimum and improvement requirements.", "The former are principles that are mandated within the FLO certification system, whereas the latter “refer to the continuous improvements certified organizations must make on average against a scoring system”, or minimum average threshold (FLO, 2011, p. 4).", "FLO has two relevant documents that were used as the basis of this analysis.", "The first document is the Fairtrade Standard for Small Producer Organizations, which outlines requirements related to producers’ economic and social wellbeing, empowerment and environmental sustainability (FLO, 2011).", "The second is the Hazardous Materials List, which lists materials and chemicals that are prohibited (the Red List), restricted and likely to be phased out (the Orange List), or under review by FLO to be potentially added to the Orange or Red Lists (the Yellow List) (FLO, 2018b).", "Each individual criterion of the four certification schemes was compared to the NGO demands for pesticides, fertilizers and GMOs.", "A coding guide was created to represent how well each criterion corresponded with the relevant NGO demand.", "Criteria that explicitly addressed the demand and mandated compliance were determined to be strong and coded green; those that explicitly addressed the demand, yet lacked obligatory implementation, were concluded as weak and coded yellow; those that only indirectly addressed the demand were coded orange; and those that failed to address the demand were coded red (see Table 2).", "After each criterion was assessed, the corresponding demand was coded according to the color coding system.", "For example, if the second demand from the pesticide issue area was weakly addressed by one criterion, indirectly addressed by another criterion, and not addressed at all by a third criterion, the demand was color coded yellow.", "Table 2 presents a comparative overview of all results and Annex 1 in Supplementary material provides the detailed results.", "Regarding pesticides, the private certification schemes address NGO demands weakly or indirectly, while only the EU Organic Regulation strongly incorporates the demands into its standard.", "The BCI principles and criteria address all of the NGO demands either weakly (demands 1, 2, 4, 5, 6 and 7) or indirectly (demands 3, 8 and 9) (BCI, 2013a, pp. 5–13).", "The demands focusing on the restriction of pesticide types (demands 1–4) are addressed more than the demands that deal with alternative means of pest control beyond conventional pesticide application (demands 5–9) (BCI, 2013a, pp. 5–13).", "Much of the ability of BCI to address NGO demands can be credited to criteria 1.1, which addresses the types of pesticides in use, as well as demands 5–9 that pertain to alternative mechanisms of pest control (BCI, 2013a, pp. 5–6).", "BCI criterion 4.1 covers the adaptation of practices that “enhance biodiversity on and surrounding the farm” (BCI, 2013a, p. 20) and is the second most pertinent of the BCI criteria to the NGO demands.", "It weakly or indirectly addresses seven of the nine demands.", "Since BCI is a proponent of integrated pest management (IPM), but “will not endeavor to prescribe what these [specific practices] should be” (BCI, 2013b, p. 7), none of the NGO demands are strongly addressed; rather, they are weakly addressed (BCI, 2013a).", "These findings were confirmed by an interview with a certification scheme representative, who emphasized that changes in pesticide use must happen gradually, rather than immediately, to give farmers time to adjust (Interview with certifier, Nov. 2 2017).", "CmiA fulfills all of the NGO demands for pesticides either weakly (demands 1, 2 and 6) or indirectly (demands 3, 4, 5, 7, 8 and 9) (CmiA, 2018b).", "The results demonstrate that demands 1–4, pertaining to restriction of types of pesticides in use, are the most focused upon by CmiA criteria.", "Demands 1 and 2 are the most widely covered, since the efficient utilization of pesticides is an overarching theme of CmiA.", "However, since synthetic pesticides are allowed under the CmiA certification scheme, and the list of HHPs banned is not inclusive of all classes included in the FAO and WHO (2016) definition of HHPs, neither of these (demand 1 and 2, respectively) are strongly addressed by the criteria.", "Demands 3 and 4 are indirectly addressed due to the restrictions placed on pesticides by CmiA exclusion criteria 9 (banned HHPs), 11 (reporting on pesticide use), and 12 (proper labelling of pesticide use), but there is no explicit mention of broad-spectrum pesticides or pesticides toxic to pollinators in the CmiA criteria matrix (CmiA, 2018b).", "Despite only weakly or indirectly addressing NGO demands, a CmiA representative expressed in an interview that he still believes that NGO perspectives were valuable in the process of formulating the certification criteria on pesticides (Interview with certifier, Nov. 13 2017).", "The EU Organic Regulation strongly addresses six of the NGO demands (1–4, 7 and 8), weakly addresses two demands (5 and 6), and indirectly addresses a single demand (9).", "The standard’s strength in addressing NGO demands largely lies in Article 4 and the explicit restriction of inputs to only those “from organic production” and “natural or naturally derived substances”, and Article 16’s similar requirements that inputs “shall be of plant, animal, microbial, or mineral origin” (European Union, 2007).", "These two articles place de facto bans on synthetic pesticides due to their chemical origin.", "This inherently meets NGO demands 2, 3 and 4 by eliminating all synthetic pesticides, HHPs, pollinator toxic pesticides like neonicotinoids, and broad-spectrum pesticides.", "FLO addresses all of the NGO demands to some degree.", "Of those directly referenced, only demand 2 is strongly addressed according to the Red List of the Hazardous Materials List that is inclusive of all types of HHPs.", "Demands 1, 3, 4, 6 and 7 are weakly addressed and 5, 8 and 9 are indirectly addressed by the requirements (FLO, 2011).", "According to an interviewee representing FLO producers, FLO’s criteria, particularly regarding pesticides, are influenced by importing countries’ concerns regarding consumer health, as well as environmental and health problems affecting producing countries.", "This may explain the emphasis on HHPs in the FLO certification scheme (Interview with certifier, Oct. 26 2017).", "In sum, the EU Organic Regulation most ambitiously fulfills the NGOs’ demands regarding pesticides, while FLO focuses on the prohibition of HHPs.", "BCI and CmiA do not explicitly address a single NGO demand on pesticides.", "Regarding fertilizers, the certification schemes all have specific criteria that address the six core NGO demands; however, of varying strengths and degrees.", "All six of the NGO demands are weakly addressed by four of the 24 BCI criteria.", "The call for practicing crop rotation (demand 5) is addressed due to explicit mention of “legume rotations” with cotton crops in criterion 3.1 (BCI, 2013b, p. 17), a suggested tactic to avoid erosion in criterion 3.3 and a mechanism for enhancing biodiversity in criterion 4.1.", "Despite nearly all demands being addressed, they are not done so strongly as the BCI certification criteria are merely options or suggestions, rather than mandatory requirements (BCI, 2013b).", "CmiA has two farm-level sustainability criteria that have implications for the management of fertilizer use.", "The direct discussion of compost in CmiA improvement criterion 3a and the explicit recommendation of crop rotation and intercropping in criterion 3b weakly address NGO demands 3, 5 and 6.", "The explicit promotion of conservation farming in CmiA farm-level criterion 3a indirectly addresses the remaining NGO demands regarding fertilizers, including demands 1, 2, 4 and 5 (CmiA, 2015, p. 6).", "According to the FAO and ICAC (2015), conservation farming seeks to achieve sustainable agriculture through the principles of minimal soil disturbance (demand 4), permanent soil cover (demand 2) and crop rotations (demand 5), along with the optimal application of plant nutrients that do not disturb biological processes (demand 1).", "In the EU Organic Regulation, there are four distinct articles pertaining to fertilizer and soil fertility.", "Articles 4 and 12 form the basis for the Regulation’s stringency in prohibiting the use of synthetic fertilizers, while Articles 5 and 16 pertain to off-farm and unnatural inputs, respectively.", "Overall, the first five of the six NGO demands are strongly addressed by these four articles.", "The exception is the demand for intercropping/multiple cropping, which is only indirectly addressed by Article 12, which suggests practices that maintain fertility and biological activity, without explicitly mentioning intercropping (European Union, 2007).", "FLO’s requirements for fertilizer use in agriculture can be found in the Fairtrade Standard for Small Producer Organizations.", "Within the Standard, there are three distinct FLO specifications relevant to fertilizer use.", "All three are improvement requirements, meaning they are categorized as only weakly addressing the NGO demands for fertilizer use.", "Although weakly, FLO explicitly recognizes five of the six NGO demands.", "Demand 4—the practice of reduced or conservation tillage—is the only demand not addressed by the Standard at all (FLO, 2011).", "Ultimately, the EU Organic Regulation most strongly addresses the NGO demands regarding fertilizers, followed by BCI, whose standard explicitly addresses all the demands, although without mandatory requirements.", "FLO’s scheme was the only certification scheme to not address demand 4, but all other demands were explicitly addressed.", "The CmiA scheme weakly or only indirectly addressed NGO demands.", "Toward GM cotton, BCI is “technology neutral” and takes the position that the organization “will neither encourage farmers to grow it, nor seek to restrict their access to it” (BCI, 2017, p. 4).", "The scheme “allows the use of GM cotton [because] it would be difficult to achieve our objective of making Better Cotton a mainstream sustainable commodity if millions of farmers were automatically excluded from our training and support” (BCI, 2017, p. 4).", "BCI claims the use of GM cotton can be an additional way to “to prevent resistant insect and/or weed populations” (BCI, 2013b, p. 6).", "The other schemes, CmiA, the EU Organic Regulation and FLO, are markedly less favorable toward GMOs.", "Instead, they take a stance that strongly addresses the NGO demand rejecting the use of GMOs in cotton production.", "CmiA explicitly forbids the “commercial growing of GMO cotton” in its exclusion criterion 14 (CmiA, 2015, p. 5).", "According to an interviewee, CmiA’s GMO restriction was attributed more to the ubiquitous anti-GMO attitude across Europe and the logistical challenge of training thousands of uneducated farmers in the proper use of GM cotton, rather than being a direct result of NGO pressure (Interview with certifier, Nov. 13 2017).", "The EU Organic Regulation devotes all of Article 9 to the dismissal of GMOs throughout every facet of organic agriculture.", "Additionally, Article 4 excludes the use of GMOs from any certified agricultural production.", "Finally, FLO maintains its “members must not intentionally use genetically engineered seed or planting stock for Fairtrade crop(s)” (FLO, 2011, p. 22).", "In sum, while textiles labelled by BCI may include GM cotton, consumers can be sure that no GMOs were used for textiles certified according to the CmiA, EU-Ecolabel and FLO standards.", "Reviewing our results, BCI fails to strongly address a single NGO demand, despite being a multi-stakeholder scheme with NGO involvement.", "CmiA, another multi-stakeholder scheme, and FLO, a ‘business only’ scheme, do not perform much better in terms of their criteria corresponding with NGO demands.", "Only FLO scores higher on prohibition of HHPs, and both CmiA and FLO forbid the use of GMOs in agriculture.", "This is in contrast to the EU Organic Regulation, which explicitly addresses the majority of all NGO demands.", "Correspondingly, one of our interviewees, who advocated against all synthetic pesticides, found the EU Organic Regulation to be the “gold standard” (Interview with certifier, Oct. 30 2017).", "In order to defend the comparatively weak performance of multi-stakeholder schemes in addressing the NGO demands, interviewees brought forward two main arguments: first, the need for NGOs to set priorities and, second, competing interests of business members in multi-stakeholder certification.", "NGOs purposefully decided to prioritize food crops and clothing production further downstream, compared to cotton cultivation in the upstream part of the supply chain, according to one interviewee (Interview with NGO representative, Nov. 3 2017):", "[When] we were focusing more on the agricultural side of things […] we were picking our priorities and at that time, our priorities were food crops.", "Indirectly, since that was our focus, cotton became a second priority.", "On the other hand, when we were looking at industry and industrial processes having an effect on water pollution in rivers, […] the textile industry became one of the priorities, [and] so cotton [became] second priority. [", "Cotton cultivation] is very important, but it’s a much longer time campaign, than is changing the production process in the textile industry.", "Moreover, interviewees emphasized that the need to maintain economic cost competitiveness, along with the role of marketing, generally influenced schemes far more than any pressure from NGOs (Interviews with certifiers, Oct. 30 2017 and Nov. 2 2017).", "Multi-stakeholder schemes do not only include NGOs, but also business members in decision-making.", "Interviewees argued that the need to be economically cost competitive “continues to be a real problem and barrier to growth…you want farmers to invest in changes, but you’re not willing to pay for it” (Interview with certifier, Oct. 30 2017; similar in interview with private person working in the cotton industry, Sept. 2 2017).", "However, this perception contrasts with our finding that FLO, as a second-party ‘business only’ certification scheme, more ambitiously addresses NGO demands, compared to BCI and CmiA, the third-party multi-stakeholder schemes, by prohibiting HHPs and GMOs.", "This article examines whether cotton certification schemes take into account NGO demands, seeking to understand whether certification provides an appealing prospect for NGOs to ameliorate environmental issues within transnational supply chains.", "All four of the studied schemes were quite extensive and detailed in their stipulations on the use of pesticides, fertilizers and GMOs in cotton agriculture.", "As a public standard, the EU Organic Regulation has some weaker requirements, thereby adhering to some corporate interests (Mutersbaugh, 2005); however, it was nevertheless referred to as the “gold standard”, and frequently cited as the ideal certification (Interview with certifier, Oct. 30 2017; interview with NGO representatives, Sept 2 2017).", "The results of our analysis, based on the NGO demand framework, confirm this.", "The fourth-party (public) certification—the EU Organic Regulation—is the only scheme that does not have a caveat for “continuous improvement”; rather, all of the Regulation’s articles must be enacted immediately.", "Despite its far weaker adherence to NGO demands in comparison to the EU Organic Regulation, we acknowledge that environmental concerns are less relevant to the FLO mission since environmental issues are of secondary concern, unless directly connected to producer health.", "In particular, one major exception was FLO’s ban of HHPs in certified agricultural production.", "Besides contributing to environmental degradation, HHPs have direct implications for smallholder health.", "Prohibition of HHPs hence shows that the diffusion of standards that aim to enhance environmental sustainability may, but do not need to, necessarily undermine the socioeconomic situation of smallholders (Brandi, 2017).", "Nevertheless, it makes sense that 65% of FLO products are additionally certified organic in order for consumers to ensure they are not unwillingly supporting exploitation in the form of environmental degradation of soil and water in producing countries.", "Our analysis demonstrates that CmiA and BCI performed similarly to FLO in addressing NGO demands regarding pesticides and fertilizer.", "While all three certification schemes addressed almost all of the NGO demands, they fail to do so explicitly in almost all cases.", "BCI and CmiA did not explicitly address any demand, aside from CmiA’s opposition to GM cotton.", "Moreover, BCI has an outlier position on the use of GM cotton in production.", "This could be simply a matter of pragmatism, to operate globally, and not just in SSA (Interview with certifier, Nov. 13 2017).", "However, BCI’s neutral stance on GM cotton is at odds with the fact that it is the scheme with a great level of international NGO involvement.", "In particular, PAN, which clearly rejects the use of GMOs, was unable to make BCI prohibit the use of GMOs when the NGO was engaged during its establishment (PAN, 2018a; BCI, 2018).", "Thus, we can conclude that private certification systems, including multi-stakeholder initiatives, are inadequate in properly addressing cotton production pollution concerns raised by international NGOs.", "Cotton certification hence contradicts earlier findings, on palm oil certification, for example, which found multi-stakeholder certification to be the most ambitious (Dingwerth and Pattberg, 2009; Kemper and Partzsch, 2018; Raynolds et al., 2007).", "Instead, following our findings, FLO, which emerged from the fair trade movement, scored comparatively better—although it is a second-party certification involving producer associations, but no NGOs (Raynolds et al., 2007).", "The multi-stakeholder certification programs are too intertwined with the interests of the actors involved in their creation and uptake, regardless of NGO presence.", "Moreover, NGOs themselves are driven by the interests of the public and potential donors (Abbott, 2012).", "In addition to the lack of substantial detail, the discretionary nature of criteria pertaining to alternative pest control and fertilizers in the certification schemes correlates with a general lack of publicized attention by NGOs on these topics.", "If an issue, such as fertilizer use or alternative methods of pest protection in this case, lack a high profile, maintaining pressure can be challenging.", "Although NGOs and the certification schemes address these two topics, their efforts are dominated by GMOs or the restriction of synthetic pesticides.", "This aligns with Bartley's (2007) and Haufler's (2003) assessment that sustained campaign pressure is necessary in order to achieve sustainability goals.", "So, it is unsurprising that minimal NGO focus on alternative pest control methods and fertilizers, besides brief mentions in publications, is mirrored by weak certification scheme criteria.", "In general, interviewees acknowledge that environmental sustainability of cotton cultivation was not an NGO priority (Interviews with NGO representatives, Nov. 3 and Sept. 2, 2017).", "Furthermore, NGOs have to compromise with business demands in multi-stakeholder certification schemes, such as BCI and CmiA.", "This has already been shown in studies addressing social issues in poverty reduction efforts (Brandi, 2017; Sneyd, 2014).", "Such compromise inherently weakens NGOs’ ability to promote their demands through certification systems.", "This revelation of the seeming importance of financial cost over environmental concern supports Sneyd’s (2014) argumentation that, although certification schemes may have more vigorous environmental standards with NGO involvement, they are still left at the mercy of large corporations.", "If NGOs cooperate with business for the sake of greater diffusion of environmental standards, and perhaps also for their own economic interests (Abbott, 2012; Bosso, 2005), they clearly compromise on the stringency of the environmental sustainability standards in the case of cotton production, and most likely in other fields as well.", "While certification schemes are meant to fill some of the gaps in social and environmental regulation (Bernstein and Cashore, 2007), this form of private governance is largely failing to do so in the case of sustainability concerns in cotton production.", "Instead, it is possible to argue that some schemes are even “diluting” (Interview with NGO representative, Nov. 3 2017) the public standard of the EU Organic Regulation.", "Bernstein and Cashore (2007) are optimistic about the ability of private governance and voluntary certification systems to regulate markets in the reality of diminished state regulatory capacity.", "However, according to our empirical findings, this form of governance only appeared as a promising approach in the case of fourth-party certification.", "A detailed content analysis of the dominant private schemes in SSA—BCI, CmiA and FLO—revealed a lack of tenacity in addressing pollution from cotton cultivation inputs.", "In conclusion, we suggest that NGOs’ campaigns should promote public standards and advocate for tightening them up, particularly in the case of EU and USDA Organic, rather than investing their limited resources in participation in multi-stakeholder initiatives.", "Moreover, with regard to SSA, NGOs might promote new bi- and multilateral state agreements; for example, regarding the recognition of public organic standards of producing countries for the EU and US market.", "Otherwise, if NGOs maintain their commitment to multi-stakeholder initiatives, they risk contributing to ‘greenwashing’ corporate conduct by mobilizing consumer support for labelling programs that actually fall behind existing public regulation on organic certification.", "Private regulation is only a second-best solution when considering environmental pollution concerns in cotton cultivation supply chains from the perspective of NGOs and their environmental sustainability demands." ]
[ "Cotton production in SSA causes a number of environmental sustainability challenges.", "Voluntary certification programs aim to influence corporate conduct along supply chains.", "The EU Organic Standard is most ambitious in addressing NGOs’ environmental demands.", "NGOs risk contributing to ‘greenwashing’ when participating in less ambitious programs." ]
Wealth inequality, family background, and estate taxation
This paper generates two main contributions. First, it provides a new theory of wealth inequality that merges two empirically relevant forces generating inequality: bequest motives and inheritance of ability across generations; and an earnings process that allows for more earnings risk for the richest. Second, it uses the resulting calibrated framework to study the effects of changing estate taxation. Increasing the estate tax reduces the wealth concentration in the hands of the richest few and the economic advantage of being born to a rich and super-rich family at the cost of reduced aggregate capital and output. However, all of these effects are quite small. In contrast, increasing estate taxation can generate a significant welfare gain to a newborn under the veil of ignorance, but this comes at a large welfare cost for the super-rich.
[ "ECON" ]
[ "Bequests", "Earnings shocks", "Estate taxation", "Parental background", "Wealth inequality" ]
[ "2002", "2003" ]
[ "Since its introduction in 1916, the estate tax has been one of the most controversial taxes in the United States tax code.", "Estate tax opponents call it the “death tax.”", "Among legislators supporting the abolition of the estate tax, former representative Ron Paul (14th district of Texas) stated: “The estate tax is immoral and counter-productive.", "…My office has received hundreds of letters and emails from individuals.", "… These people are not rich, but they have worked hard and saved to create an inheritance for their children…”1", "Estate tax supporters see the estate tax as an extremely progressive tax and a very effective way to tax the richest (and dead) few.", "Former representative Bob Etheridge (2nd district of North Carolina) stated “the so-called Death Tax Elimination Act should be called the Multi-millionaire Protection Act.”2", "There is also much debate about wealth inequality and the importance of parental background in determining one׳s lot in life.", "Many papers measure and document the importance of parental background and initial conditions at the individual level.", "At the aggregate level, the large amount of wealth that is transmitted across generations, including physical wealth and human capital, has been extensively measured and debated.", "There is also a lot of discussion about the role of taxation and estate taxation in particular.", "This paper provides two main contributions.", "First, it provides a new theory of wealth inequality that merges two sources of inequality previously proposed: bequest motives and inheritance of ability across generations; and an earnings process that allows for more earnings risk for the richest.", "Second, it uses our calibrated framework to study the importance of parental background in our benchmark economy and the effects of changing estate taxation on aggregate capital accumulation, inequality, parental background as a source of inequality, and welfare.", "We calibrate our framework to the 1990s, a period during which the estate tax was relatively stable, and we match key moments of earnings mobility and earnings inequality, aggregate savings, the fraction of wealth transmitted across generations, one moment of the observed bequest distribution, the fraction of estates that pay the estate tax, and the total estate tax revenue as a fraction of output.", "Our implied effective exemption level is $756,000 (compared with the statutory level of $675,000) and our implied effective marginal tax rate is 21% (compared with the highest statutory rate of 55%).", "These numbers are consistent with other estimates of the effective estate tax rate, including across the estate size distribution (see, for instance, Gale and Slemrod, 2001).", "Our calibrated model also generates realistically skewed distributions for wealth, earnings, and bequests, and is thus a good laboratory to use to study the effects of estate taxation; and it yields several interesting findings.", "First, our benchmark model allows for four types of parental backgrounds – low-earnings, middle-earnings, high-earnings (rich), and super-high earnings (super-rich) – and implies that one׳s parental background is an important determinant of one׳s expected lifetime utility, especially for the rich and super-rich.", "Second, we consider revenue-neutral reforms and study the effects of balancing the government budget constraint using either a labor income or capital income tax adjustment.", "We find that both taxes have very similar effects in terms of inequality, importance of parental background, and aggregate capital and inequality, but that adjusting the labor income tax when the estate tax is raised yields larger welfare gains for most of the population.", "Third, changing estate taxation from their effective levels to levels of the order of the statutory ones that were common around the year 2000 (an exemption level of $675,000 and a marginal tax rate of 55%) would lower aggregate capital and output, but would also reduce wealth inequality (and especially the concentration of wealth in the hands of the wealthiest 1%) and the advantage to being born to a rich and super-rich family.", "However, these effects are quite small.", "In terms of welfare, this reform generates a significant welfare gain from the standpoint of a newborn under the veil of ignorance, which comes at a huge welfare cost for the super-rich.", "Fourth, our results about the effects of higher estate taxation on the aggregates, on inequality, and on the importance of parental background are surprisingly consistent regardless of whether bequests net or gross of estate taxes enter the utility function (more of a “wealth in the utility function formulation”), or even in the case of bequests due to completely altruistic parents, once these models are calibrated to match the same facts as closely as possible.", "In addition, even the ex-ante welfare measures are surprisingly similar.", "To be consistent with the observed distribution of bequests, our calibrated bequest motives are of the luxury goods kind; that is, people desire to leave bequests only when they are rich enough.", "Hence, households that get rich because they received positive earnings shocks and/or large bequests want to share their luck with their descendants.", "The bequest motive thus raises the saving rate of the already rich and endogenously generates a positive correlation of saving rates across generations.", "In addition, more successful parents tend to have higher-earning offspring, which makes for an even more concentrated distribution of wealth and a higher correlation of savings across generations.", "The paper is organized as follows.", "Section 2 frames our contribution in the context of the previous literature.", "Section 3 presents the model.", "Section 4 discusses the model׳s calibration choices.", "Section 5 highlights the calibrated model׳s implications.", "Section 6 investigates the effects of various estate tax reforms.", "Section 7 compares our results with those in the previous literature.", "Section 8 concludes and discusses directions for future research.", "Our analysis builds on the model developed by De Nardi (2004) (and further refined by Yang, 2013; and De Nardi and Yang, 2014) by introducing an earnings process calibration based on the one proposed by Castañeda et al. (2003), which helps in matching the observed wealth concentration (see Cagetti and De Nardi, 2008 for a survey on wealth inequality), and by considering two different bequest motives.", "We use this improved framework to study the effects of parental background and estate taxation.", "An extensive literature, both empirical and theoretical, shows that the transmission of physical and human capital from parents to children is a very important determinant of household wealth in the aggregate economy (see Kotlikoff and Summers, 1981; Modigliani, 1988, and Gale and Scholz, 1994) and of wealth and earnings ability over the household׳s life cycle (see Hurd and Smith, 1999; Becker and Tomes, 1986).", "As a result, they are also prime forces to include to study the effects of parental background on inequality and the effects of estate taxation.", "Another set of papers has pointed out the importance of initial conditions at labor market entry in determining lifetime inequality (and one׳s success in the labor market and expected lifetime utility); see Keane and Wolpin (1997) for an earlier contribution and Huggett et al. (2011) for a more recent one.", "We also study this dimension, as well as the effects of parental background on lifetime utility and inequality.", "The literature studying the effects of estate taxation in quantitatively calibrated models that match observed wealth inequality includes Cagetti and De Nardi (2009) and Castañeda et al. (2003).", "While Cagetti and De Nardi (and their previous paper Cagetti and De Nardi, 2006) do so in a model with entrepreneurial choice and Castañeda et al. do not, both use a simplified life cycle with stochastic aging and assume completely altruistic parents.", "In contrast, we model the life cycle structure and two types of intergenerational links carefully, in a framework that also matches the observed distribution of bequests and generates a realistic increase of wealth inequality over the life cycle.", "We compare our results with those reported in these previous papers in Section 7.", "Our analysis is also connected to the qualitative literature on the effects of estate taxation in the presence of different bequest motives (see, for example, Gale and Perozek, 2001; Cremer and Pestieau, 2003; Pestieau and Sato, 2006; Hines, 2013).", "Our contribution is quantitative in nature, and we address the issue of the sensitivity of the results to the assumed bequest motives in two ways.", "First, we consider two different formulations of bequest motives: one formulation (our main one) in which parents care about bequests net of estate taxes, which is closer to an altruistic formulation; and another formulation in which parents care about the bequest left gross of taxes, a less “altruistic” formulation, which is closer to the “wealth in the utility function” formulation advocated by Carroll (1998).", "Second, we compare our findings with those of Cagetti and De Nardi (2009) and Castañeda et al. (2003) papers that assume perfectly altruistic dynasties.", "There is also a literature testing the empirical implications of parental altruism or trying to infer bequest motives using rich micro-level data sets.", "This branch of the literature has some bearing on the choices we might want to make when modeling bequest motives.", "For instance, the completely altruistic model, in which children׳s utility enters parent׳s utility, has implications for intergenerational risk sharing that have been rejected by Altonji et al. (1997), among others.", "An interesting paper by Laitner and Juster (1996) finds heterogeneity in bequest motives for the relatively affluent retirees in his sample.", "A contribution by Kopczuk and Lupton (2007) estimates a bequest motive that might or might not be present, depending on some households׳ characteristics, both observable and unobservable.", "Our view is that, while the jury is still out on how to best model bequests, a minimum requirement that a reasonable bequest motive should satisfy is that it should generate a realistic distribution of bequests, including the observation that many households die leaving bequests of negligible value.", "In addition, given that the intergenerational risk-sharing implications of complete altruism have been rejected, the bequest motive should not be of the perfectly altruistic type.", "Given these considerations and the empirical success of our paper (and its variations in bequest motives) in matching wealth and bequest inequality, we see our exercise as a valid contribution to evaluating the effects of parental background and estate taxation.", "Finally, our paper proposes a positive analysis of estate taxation, as opposed to a normative one (for a relatively recent contribution of this kind, see Farhi and Werning, 2010).", "The model is a discrete-time, incomplete-markets, overlapping-generations economy with an infinitely lived government.", "People derive utility from holding onto assets because they turn into bequests upon death.", "This form of ‘impure’ bequest motive implies that an individual cares about total bequests left to his/her children, but not about the consumption of his/her children.", "We focus on a stationary equilibrium concept in which factor prices and age-wealth distribution are constant over time.", "Due to space constraints, the definition of a stationary equilibrium for our economy is in the online appendix.", "Unless stated otherwise, we report parameters at an annual frequency.", "The calibration table in the online appendix summarizes the parameters that are either taken from other studies or can be solved independently of the endogenous outcomes of the model.", "Regarding the latter, due to the assumption of exogenous labor supply and retirement decisions, the tax rate on Social Security only depends on the earnings shocks and the population demographics, which are exogenous to the model.", "We set the population growth rate, n, to be 1.2%, the average value of population growth from 1950 to 1997 from the Council of Economic Advisors (1998).", "The pt׳s are the vectors of conditional survival probabilities for people older than 65 and are set to the survival probabilities for people born in 1965 (Bell et al., 1992).", "We take the risk aversion coefficient, γ, to be 1.5.", "First, it assumes four possible earnings states: low, middle, high, and super-high.", "The support of the earnings shocks comes from Castañeda et al. (2003) and the resulting grid points for ψ are [1, 3.15, 9.78, 1,061].", "Second, it takes the persistence ρh of the earnings inheritance process from Zimmerman (1992) and Solon (1992) and the variance σh2 from De Nardi (2004).", "The earnings inheritance process is then discretized as proposed by Tauchen (1986).", "Third, it takes PSID estimates on the persistence (0.92) and variance (0.38) over five-year periods from Table A1 in Appendix A in De Nardi (2004); and discretizes this process for the lowest three grid points using Tauchen (1986) to make sure that our process accurately represents the estimated earnings dynamics for much of the population.", "This gives a three by three transition matrix.", "Fourth, the remaining six elements of the four by four transition matrix are picked to match the following aspects of the earnings distribution: the Gini coefficient and the share of total earnings earned, respectively, by the top 1%, 5%, 20%, 40%, 60%, and an earnings persistence at the top of 80%.", "The latter is consistent with work by De Backer et al. (2012), which reports that the persistence of both labor and business income at the top of labor and business income distributions is high and that, in particular, the probability of staying there, both after one year and five years (the latter results are available from the authors on request), is around 80%.", "We also impose adding-up restrictions.", "The online appendix (on Science Direct) shows that our calibration generates a cross-sectional earnings distribution that is very close to that computed from the Survey of Consumer Finances (SCF) data.", "It also reports the transition matrices for the earnings process over time and across generations and the invariant distribution over earnings states upon entering the economy.", "The share of income that goes to capital, α, is set at 0.36 (Cooley and Prescott, 1995), and depreciation is 6% (Stokey and Rebelo, 1995).", "The discount factor affects savings and average wealth in the economy.", "The term ϕ1 measures the strength of bequest motives, thus we choose the aggregate bequest as a moment.", "The term ϕ2 reflects the extent to which bequests are luxury goods, thus affecting the upper tail of the bequest distribution.", "Our calibration for the model with net bequests in the utility function implies that, during the last period of life, when the individual knows that he/she will die for sure next period, the marginal propensity to bequeath out of an additional dollar above the estate tax threshold is 56%, while the threshold above which the person wants to start bequeathing is $1.095 million (normalized using $57,135 as average income in 2000).", "The corresponding numbers for the gross bequests model are, respectively, 53% and $1.376 million.", "We discuss the interpretation of the bequest parameters in the online appendix.", "Although many experts agree that effective estate taxation can be reduced substantially by appropriate estate management and valuation, there is considerable uncertainty about how much people can and do reduce the estate tax burden through both legal and illegal means.", "There is, in contrast, no dispute about the observed revenues from the estate and gift tax and the fraction of estates that do pay estate taxes.", "We choose the tax parameters τb and xb to match the fraction of estate tax revenue to output (0.33%, Gale et al., 2001 and Gale and Slemrod, 2001) and the fraction of estates that pay estate taxes (2.0%, Gale et al., 2001 and Gale and Slemrod, 2001).", "The implied exemption level expressed in terms of year 2000 dollars turns out to be $756,000, which is only modestly higher than the $675,000 exemption that was in place at that time.", "Our calibrated numbers fall well within the bounds proposed by the previous literature.", "Given that our model matches asset holdings so well, and given the considerable uncertainty about effective estate tax avoidance and evasion, we see this as a useful way to proceed.", "We choose τl to balance the government budget constraint, given a ratio of government spending to output of 18% (Council of Economic Advisors, 1998).", "In the model without bequest motives, we choose the parameters β, τb, xb, and τl to match the capital output ratio, the fraction of estate tax revenue to output, the fraction of estates that pay estate taxes, and the ratio of government spending to output.", "An inspection of Table 1 reveals that, unlike our calibrated model with voluntary bequest motives, the model without bequest motives cannot match the flow of aggregate bequests to aggregate wealth.", "In fact, it only captures 66% of it, and thus overstates the estate tax rate (63%), setting it even higher than the statutory tax rate (55%), which applies only to the largest bequests.", "The higher tax rate mechanically comes from the fact that the flow of bequests is too small and yet the estate tax revenue has to match the observed revenue in the data.", "We present our numerical results as follows.", "In Section 5, we discuss three versions of the model and their implications, how they compare with the actual data, and how they differ across models.", "We also discuss the importance of parental background in affecting lifetime expected utility, and we evaluate the intergenerational persistence of earnings and wealth in our benchmark economy.", "The online appendix also discusses the distribution of the tax burden and how it changes with estate taxation reform.", "In Section 6, we study the long-run effects of various estate taxation reforms, in which we use either the tax on capital or the tax on labor income to re-establish budget balance.", "In each run, unless otherwise indicated, we solve for the dynamic programming problem, impose budget balance for the government, and adjust prices to re-establish market clearing.", "This section highlights the implications of the calibrated model.", "Table 2 reports values of the wealth distribution.", "The first line refers to data from the 1998 SCF taken from Budría et al. (2002) and shows that, in the data, wealth is highly unevenly distributed.", "The wealthiest 1% of people hold 35% of net worth, while the wealthiest 5% hold 58% of total net worth.", "The second line of data reports the corresponding numbers for the benchmark model with intergenerational links and bequest motives and bequests net of taxes entering the utility function.", "The third line of data reports the corresponding numbers for the model with intergenerational links and bequest motives and bequests gross of taxes entering the utility function.4", "Both versions of the model with voluntary bequests, whether the utility from bequests is net or gross of taxes, when appropriately calibrated to match our target moments, succeed in generating the observed wealth concentration.", "The fourth line of data reports values for the wealth distribution generated by a model without voluntary bequests that is calibrated as discussed previously.", "This version of the model succeeds in generating wealth holdings in the hands of the richest 1% that are larger than the share of earnings of the richest 1%.", "The key mechanism generating this is that the earnings super-rich have a 20% probability of sliding into a much lower earnings state each period and thus save at very high rates to smooth consumption over time.", "Despite this additional saving motive for the high earners, however, the model falls short of matching the observed fractions of wealth held by the richest.", "The comparison between the model with voluntary bequest motives and the one without voluntary bequest motives highlights the role of the voluntary bequest motive, calibrated as a luxury good, in generating a higher concentration of wealth in the hands of the richest few and raising overall wealth inequality as measured by the Gini coefficient.", "The intuition is that this kind of bequest motive raises the saving rate of the rich, who thus leave larger estates to their children; they in turn also save more, thus increasing wealth concentration.", "Fig. 1 displays the evolution of a summary measure of wealth concentration, the Gini coefficient, by age.", "The figure reports two different series for the data.", "The first one, from Huggett (1996), displays a U-Shaped form by age (dash-dot line); while the second one, from Kuhn (2014), is flatter (solid line).", "Both lines imply a high concentration of wealth at all ages.", "The Gini coefficient of wealth by age produced by our benchmark model with net bequests in the utility function (line with circles) coincides with the one in the model with gross bequests in the utility function (line with triangles), as the two models, appropriately re-calibrated, fit the data very similarly.", "All of our models produce Gini coefficients by age in the ballpark of the data, but the models with voluntary bequests better match the observed Gini coefficient for wealth at all ages compared with the implications of the model without voluntary bequests (line with squares).", "This indicates that the model with voluntary bequests not only better matches the cross-sectional wealth inequality at all ages, but also better reproduces some of its evolution over the life cycle.", "The evolution of the Gini coefficient, and more generally wealth inequality, as people age, is a promising avenue to help identify bequest motives.", "Parental background affects one׳s prospects in life through two channels.", "First, since richer parents leave larger bequests, it influences the amount of expected bequests that one will receive.", "Second, since one׳s initial earnings draw is correlated to one׳s parental earnings and is then persistent over time, it also influences one׳s lifetime earnings.", "In this subsection, we discuss the value of being born to a family with different parental backgrounds (or earnings).", "Later, when evaluating various policy reforms, we assess to what extent estate, capital income, and labor income taxes can affect the luck (or lack thereof) of being born with a certain parental background rather than another one in our framework.", "In our calibration, the earnings of both parents and children can assume four values: low-earnings, middle-earnings, high-earnings, and super-high earnings.", "We perform our calculations of the value of being born in a certain parental socio-economic class as follows.", "Take a new worker with parental background i. Find the median of the other parental state variables (assets and associated lifetime earnings) with current parental earnings or background at the time when the child enters the labor market.", "Also find the median of the new workers׳ state variables (initial earnings) conditional on their parental earnings or background.", "Take the corresponding value function for all of these state variables.", "Repeat this process for a new worker with parental background j. Compare the two value functions, and compute the one-time asset compensation requested to make the newborn worker born to a given family background indifferent to being born to a family with another level of parental earnings or background and divide by average income to normalize.", "One way of interpreting this comparison is that it calculates the value of being born to a typical background, conditional on parental socio-economic status, and all of the median associated state variables that go along with it, with a different parental socio-economic status and all associated other median state variables.", "In Table 3, we first switch both initial human capital and expected bequests among the offspring of families from different parental backgrounds, thus evaluating the importance of bequests and initial productivity, or human capital, together (top panel).", "In the bottom panel, we only switch expected bequests, thus evaluating only the importance of wealth transmission across generations, for a given level of initial productivity or human capital.", "The top panel of Table 3 shows that newborn workers whose parents are at the highest earnings at age 55 need to be compensated, respectively, by 35.7, 35.5, and 28.4 times average income to be moved to the state of being born to a family with the low (1st), middle (2nd), or high level (3rd) of parental earnings.", "A newborn worker with parents in the 3rd (high) earnings background has to be compensated by over five times average income to be born to a family with middle- or low-earning parents.", "Finally, low (1st) and middle (2nd) earning families are quite similar in terms of the lifetime utility that they provide to their children, compared with the high or super-high earners.", "The bottom panel shows that, for those born to a super-rich family, 40% of the compensation is related to expected bequests, while 60% is related to a higher initial level of productivity, or human capital inheritance.", "Comparing the second line of both panels shows that the importance of bequests shrinks very quickly as we move from a super-rich family background to a merely rich family background.", "These calculations thus suggest that the value of being born into a family with a high or super-high socio-economic background is very large, and that parental background (and especially expected bequests) is an important determinant of the lifetime utility of the richer households in our calibrated model.", "In contrast, the effects of parental background are much smaller for the households born to the low- and middle-income parental level, with the importance of bequests becoming much smaller as the level of parental background decreases.", "Since in our economy the fraction of people born with a high- and super-high parental earnings background is very small (about 2%), for the majority of people in our economy the effect of parental background on lifetime expected utility is small.", "We study two key margins of estate taxation: the threshold above which estates start being taxed, and the marginal tax rate above which estates are taxed above the exemption threshold.", "Modifying the estate taxation exemption levels affects both the size of the estates that are hit by estate taxes and the burden of estate taxation.", "For example, reducing the exemption level implies that smaller estates start being taxed, but also that the previously taxed estates pay more taxes, because their exemption level is smaller.", "In contrast, changing the estate tax rate for a given exemption level just increases or decreases the burden of estate taxes on estates of the same size.", "Changing estate taxation also has an effect on the estate distribution.", "This can happen for two reasons.", "First, if the people leaving estates care about the estate net of bequest taxes (a more altruistic form of bequest motive), they will change their saving behavior and desired bequest when estate taxation changes.", "This effect will be missing in the case of gross bequests in the utility function.", "Second, people might receive different amounts of bequests net of taxes, which will affect their saving behavior and desired bequests in turn, because the model with realistically calibrated bequest motives generates a non-homotheticity of savings in income and wealth.", "We now turn to presenting the effects of various estate taxation policy reforms on the aggregates, on inequality, on the importance of parental background, and on welfare, in the cases of adjusting either the capital income (Table 4) or the labor income tax (some of the results for this case are in the online appendix) to re-establish government budget balance.", "In some instances, to better discuss the various effects, we also report results for fixed prices and thus partial equilibrium.", "Table 4 reports the results for the estate tax reforms in which the capital income tax is used to re-establish budget balance.", "Due to space constraints, we report the aggregate and distributional results for the estate tax reforms in the case in which the labor income tax is used to re-establish government budget balance in the online appendix.", "Changing the tax on capital income changes the incentives to save by affecting the net rate of return on capital.", "Changing labor income taxation does not distort labor supply decisions because, for tractability, we assume exogenous labor supply; it does affect net lifetime income, and hence the importance of human capital inheritance across generations.", "In fact, raising the tax on labor earnings reduces the advantage of being born to more able parents and having a higher expected lifetime income.", "The top panel of the table shows the aggregate effects of changing the estate tax rate, while the second panel reports the results for changes in the estate tax exemption level.", "The third panel changes both the estate tax rate and its exemption level at the same time.", "The line in bold refers to our benchmark economy.", "The bottom panel reproduces some of the analysis for the case of utility from bequests gross of taxes.", "Lowering the estate tax rate (τb) below our calibrated level of 21% increases the return to leaving a bequest for people who are rich enough to have an active bequest motive but requires an increase in another tax instrument to re-establish budget balance.", "Increasing the tax rate on capital income decreases the incentive to save for everyone and especially for those who are not actively saving to leave a bequest.", "The net effect for the richest in our framework is that the increased return from leaving a bequest is larger than the disincentive coming from the lower interest rate.", "In addition, in the aggregate, the increased savings of the richest are large enough to counterbalance the decreased savings of everyone else and, on net, aggregate capital and income go up as the estate tax is lowered.", "When the labor income tax is used to balance the government budget constraints, for given prices, reducing estate taxation does not reduce the rate of return to savings for anyone in the population and still increases the return to leaving a bequest for the rich.", "As a result, aggregate capital goes up a bit more (which tends to reduce the interest rate by more in general equilibrium) and so does aggregate output.", "This is not very surprising because not only does taxing labor not discourage savings as taxing capital income does, but in our economy labor supply is fixed, and therefore there is no disincentive of labor supply coming from increasing the labor tax.", "Increasing the tax rate on estates: Up to a tax rate on estates of about 50%, raising the estate tax rate and lowering the capital income tax reduce both aggregate capital and output due to the fact that the return to leaving a bequest goes down when the estate tax goes up, and the saving rate of the rich goes down by more than the increased savings of everyone else (now facing higher returns due to a lower tax rate on capital and higher equilibrium interest rates and thus saving more).", "However, around a tax rate on estates of about 60%, bequests net of the estate tax become smaller and smaller and the richest keep up their saving to avoid a large reduction in net bequests.", "The rest of the population faces a lower capital income tax and desire to save more, and aggregate capital and income go up.", "In the model with gross bequest motives, the rich do not adjust savings up to avoid a large reduction in net estates, as the tax rate on estates keeps going up and this nonlinearity is absent.", "This nonlinearity is also absent when we increase the estate tax while lowering the tax rate on labor.", "In this case, the rich keep getting less rich due to smaller net bequests as we increase the estate tax, but the effect of increasing the returns to savings due to lower capital income taxes is no longer present across the whole population.", "However, most of the population experiences a positive wealth effect due to lower labor income taxes, and thus saves a little more as a result.", "Lowering the exemption level has two effects.", "First, it introduces estate taxes for smaller estates that were not taxed previously; and second, it taxes more heavily the estates that were already taxed previously.", "When the exemption level is lowered, aggregate capital and income decrease.", "When it is increased, the effects go in the opposite direction but are very small.", "This holds regardless of whether the capital income or the labor income tax is adjusted.", "In the model in which people derive utility from bequests gross of taxes rather than net of taxes, when the estate tax rate goes down, people do not decrease their desired gross bequests and capital and, as a result, output goes up by a little more than the case of net bequests in the utility function (more results than those reported are available on request).", "In contrast, when the estate tax rate goes up, people do not save more to leave larger bequests net of taxes to their children.", "As a result, aggregate capital and income drop slightly more than in the model with utility from bequests net of taxes.", "Interestingly however, both the calibrated models with net and gross bequests in the utility function imply a drop in aggregate capital and income when estate taxation is raised; and the differences in the effects generated by these two models are quite small, once the two models are calibrated to match the same facts in their respective benchmark calibrations.", "We also computed the elasticity of the estate tax base to changes in the estate tax in both the net bequest model and gross bequest model, adjusting either the capital income or the labor income tax, in either a general equilibrium or a partial equilibrium setting.", "For changes of the estate tax rate between 10% and 60%, the elasticity of the tax base ranges between −0.158 and 0.082 and it is thus very small.", "Overall, the results of changing the estate tax are thus remarkably similar whether or not the tax on capital or labor income is adjusted and whether households derive utility from leaving bequests gross or net of estate taxes.", "Hence, putting together the aggregate and distributional effects of these reforms, we find that reducing estate taxation increases aggregate output and capital but also increases wealth inequality, while increasing the estate tax rate has the opposite effect; and the results are remarkably similar when the capital or labor income tax is adjusted and for gross and net bequests in the utility function.", "These tables yield several findings.", "First, the value of being born to a family with the highest parental earnings background is significantly reduced when estate taxes are increased.", "For instance, the compensation requested for moving from the top to the bottom of parental earnings is 35.7 times average income in the benchmark economy, while it goes down to 33.7 times when estate taxation is increased.", "Second, these compensations are very similar regardless of which tax is used to balance the government budget (the results for the labor income tax are in the online appendix), and this indicates that it is the reduction in the net bequests received that dominates the effects of this reform, rather than the tax used to balance the government budget constraint.", "Third, the effects of the importance of parental background and its changes are very similar both for net and gross bequests in the utility function, with only a slightly bigger reduction for the case of gross bequests in the utility function.", "Fourth, the importance of parental background to determine one׳s lot in life is basically unchanged for all other people who are not born in a super-rich family, first reflecting the very high progressivity of estate taxation both before and after the reform; second, reflecting the fact that this tax raises little revenue that can be used to rebate other taxes; and third, reflecting the fact that only a small fraction of the population receive a very large bequest.", "Our incomplete market framework generates, absent any policy or exogenous changes, a stationary distribution of wealth.", "As done by Conesa et al. (2009), we employ an ex-ante welfare criterion (before ability is realized) that measures expected (with respect to idiosyncratic shocks and parental background) lifetime utility of a newborn worker in a stationary equilibrium.", "To better understand the welfare costs and benefits of this reform, we also report the ex-ante expected lifetime utility of a newborn worker, conditional on their initial productivity.", "Table 8 reports one-time welfare compensations and the fraction of people gaining from a reform.", "The welfare compensation computes the amount of assets that we need to give agents in the economy before a reform, as a fraction of average income, so that each agent is indifferent between living in the economy before and after a reform.", "For simplicity, we then switch the signs so that a positive number means a welfare gain of switching from the benchmark economy to the economy with higher estate taxation.", "To isolate the general equilibrium effects, we present the partial equilibrium results in which we fix the prices at their level in the initial steady state (top panel).", "The column “All” refers to the ex-ante welfare measure computed under the veil of ignorance.", "The columns labeled “Initial Earnings” condition on the newborn workers׳ initial earnings draw, while the last three columns report, respectively, the fraction of households benefiting from the reform, the average gains of those who gain, and the losses of those who lose.", "A few things are worth noticing.", "For fixed prices, first the vast majority of people gain from switching to the year 2000 statutory estate taxation.", "The fraction is highest when the labor income tax is lowered to balance the government budget, because many people save little and thus do not benefit from a tax break on capital income.", "Second, the fraction of people gaining from increasing estate taxation is very similar regardless of whether net or gross bequests enter one׳s utility function.", "Third, the first three columns of the table report the average gain or loss conditional on one׳s initial earnings upon entering the labor market.", "Conditioning on this reveals that the average gains conditional of being born in a given productivity level are positive, with the exception of the largest earnings realization.", "Conditional on being in that state, the utility loss from increased estate taxation as a fraction of average income is large, especially in the case in which net bequests enter the utility functions, because households in that case lose utility both because they get lower net estates (which happens with both utility functions) and because they receive smaller utility from gross bequests due to increased estate taxation (an effect present only with net bequests in the utility function).", "Interestingly, however, the results are surprisingly close with gross and net bequests in the utility function.", "For endogenous prices, things change.", "First, the interest rate goes up, but the wage rate goes down, regardless of whether capital or labor income tax is lowered.", "A higher interest rate increases the rate of return to savings and thus tends to improve the welfare of the savers.", "A reduction in the wage rate, in contrast, decreases the earnings of all workers, thus generating a welfare loss.", "The negative wage effect dominates, thus resulting in a much smaller fraction of people benefiting from an increase in estate taxes than for fixed prices.", "Second, in the case the labor income tax is lowered due to the increased revenue from estate taxation, the welfare loss from lower wages is partly offset in wages net of taxes, thus generating a larger fraction of people gaining from the reform.", "Third, the welfare gains can be nonlinear as a function of one׳s initial earnings because wages go down but the interest rate goes up.", "As a result, low earners who do not save much lose due to lower wages.", "As we move up the earnings distribution, savings increase and people start to gain due to the higher return to saving.", "This holds true until we get to the highest earnings level at which leaving and receiving bequests become very important; hence, the welfare of people in this state is hurt by higher estate taxes.", "Lastly, for almost all reforms, with the exception of the one with gross bequests in the utility function, in which the capital income tax is adjusted and prices adjust (which implies an even larger drop in wages), increasing estate taxation results in an ex-ante welfare gain from the standpoint of the unborn person who is under the veil of ignorance.", "The literature studying the effects of estate taxation in quantitatively calibrated models that match the observed wealth inequality includes Cagetti and De Nardi (2009) and Castañeda et al. (2003).", "Both papers use a simplified life cycle structure and altruistic households, but Cagetti and De Nardi do so in a model with entrepreneurial choice in which entrepreneurs are potentially very productive and credit constrained, while in Castañeda et al. the households face high earnings risk once they become super-rich (a mechanism that we also include in our analysis).", "Compared with these two papers, we model the life cycle structure and two types of intergenerational links carefully.", "Both Cagetti and De Nardi and Castañeda et al. only study the case of abolishing estate taxation.", "Interestingly, both papers find, as we do, that abolishing estate taxation would generate small increases in aggregate capital and output.", "More specifically, for instance, all three papers, including ours, generate increases in the range of 0.7–1.5% for aggregate capital, 0.1–0.6% of aggregate output, and 1.0–1.7 percentage point increases in the share of wealth held by the richest 1%.", "Cagetti and De Nardi also compute welfare gains and losses and find that abolishing estate taxation would generate large welfare losses for a large fraction of the population, a finding that is also broadly consistent with ours.", "While it is reassuring that the results are quite similar for the specific case of abolishing estate taxation, we study a much broader range of estate tax reforms and we flesh out the effects of these reforms on many important outcomes, including the importance of parental background.", "In addition, we also study the robustness of our results to two different types of voluntary bequest motives that match important aspects of the observed estate distribution.", "The framework we use includes both voluntary and accidental bequests and the transmission of ability (or human capital) across generations and earnings risks which allows us to study wealth inequality, the importance of parental background, and the effects of reforming estate taxation.", "Our model fits key aspects of the data very well and is quite rich, but it makes some important assumptions.", "First, we limit ourselves to steady state analysis.", "This is due to both computational costs and to the fact that we see understanding steady state inequality as a necessary step that comes before studying the transitions and evolution of inequality over time.", "Second, for tractability, we assume exogenous labor supply, and we thus abstract from labor supply distortions coming from taxation.", "It would be interesting to study this channel, both in stationary environments with different taxation structures (see, for example, De Nardi et al., 2000, for a discussion of the effects of government policies on income across countries) and in the context of the observed rise in wage inequality in the United States (see Heathcote et al., 2010, for a discussion of the macroeconomic effects of these changes).", "Third, we assume an exogenous transmission of human capital across generations, thus not modeling this interesting channel, its formation, and its reaction to policy reforms.", "While it would be interesting to endogenize human capital formation, this is a major undertaking in this framework.", "In addition, because the richest 1% of households pay 99% of the estate tax, and because they are more than rich enough to invest optimally in their children׳s human capital, it is unlikely that they would change their human capital investment in children when faced with realistic changes in the estate tax rates and exemption levels; and because the revenue generated by the estate tax is very small, so are the adjustments implied in the labor or capital income tax.", "However, there are many other reforms that are likely to impact human capital formation, including, for instance, reforms that drastically both lower the estate tax exemption level and raise its tax rate, thus heavily taxing also the estates of the upper and middle-income families, and reforms that change the level and the progressivity of the labor income tax.", "For examples of frameworks modeling parental investment and the effects of family structure on income, see Aiyagari et al. (2002), Greenwood et al. (2003), Scholz and Seshadri (2012), Lee et al. (2015), and Lee and Seshadri (2014).", "Fourth, we abstract from complementary important reasons to save, such as medical expense risks after retirement, heterogeneity in life expectancy, and health investments across generations.", "De Nardi et al. (2009, 2010, 2013) and French et al. (2006) point to the importance of heterogeneity in longevity and out-of-pocket medical expenses risk that rise with age and income, and show that these factors go a long way toward explaining the lack of assets decumulation by the high-income elderly in old age.", "Eriksson et al. (2005) find that in the Danish data there is strong correlation in health across generations and that accounting for health reduces the intergenerational correlation of earnings by 25–28%.", "However, while it is important to explicitly model health and health investment, medical risk, and heterogeneity in longevity, the fact remains that substantial physical wealth is transmitted across generations.", "More generally, the important related question concerning the ability of estate taxation to reduce intergenerational transmission of wealth has to do with the identification of the strength of bequest motives as opposed to precautionary savings.", "While more work along these lines is a promising avenue for future research, it is reassuring that our results on the effects of estate taxation in the context of realistic models of wealth inequality are robust to a variety of bequest motives and reasons to save (from warm glow bequests and high earnings risk for the richest, to completely altruistic households and entrepreneurial savings).", "Lastly, our effective estate taxation is lower than the statutory one, and it would be interesting to explicitly model the costs of estate tax avoidance and to properly account for their endogenous changes in the presence of estate tax reforms." ]
[ "We model bequest motives and inheritance of ability across generations.", "We incorporate large earnings risk for the top earners.", "We study the aggregate and welfare implications of estate taxation reforms.", "Increasing estate taxation results in ex-ante welfare gain to the unborn child.", "This welfare gain comes at a large welfare cost for the super-rich." ]
3,5-Dialkoxypyridine analogues of bedaquiline are potent antituberculosis agents with minimal inhibition of the hERG channel
Bedaquiline is a new drug of the diarylquinoline class that has proven to be clinically effective against drug-resistant tuberculosis, but has a cardiac liability (prolongation of the QT interval) due to its potent inhibition of the cardiac potassium channel protein hERG. Bedaquiline is highly lipophilic and has an extremely long terminal half-life, so has the potential for more-than-desired accumulation in tissues during the relatively long treatment durations required to cure TB. The present work is part of a program that seeks to identify a diarylquinoline that is as potent as bedaquiline against Mycobacterium tuberculosis, with lower lipophilicity, higher clearance, and lower risk for QT prolongation. Previous work led to the identification of compounds with greatly-reduced lipophilicity compounds that retain good anti-tubercular activity in vitro and in mouse models of TB, but has not addressed the hERG blockade. We now present compounds where the C-unit naphthalene is replaced by a 3,5-dialkoxy-4-pyridyl, demonstrate more potent in vitro and in vivo anti-tubercular activity, with greatly attenuated hERG blockade. Two examples of this series are in preclinical development.
[ "BIOC", "CHEM", "PHAR" ]
[ "CEKMWLSBGRPKES-UHFFFAOYSA-N", "CFU, colony-forming units", "FROBLYAPPGEAGY-UHFFFAOYSA-N", "GKYYBCNOEUQDTQ-UHFFFAOYSA-N", "GQTLJYIGFBZGEF-UHFFFAOYSA-N", "GRADMLRLWZORGX-UHFFFAOYSA-N", "GUWFEHMMBHOINR-UHFFFAOYSA-N", "HARUWTIOWZSVJY-UHFFFAOYSA-N", "HEUAVAYKJDRKNK-UHFFFAOYSA-N", "HHDDKDPLFXIPBX-UHFFFAOYSA-N", "HPEZANWJRVQQPY-UHFFFAOYSA-N", "HPLC, high-performance liquid chromatography", "IEDHWPCLYLYTME-UHFFFAOYSA-N", "IKSKNYJMDKYTOW-UHFFFAOYSA-N", "JJEGOJPMKLRSPJ-UHFFFAOYSA-N", "KLIQFVIIBDEFSR-UHFFFAOYSA-N", "LCXPUYSULUUQTQ-UHFFFAOYSA-N", "LDA, lithium diisopropylamide", "LORA, low oxygen recovery assay", "LiTMP, lithium tetramethylpiperidide", "M. tb, mycobacterium tuberculosis", "MABA, microplate alamar blue assay", "MAHMFPJJKAUVMB-UHFFFAOYSA-N", "MDR, multidrug-resistant", "MIC90, minimum concentration for 90% inhibition of growth", "NBUAEURVOPSXCJ-UHFFFAOYSA-N", "OYDTTXUPMJGJQF-UHFFFAOYSA-N", "PDVNUOJSRPPQNZ-UHFFFAOYSA-N", "PEJQWNBUKMHGGO-UHFFFAOYSA-N", "PTUDVOVIFTXCHK-UHFFFAOYSA-N", "QFVACMXSSGTRNB-UHFFFAOYSA-N", "QZVLJNAOIGLPKW-UHFFFAOYSA-N", "SNVWFSQDKYVAAG-UHFFFAOYSA-N", "SNWMCNSZCLYXCK-UHFFFAOYSA-N", "SVYKJBBFRWVRKZ-UHFFFAOYSA-N", "TB, tuberculosis", "UGUNGVBWXUEQAQ-UHFFFAOYSA-N", "UXLVGNMLOBCENA-UHFFFAOYSA-N", "VCZLKKRCINFNGE-UHFFFAOYSA-N", "VDAOJOKLIXNFRY-UHFFFAOYSA-N", "VRRHQOVTENICRT-UHFFFAOYSA-N", "XDKSYOXEWIYWFX-UHFFFAOYSA-N", "YCGHXWPDGDGPKQ-UHFFFAOYSA-N", "YEGWEZNQGSXHHQ-UHFFFAOYSA-N", "YJQCLWPGMHGBLU-UHFFFAOYSA-N", "ZIMZHZVDHBTFKS-UHFFFAOYSA-N", "hERG (human Ether-a-go-go Related Gene)" ]
[ "1303", "1308", "1312", "1313", "1605", "3002", "3003" ]
[ "Bedaquiline (TMC207, Sirturo, Janssen Pharmaceuticals; 1; Table 2), a diarylquinoline, is the first example of a new class of drug that has proven clinically effective against drug-resistant tuberculosis (TB).1", "It has a novel mechanism of action, selectively inhibiting the mycobacterial ATP synthase enzyme.2", "In attempts to develop improved second-generation analogues of 1, we have focused on two areas.", "One is to lower its very high lipophilicity (clogP 7.25), which may contribute to its long terminal elimination half-life and tissue accumulation at high doses.3", "The other aim is to attenuate its inhibition (IC50 1.6 µM) of the cardiac potassium channel protein coded by the human ether-a-go-go-related gene (hERG).4", "This forms the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr), which is important for cardiac repolarization.", "Dysfunction of hERG causes long QT syndrome and can increase the risk of sudden death in patients with cardiac ischemia - a potential safety issue that all drug candidates seeking regulatory approval must currently address.", "In our attempts to develop analogues of lower lipophilicity, we have reported on the utility of replacing the 6-Br substituent on the A-unit quinoline with a more polar cyano group,5 replacing the B-unit phenyl with heterocycles,6 and replacing the C-unit naphthalene with either bicyclic heterocycles7 or substituted pyridines.8", "These studies provided a diverse range of potent diarylquinolines with much lower lipophilicity, and defined structure-activity relationships between antitubercular activity and lipophilicity.", "While they were, in the main, less successful in identifying active compounds with significantly attenuated potency against the hERG channel, we recently8 reported encouraging results (hERG IC50 values around or >10 µM) in a small number of compounds containing a 4-pyridyl-3,5-dialkoxy C-unit, suggesting this substitution is a promising one for mitigation of hERG potency.", "In the present paper, we follow up on that observation with a more extensive structure-activity study of this motif, which has allowed selection of two candidate compounds for preclinical development.", "The bedaquiline analogues of Table 2 were prepared as previously described,5–9 by LDA-mediated coupling of the appropriate benzylquinoline A/B-units and 1-(2,6-dialkoxypyridin-4-yl)-3-(dimethylamino)propan-1-one (Mannich base) C/d-units (Scheme 1).", "The 6-cyano derivatives were prepared by palladium-catalyzed cyanation of the corresponding bromo analogues.", "The resulting diarylquinolines were formed as a racemic mixture of four diastereomers, and the desired 1R,2S diastereomer (depicted) was isolated by super-critical fluid HPLC at BioDuro LLC (Beijing).", "Having previously established the utility of a range of substituted pyridyl B- and/or C-units6,8 in lowering overall clogP values, in the present SAR study we report 3,5-dialkoxy-4-pyridyl C-unit analogues bearing a wide range of different B-unit substitutions, with the focus on mitigation of hERG potency.", "Thirty-four different A/B-units were used, apart from the unsubstituted parent; syntheses of many of these (for compounds 7, 9, 10, 13, 14, 16, 17, 23–26, 34, 40–42, 48, 50 of Table 2) have already been reported.5–8", "The remainder were prepared as outlined in Scheme 1 and Table 1.", "The acid chlorides derived from I were reacted with N,O-dimethylhydroxylamine to give the N-methoxyacetamides (Weinreb amides) (II), which were converted to the required Mannich bases (III) in high yields by reaction with vinyl magnesium bromide to generate an intermediate (IIa), followed by reaction with dimethylamine.", "Mannich bases IV to VII were prepared to evaluate the role of varying D unit pKa on hERG inhibition (see Table 4).", "In the absence of an additional dialkylamine source, the N,O-dimethylhydroxylamide liberated in the formation of IIa itself reacts with IIa to give IV.", "The imidazole (VI) and triazole (VII) derivatives required the isolation of crude IIa before subsequent reaction with imidazole or triazole, this avoided the competitive reaction between IIa and the more nucleophilic N,O-dimethylhydroxylamine.", "In previous work,8 we demonstrated the ability of a range of analogues with substituted pyridyl C-units to significantly lower lipophilicity (clogP values between about 5.5–4.0) while producing compounds only slightly less effective than 1 against both replicating and non-replicating cultures of M.tb in vitro, and in a mouse TB infection model.", "While these compounds, as a class, did not show useful reductions in potency in hERG channel blockade, the 3,5-dimethoxy-4-pyridyl unit did appear to have some utility, prompting the present study of 3,5-dimethoxy-4-pyridyl and 3,5-diethoxy-4-pyridyl analogues with a wide range of B-units.", "Table 2 provides data on analogues of 1 (2–52), where the naphthalene C-unit has been replaced by either a 3,5-dimethoxy-4-pyridyl or a 3,5-diethoxy-4-pyridyl unit.", "The compounds were evaluated for bacterial growth inhibition (MIC90 values) in cultures of M.tb (strain H37Rv) under both replicating (MABA assay10) and non-replicating (LORA assay11) conditions.", "The new bedaquiline analogues characterized in the present work have calculated clogP values between 7.50 and 3.36, with the majority between 6.25 and 4.25 (Table 2).", "Nearly all have MIC90 values significantly superior (many by>10-fold) to 1 against both replicating and non-replicating cultures of M.tb.", "As shown previously,7,8 the more lipophilic compounds appear more potent, but as so many had indeterminate endpoint values (below the testing range of the assay), this could not be quantified.", "We have previously shown8 that different B-units have little specific effect on inhibitory activity (MIC90s) apart from their contribution to overall lipophilicity.", "This appears to also be the case for the analogues tested here.", "Representative compounds in the series were evaluated in vitro for a range of ADME and toxicological properties (Table 3).", "Compounds were tested for cytotoxicity in Vero green monkey-derived epithelial kidney cells,12 and for inhibition of CYP 3A4, the major metabolising enzyme for 1.12", "All tested compounds had IC50s > 10 µg/mL in the Vero assay, except for compound 9, which had a value of 7.3, and compounds 28 and 34, where this was not measured.", "In comparison, the value for 1 in repeat assays was between 4 and 16 µg/mL.", "Where tested, compounds had IC50s > 10 µM for CYP3A4 inhibition (bedaquiline IC50 > 40 µM), however, compounds 3–7, 11, 19–21, 24, 27–28, 30–31, 33, 36, 41–42, 45–49, 51–52 were not tested in this assay.", "The most interesting result of this study was the very positive effect of the 3,5-dialkoxy-4-pyridyl C-unit substitution on hERG potassium channel inhibition.", "Of the 33 compounds evaluated, 19 had IC50 values of ≥ 10 µM, in strong contrast with previous studies of different sets of analogues,6–8 where no compound had IC50 values even approaching 10 µM.", "The data suggest that, for this series, potent hERG inhibition is broadly related to higher compound lipophilicity; the 14 analogues with hERG values<10 µM had an average clogP of 4.57, whereas the 19 analogues with hERG values > 10 µM had an average clogP of 6.17 (Table 2).", "Put another way, 15/17 compounds with clogP > 5.5 had hERG IC50s > 10 µM, compared to only 4/16 with clogP < 5.5.", "Table 3 also provides data on the in vitro clearance of the analogues from human and mouse liver microsomes.", "The very slow clearance (CLint 3 μL/min/mg) and concomitantly long half-life (231 min) of 1 in human liver microsomes is improved upon 2-fold by many of the analogues but a 3-fold or more increase in human microsomal clearance was seen for a handful of analogues only.", "The mouse liver microsome data did not always reflect the corresponding human data.", "For example, for several compounds (16, 18, 22, 28, 41, 45, 46 and 50), the Clint in human microsomes is similar to, or 2-fold higher than, that of 1, but in mouse microsomes, they show at least 3-fold higher clearance than 1.", "This may be due to differing relative contributions to metabolism of these compounds versus 1 for CYP isoforms in human versus mouse microsomes.", "The human microsome data may better predict the human clearance for these compounds, if microsomal metabolism is representative of metabolism overall, for these compounds.", "Studies of pharmacokinetics in several species, followed by modelling work, will be needed to provide a better prediction of human PK.", "The pharmacokinetics in mice were evaluated after administration of a single oral dose and a single intravenous injection.", "Clearance in vivo ranged from low (similar to that of 1) to moderate, around 50 mL/min/kg, and did not always reflect the in vitro Clint values.", "In particular, a significantly higher in vitro Clint than 1 did not always lead to much higher in vivo clearance (eg compounds 31 and 41).", "Although all compounds shown demonstrated high volumes of distribution (12 L/kg and higher), compounds such as 31 and 41 showed particularly high volume of distribution values, and higher tissue levels in these cases may have contributed to lower in vivo clearance than predicted by in vitro Clint.", "Bioavailability (F) ranged from moderate (20% for compound 50) to very high (94% for compound 11) and, after oral administration, a range of AUC values was seen.", "The AUC values correlated broadly with clearance values, suggesting that for the large part, plasma exposures varied depending on relative clearance.", "Regarding bioavailability, for the compounds with F of 30% or lower, while they showed clearance in mice of 20 mL/min/kg and higher (except compound 51, which showed lower clearance), they did not necessarily show the highest in vivo clearance values overall.", "Of note, compounds with F of 30% and below had clogP values of 5.53 and below, except for compound 51, suggesting perhaps lower clogP was one factor associated with lower %F for this series.", "All compounds tested demonstrated human plasma protein binding of >99.9%, with the exceptions of compounds 18, 28, and 30, which were 99.85%, 99.78%, and 99.86% bound, respectively (data not shown).", "Assuming mouse plasma protein binding is similar, relative PPB presumably does not play a role in relative PK for these compounds.", "Evaluation of the compounds for in vivo efficacy used a mouse model of TB.", "Female BALB/c mice (n = 7 per group) were dosed at 20 mg/kg daily, by oral gavage, for 12 days, beginning 10 days after M.tb H37Rv inoculation by the aerosol route.", "Compounds were formulated in hydroxy-beta-cyclodextrin 20%.", "The relative in vivo activity was assessed by the log reduction in colony-forming units (CFU) from lung homogenates, an indicator of bacterial burden in the lungs, following treatment with a test compound compared to that effected by vehicle administration only, and to that demonstrated by bedaquiline, dosed at 20 mg/kg in the same assay run.", "In this mouse model of TB, the CFU reach approximately 5–6 log in the lungs in the vehicle-treated control group.", "Hence a reduction in lung CFU of 5 of more log units implies complete clearance of the recoverable lung CFU following 12 days of treatment.", "Therefore, for compounds in Table 3 where a reduction of approximately 5 log CFU is shown, complete clearance was achieved, and it is not possible to further distinguish the relative efficacy between these compounds.", "Reductions of 4 log CFU and higher mean only very few bacteria were remaining in the lungs of mice and these compounds can therefore be considered to show similar efficacy to bedaquiline within the range of these experiments.", "Gratifyingly, and in contrast to a previous study of bedaquiline analogues with differently-configured substituted C-unit pyridyl substituents,8 most of the compounds showed in vivo activities similar to that of 1, at the same dose, with only compounds 12, 14 and 48 being less active, showing CFU reductions of <4 log units (Table 3).", "It was interesting to note that the compounds demonstrating smaller CFU reductions in vivo were not the compounds with the lowest AUC values and, indeed, several compounds demonstrated CFU reductions that were at least similar to that effected by bedaquiline (within the limits of the assay), with substantially lower AUC values.", "For example, compounds 8, 30, 31, and 41 demonstrated CFU reductions within one log unit of the CFU reduction effected by bedaquiline, with AUC values of 2.69 µg*hr/mL or below, compared to 20.9 µg*hr/ml for 1.", "In general, for the tested compounds, where similar efficacy was achieved with lower AUCs, this is likely due to the lower MICs for these compounds than for bedaquiline, and consistent with previous work that has demonstrated AUC/MIC as the driver of efficacy against murine TB for bedaquiline.", "It may also be due to formation of metabolites that are active against M.tb, as is seen for bedaquiline.13", "One exception was compound 10, where efficacy in the range of that of bedaquiline was seen, with a 4-fold lower AUC, and a similar MIC.", "In this case, it is possible that some other factor such as tissue distribution, contributed to the CFU reduction seen at the lower AUC.", "Several compounds presented here, namely 3, 8, 10, 18, 22, 24, 27, 28, 30, 31, 33, 41 45, 46, 50 and 51 demonstrated 4 log unit CFU reductions in mice, at an AUC at least approximately 4-fold lower than that of bedaquiline.", "This suggests these analogues may have potential to demonstrate efficacy against TB in patients at lower plasma levels than bedaquiline.", "Of these, compounds 8, 10, 24, 41, 45, 46, 50 and 51 exhibited hERG IC50s of 9.9 µM or higher, compared to 1.6 µM for bedaquiline.", "Taken together, a higher IC50 against hERG along with a lower efficacious exposure may predict a lower risk of QTc prolongation in patients for these analogues compared to bedaquiline.", "Although not all of these compounds showed higher human microsome Clint than bedaquiline, the possible lower risk of QTc prolongation along with potential for lower overall adverse effects based on a lower efficacious exposure, resulted in selection of these compounds, as well as others in Table 3 that demonstrated similar efficacy to bedaquiline with lower potency against hERG, for further evaluation.", "Electrocardiography studies in animals and ultimately in human subjects will be needed to determine whether the decreases in potency against hERG seen here, along with the decreased plasma levels needed for efficacy for some of these compounds, translates into an absence of QTc prolongation at a therapeutic dose and exposure.", "Finally, the overall good activity profile of this series of 3,5-dialkoxy-4-pyridyl analogues was utilized to explore the results of changes in the pKa values of the d-unit side chain.", "This was explored previously by Guillemont et al.9 in their original paper, where they concluded that a sidechain with a pKa of >8 was needed to retain activity against the related mycobacterium, M. smegmatis.", "We were thus motivated by the idea that utilising a much weaker base in the side chain in analogue series with intrinsically higher potency might further attenuate hERG blockade while retaining good M.tb potency.", "The results in Table 4 suggest that a weaker base did lead to reduced hERG inhibition (compare hERG data for 12 and 54, 33 and 61), but there was also a large increase in the MICs for inhibition of M.tb.", "In previous publications in this series, we have shown that less lipophilic analogues of 1 (clogP 7.25) retain substantial in vitro and in vivo activity against M.tb, down to compounds with a clogP of about 4; this shows that one of the potential drawbacks of 1 can be ameliorated.", "In the present paper, we show that substitution of the C-unit naphthalene of 1 with a 3,5-dialkoxy-4-pyridyl group provide analogues with not only higher potency and substantially lower clogP than 1, but also with a greatly reduced potential cardiotoxicity risk profile, due to significantly reduced blockade of the hERG potassium channel.", "This work has recently allowed the selection of analogues 8 (TBAJ-587)14,15 and 46 (TBAJ-876)16 as compounds for pre-clinical development.", "Based on the preliminary data presented in this paper, several compounds (including 8 and 46), with activity against murine TB similar to bedaquiline at the same dose, with lower clogP, higher IC50 against hERG, and, in most cases, higher Cl values in human microsomes, were selected to progress to further studies.", "Those advanced studies evaluated the PK in rodents and non-rodents, MICs against clinical isolates of M. tb, efficacy against a model of chronic murine TB, and toxicity in a preliminary rat toxicity and toxicokinetics study.", "Based on that data, compounds 8 and 46 were selected due to their lower MICs than bedaquiline against clinical isolates of M.tb, efficacy demonstrated against murine TB at lower exposures than bedaquiline, lower potency against hERG, predicted higher human clearance, and an acceptable safety margin, based on the safe exposure in rats compared to the efficacious exposure in mice.", "Further reports on the development of these two compounds will be published in due course.", "Final products were analysed by reverse-phase HPLC (Alltima C18 5 µm column, 150 × 3.2 mm; Alltech Associated, Inc., Deerfield, IL) using an Agilent HP1100 equipped with a diode-array detector.", "Mobile phases were gradients of 80% CH3CN/20% H2O (v/v) in 45 mM NH4HCO2 at pH 3.5 and 0.5 mL/min.", "Purity was determined by monitoring at 330 ± 50 nm and was ≥ 95% for all final products.", "Melting points were determined on an Electrothermal 9100 melting point apparatus.", "NMR spectra were obtained on a Bruker Avance 400 spectrometer at 400 MHz for 1H.", "Low-resolution atmospheric pressure chemical ionization (APCI) mass spectra were measured for organic solutions on a ThermoFinnigan Surveyor MSQ mass spectrometer, connected to a Gilson autosampler.", "To a solution of 2,3-dihydro-1H-indene-4-carboxylic acid (6.12 g, 28.4 mmol) in THF (100 mL, dist.", "Na) at 0 °C was added lithium aluminium hydride (8.09 mL, 85.3 mmol) in small portions.", "The reaction mixture was stirred at 0 °C for 30 min and then at 20 °C for a further 24 h.", "The reaction mixture was washed with water (100 mL) and extracted with EtOAc (3 × 50 mL).", "The organic phase was dried with Na2SO4 and concentrated under reduced pressure to obtain 2,3-dihydro-1H-inden-4-yl)methanol (2.45 g, 99%) as a yellow oil.", "1H NMR (CDCl3) δ 7.22–7.12 (m, 3H), 4.67 (s, 2H), 2.93 (t, J = 7.6 Hz, 2H), 2.91 (t, J = 7.4 Hz, 2H), 2.09 (p, J = 7.6 Hz, 2H).", "Found: [M + H-18] = 131.5.", "To a solution of 2,3-dihydro-1H-inden-4-yl)methanol (4.33 g, 36.4 mmol) in DCM (50 mL) at 0 °C was added thionyl chloride (2.45 g, 16.5 mmol).", "The reaction mixture was stirred at 20 °C for 24 h and solvent was removed under reduced pressure.", "The residue was diluted with DCM (100 mL) and quenched with ice-water (100 mL).", "The organic phase was washed with sat.", "aq.", "NaHCO3, dried with Na2SO4 and concentrated to give a yellow residue.", "Purification by flash column chromatography using hexanes:EtOAc (1:1) gave 4-(chloromethyl)-2,3-dihydro-1H-indene (2.36 g, 86%) as a colourless oil.", "1H NMR (CDCl3) δ 7.22–7.13 (m, 3H), 4.59 (s, 2H), 2.99 (t, J = 7.5 Hz, 2H), 2.94 (t, J = 7.5 Hz, 2H), 2.11 (p, J = 7.6 Hz, 2H).", "Found: [M + H] = 167.5.", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (3.69 g, 12.9 mmol), 4-(chloromethyl)-2,3-dihydro-1H-indene (2.36 g, 14.2 mmol) and Cs2CO3 (9.67 g, 29.7 mmol) in toluene:DMF (60 mL, 2:1) was degassed under N2, then Pd(PPh3)4 (0.745 g, 0.645 mmol), was added and the mixture was heated at 90 °C for 3 h.", "The reaction mixture was cooled to 20 °C, filtered through a plug of Celite, water (150 mL) was added the mixture was extracted with EtOAc (3 × 100 mL).", "The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to obtain a yellow residue.", "Purification by flash column chromatography using hexanes:EtOAc (9:1) gave 6-bromo-3-((2,3-dihydro-1H-inden-4-yl)methyl)-2-methoxyquinoline (AB-1) (4.70 g, 99%) as a yellow oil.", "1H NMR (CDCl3) δ 7.71 (d, J = 2.2 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.60 (dd, J = 8.9, 2.2 Hz, 1H), 7.33 (s, 1H), 7.18–7.11 (m, 2H), 6.95 (d, J = 7.2 Hz, 1H), 4.11 (s, 3H), 3.98 (s, 2H), 2.96 (t, J = 7.5 Hz, 2H), 2.79 (t, J = 7.4 Hz, 2H), 2.04 (p, J = 7.6 Hz, 2H).", "Found: [M + H] = 368.5.", "To a solution of 1-tetrahydronaphthoic acid (5.70 g, 32.3 mmol) in THF (100 mL, dist.", "Na) at 0 °C was added lithium aluminium hydride (2.46 g, 64.7 mmol) in small portions.", "The reaction mixture was stirred at 0 °C for 30 min and then stirred for further 24 h at 20 °C.", "The reaction mixture was washed with water (100 mL) and extracted with EtOAc (3 × 50 mL).", "The organic phase was dried with Na2SO4 and concentrated under reduced pressure to obtain (5,6,7,8-tetrahydronaphthalen-1-yl)methanol (5.23 g, 99%) as colourless oil.", "1H NMR (CDCl3) δ 7.18 (d, J = 7.4 Hz, 1H), 7.11 (d, J = 7.5 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 4.67 (s, 2H), 2.80 (t, J = 6.2 Hz, 2H), 2.76 (t, J = 6.4 Hz, 2H), 1.88–1.77 (m, 4H).", "Found: [M + H-18] = 145.5.", "To a solution of (5,6,7,8-tetrahydronaphthalen-1-yl)methanol (5.24 g, 32.3 mmol) in DCM (200 mL) at 0 °C was added thionyl chloride (8.45 g, 71.1 mmol).", "The reaction mixture was stirred at 20 °C for 24 h, then solvent was removed under reduced pressure.", "The residue was diluted with DCM (100 mL) and quenched with ice-water (100 mL).", "The organic phase was washed with sat.", "aq.", "NaHCO3, dried with Na2SO4 and concentrated to give 5-(chloromethyl)-1,2,3,4-tetrahydronaphthalene (3.25 g, 56%) as a brown oil.", "1H NMR (CDCl3) δ 7.16–7.05 (m, 3H), 4.59 (s, 2H), 2.86 (t, J = 6.3 Hz, 2H), 2.79 (t, J = 6.4 Hz, 2H), 1.88–1.77 (m, 4H).", "Found: [M + H] = 181.6.", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (4.69 g, 16.4 mmol), 5-(chloromethyl)-1,2,3,4-tetrahydronaphthalene (3.25 g, 18.0 mmol) and Cs2CO3 (12.29 g, 37.7 mmol) in toluene:DMF (60 mL, 2:1) was degassed under N2, then Pd(PPh3)4 (0.948 g, 0.82 mmol) was added and the mixture was heated at 90 °C for 3 h.", "The reaction mixture was cooled to 20 °C, filtered through a plug of Celite, water (150 mL) was added and the mixture was extracted with EtOAc (3 × 100 mL).", "The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to obtain a yellow residue.", "Purification by flash column chromatography using hexanes:EtOAc (9:1) gave 6-bromo-2-methoxy-3-((5,6,7,8-tetrahydronaphthalen-1-yl)methyl)quinolone (AB-2) (4.64 g, 74%) as a white solid.", "1H NMR (CDCl3) δ 7.71–7.69 (m, 2H), 7.60 (dd, J = 8.9, 2.2 Hz, 1H), 7.23 (s, 1H), 7.12–7.04 (m, 2H), 6.92 (d, J = 6.9 Hz, 1H), 4.11 (s, 3H), 3.96 (s, 2H), 2.83 (bs, 2H), 2.57 (bs, 2H), 1.76 (p, J = 3.5 Hz, 4H).", "Found: [M + H] = 382.1.", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (3.00 g, 10.5 mmol), 1-(bromomethyl)-2-fluoro-3-methylbenzene (S7) (4.24 g, 20.9 mmol) and Cs2CO3 (7.87 g, 24.2 mmol) in toluene:DMF (60 mL, 2:1) was degassed under N2, then Pd(PPh3)4 (0.607 g, 0.525 mmol) was added, and the mixture was heated at 90 °C for 2 h.", "The reaction mixture was cooled to 20 °C, filtered through a plug of Celite, water (150 mL) was added and the mixture was extracted with EtOAc (3 × 100 mL).", "The organic layer was washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to obtain a yellow residue.", "Purification by flash column chromatography using hexanes:EtOAc (9:1) gave 6-bromo-3-(2-fluoro-3-methylbenzyl)-2-methoxyquinoline (AB-3) (2.87 g, 76%) as a white solid.", "1H NMR (CDCl3) δ 7.75 (d, J = 2.2 Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.60 (dd, J = 8.9, 2.2 Hz, 1H), 7.50 (s, 1H), 7.11–6.96 (m, 3H), 4.09 (s, 3H), 4.03 (s, 2H), 2.28 (d, J = 2.1 Hz, 3H).", "Found: [M + H] = 360.6.", "Borane − dimethylsulfide complex (2.79 mL, 29.40 mmol) and trimethyl borate (3.34 mL, 29.40 mmol) were added to a solution of 3-fluoro-4-methoxybenzoic acid (2.50 g, 14.69 mmol) in THF (80 mL, dist.", "Na) at 0 °C, and the solution warmed to 20 °C and stirred overnight.", "The mixture was then cooled to 0 °C, and quenched with MeOH (10 mL).", "The solvent was then evaporated and the residue was partitioned between EtOAc and water.", "The organic layer was then dried and evaporated to afford (3-fluoro-4-methoxyphenyl)methanol, (2.35 g, 100%).", "1H NMR (CDCl3) δ 7.11 (1H, dd, J = 2.0, 11.9 Hz), 7.06 (1H, ddd, J = 0.9, 2.0, 9.1 Hz), 6.94 (1H, dd, J = 8.4, 8.4 Hz), 4.61 (2H, s), 3.89 (3H, s), 1.73 (1H, s).", "Found: [M−OH] = 139.7.", "To a solution of (3-fluoro-4-methoxyphenyl)methanol (2.35 g, 15.05 mmol) and triethylamine (3.15 mL, 22.58 mmol) in DCM (50 mL, anhydrous) at 20 °C was added mesyl chloride (1.414 mL, 18.06 mmol) dropwise.", "After 15 min, the reaction was diluted with DCM (50 mL) and the organic layer washed with sat.", "aq.", "NaHCO3, dried and evaporated.", "The residue was dissolved in acetone (100 mL, anhydrous), lithium bromide (excess) added, and the mixture heated at reflux for 30 min.", "The solution was then cooled and the solvent evaporated, and the residue partitioned between EtOAc and water.", "The aqueous layer was extracted twice with EtOAc and the organic layer was dried and evaporated to afford 4-(bromomethyl)-2-fluoro-1-methoxybenzene (3.00 g, 91%).", "1H NMR (CDCl3) δ 7.16–7.07 (2H, m), 6.90 (1H, dd, J = 8.3, 8.4 Hz), 4.45 (2H, s), 3.89 (3H, s).", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (3.51 g, 12.45 mmol), 4-(bromomethyl)-2-fluoro-1-methoxybenzene (3.00 g, 13.69 mmol) and cesium carbonate (8.93 g, 27.40 mmol) in toluene (60 mL, anhydrous) and DMF (30 mL, anhydrous) was purged with nitrogen.", "Pd(PPh3)4 (0.58 g, 0.50 mmol) was then added, the mixture purged with nitrogen then heated to 80 °C under nitrogen for 5 h.", "The reaction was partitioned between EtOAc and water and the organic fraction was dried and evaporated.", "Column chromatography (19:1 hexanes/EtOAc) gave 6-bromo-3-(3-fluoro-4-methoxybenzyl)-2-methoxyquinoline (AB-4) (2.10 g, 41%).", "1H NMR (CDCl3) δ 7.76 (1H, d, J = 2.2 Hz), 7.69 (1H, d, J = 12.3 Hz), 7.62 (1H, dd, J = 2.2, 8.9 Hz), 7.49 (1H, s), 7.00–6.86 (3H, m), 4.07 (3H, s), 3.95 (2H, s), 3.88 (3H, s).", "Found: [M + H] = 376.0", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (1.5 g, 5.32 mmol), 4-(bromomethyl)benzo[d][1,3]dioxole (1.2 g, 5.59 mmol) and 2 M Na2CO3 (5 mL) in DME (25 mL) was purged with nitrogen.", "Pd(PPh3)4 (0.31 g, 0.27 mmol) was added, the mixture was purged with nitrogen then heated to 95 °C under nitrogen for 4 h.", "The reaction was partitioned between EtOAc and water and the organic fraction was dried and evaporated.", "Column chromatography with hexanes:EtOAc (100:0 to 95:5) gave 3-(benzo[d][1,3]dioxol-4-ylmethyl)-6-bromo-2-methoxyquinoline (AB-5) (1.52 g, 76%).", "1H NMR (CDCl3) δ 7.77 (d, J = 2.2 Hz, 1H), 7.69 (d, J = 8.9 Hz, 1H), 7.61 (dd, J = 8.9, 2.2 Hz, 1H), 7.53 (s, 1H) , 6.82–6.73 (m, 2H), 6.70 (dd, J = 7.4, 1.7 Hz, 1H), 5.94 (s, 2H), 4.09 (s, 3H), 3.98 (s, 2H).", "Found: [M + H] = 373.1", "A solution of methyl benzofuran-7-carboxylate(3.59 g, 20.4 mmol) in Et2O (100 mL, dist.", "Na) at 0 °C was treated with LiAlH4 (1.54 g, 40.6 mmol) then stirred at 20 °C for 3 h and quenched with ice.", "The mixture was partitioned between Et2O and sat.", "aq.", "sodium potassium tartrate and then filtered through Celite.", "The aqueous layer was extracted with Et2O and the organic phases were combined and dried.", "Column chromatography (0–5% EtOAc:DCM) gave benzofuran-7-ylmethanol (2.60 g, 86%).", "1H NMR (CDCl3) δ 7.65 (d, J = 2.2 Hz, 1H), 7.56 (dd, J = 7.7, 1.2 Hz, 1H), 7.31 (dd, J = 7.3, 0.6 Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H), 6.80 (d, J = 2.2 Hz, 1H), 5.02 (d, J = 6.2 Hz, 2H), 1.93 (t, J = 6.2 Hz, 1H).", "A solution of benzofuran-7-ylmethanol (4.72 g, 31.8 mmol) in DCM (100 mL, anhydrous) at 0 °C was treated sequentially with triethylamine (8.9 mL, 63.9 mmol) then mesyl chloride (3.70 mL, 47.8 mmol), the mixture was stirred at 0 °C for 1 h then partitioned between DCM and water.", "The organic fraction was dried and evaporated and the residue was dissolved in acetone (200 mL), LiBr (27.6 g, 318 mmol) was added and the mixture was refluxed for 0.5 h and then evaporated.", "The residue was partitioned between DCM and water; the organic fraction was dried and evaporated.", "Column chromatography (DCM) gave 7-(bromomethyl)benzofuran (6.08 g, 90%).", "1H NMR (CDCl3) δ 7.69 (d, J = 2.2 Hz, 1H), 7.57 (dd, J = 7.7, 1.2 Hz, 1H), 7.32 (dd, J = 7.4, 0.7 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 6.80 (d, J = 2.2 Hz, 1H), 4.81 (s, 2H).", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (8.00 g, 28.4 mmol), 7-(bromomethyl)benzofuran (5.99 g, 28.4 mmol) and Cs2CO3 (18.5 g, 56.7 mmol) in toluene (100 mL) and DMF (50 mL) was purged with nitrogen.", "Pd(PPh3)4 (0.66 g, 0.57 mmol) was added, the mixture was purged with nitrogen then heated to 80 °C under nitrogen for 3 h.", "The reaction was partitioned between EtOAc and water and the organic fraction was dried and evaporated.", "Column chromatography with 3:1 hexanes:DCM eluted impurities, then elution with 1:1 hexanes:DCM then DCM gave 3-(benzofuran-7-ylmethyl)-6-bromo-2-methoxyquinoline (AB-6) (6.95 g, 67%).", "1H NMR (CDCl3) δ 7.71 (d, J = 2.2 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.58–7.62 (m, 2H), 7.50–7.54 (m, 2H) , 7.20 (t, J = 7.4 Hz, 1H), 7.13 (dd, J = 7.4, 0.6 Hz, 1H), 6.79 (d, J = 2.2 Hz, 1H), 4.32 (s, 2H), 4.10 (s, 3H).", "Found: [M + H] = 368.8", "To a solution of freshly distilled N,N,N,N,-tetramethylpiperidine (1.60 mL, 9.56 mmol) in freshly distilled THF (13 mL) was added at −30 °C under nitrogen , n-BuLi (4.40 mL, 8.76 mmol) dropwise.", "The mixture was maintained at about −30 °C for 15 min, then cooled to −78 °C.", "A solution of 6-bromo-2-methoxyquinoline (1.90 g, 7.96 mmol) in dry THF (15 mL) was added dropwise at −78 °C.", "The resultant organic mixture was stirred at the same temperature for 75 min.", "A solution of 6-(diethylamino)nicotinaldehyde (1.42 g, 7.96 mmol) in dry THF (6 mL) was added dropwise at −78 °C, the reaction mixture remained orange brown, stirred at −78 °C for 2.5 h.", "The mixture was quenched with acetic acid (0.68 mL) at −65 °C.", "Water was added, the aqueous mixture was extracted with ethyl acetate (2x), and the combined extract was washed with brine, dried (MgSO4) and concentrated in vacuo to give the crude product as a yellow solid.", "Flash chromatography of the crude product using 10–100% ethyl acetate in hexane as eluent afforded product (6-bromo-2-methoxyquinolin-3-yl)(6-(diethylamino)pyridin-3-yl)methanol (1.90 g, 57%) as an off-white solid.", "1H NMR (CDCl3) δ 8.15 (d, J = 2.4 Hz, 1H), 8.01 (s, 1H), 7.88 (d, J = 2.1 Hz, 1H), 7.70–7.64 (m, 2H), 7.39 (dd, J = 8.9, 2.5 Hz, 1H), 6.43 (d, J = 8.9 Hz, 1H), 5.94 (d, J = 3.7 Hz, 1H), 4.04 (s, 3H), 3.53–3.47 (m, 4H), 2.65 (d, J = 3.9 Hz, 1H), 1.17 (t, J = 7.0 Hz, 6H).", "To a sparingly soluble solution of 2-methoxyquinolin-3-yl)(6-(diethylamino)pyridin-3-yl)methanol (1.90 g, 4.57 mmol) in freshly distilled THF (19 mL) was added at 2 °C under nitrogen sodium borohydride (0.86 g, 23.0 mmol) in 3 portions over 10 min.", "The mixture was stirred at 2–4 °C for 1 h.", "The mixture was cooled to 2 °C again, aluminium chloride (1.83 g, 13.7 mmol) was added in 4 batches over 15 min.", "The mixture was stirred at 2 °C for 10 min, then refluxed for 2 h.", "The mixture was then quenched with water cautiously at 2 °C, until gas evolution ceased.", "The white slurry was filtered through celite.", "The milky white filtrate was diluted in water, and the organic phase was collected.", "The aqueous phase was extracted with ethyl acetate (3x).", "The organic extract was washed with brine, dried (MgSO4) and concentrated in vacuo to furnish the crude product as a brownish residue.", "Flash chromatography of the crude product using 10% ethyl acetate in hexane as eluent afforded product 5-((6-bromo-2-methoxyquinolin-3-yl)methyl)-N,N-diethylpyridin-2-amine (AB-7) (1.39 g, 76%) as a white solid.", "1H NMR (CDCl3) δ 8.06 (d, J = 2.1 Hz, 1H), 7.76 (d, J = 2.2 Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.60 (dd, J = 8.8, 2.2 Hz, 1H), 7.52 (s, 1H), 7.28 (dd, J = 8.7, 2.4 Hz, 1H), 6.42 (dd, J = 8.7, 0.3 Hz, 1H), 4.09 (s, 3H), 3.85 (s, 2H), 3.50 (q, J = 7.0 Hz, 4H), 1.18 (t, J = 7.0 Hz, 6H).", "A mixture of 5-hydroxy-2-methoxynicotinaldehyde (1.00 g, 6.53 mmol) and potassium carbonate (1.35 g, 9.80 mmol) in DMF (30 mL, anhydrous) was heated at 50 °C for 10 min.", "Isopropyl iodide (0.78 mL, 7.84 mmol) was then added and the mixture stirred at this temperature for 2 h.", "The resultant solution was diluted with EtOAc and washed with brine three times.", "The organic layer was dried and evaporated to afford 5-isopropoxy-2-methoxynicotinaldehyde (0.90 g, 71%).", "1H NMR (CDCl3) δ 10.34 (1H, s), 8.07 (1H, d, J = 3.2 Hz), 7.66 (1H, d, J = 3.2 Hz), 4.48 (1H, sp, J = 6.1 Hz), 4.03 (3H, s), 1.33 (6H, d, J = 4.8 Hz).", "A mixture of isopropoxy-2-methoxynicotinaldehyde (0.90 g, 4.61 mmol) and sodium borohydride (0.35 g, 9.22 mmol) in MeOH (15 mL, anhydrous) was stirred at 20 °C for 1 h.", "The solvent was then removed and the residue partitioned between EtOAc and water.", "The organic layer was dried and evaporated.", "Column chromatography with 9:1 hexanes/EtOAc afforded (5-isopropoxy-2-methoxypyridin-3-yl)methanol (0.68 g, 75%).", "1H NMR (CDCl3) δ 7.72 (1H, s), 7.25 (1H, d, J = 3 Hz), 4.61 (2H, s), 4.42 (1H, sp), 3.93 (3H, s), 2.92–2.19 (1H, br s), 1.32 (6H, d, J = 6.1 Hz).", "To a solution of (5-isopropoxy-2-methoxypyridin-3-yl)methanol (0.68 g, 3.45 mmol) and triethylamine (0.72 mL, 5.18 mmol) in DCM (10 mL, anhydrous) at 20 °C was added mesyl chloride (0.32 mL, 4.14 mmol) dropwise.", "After 15 min, the reaction was diluted with DCM (10 mL) and the organic layer washed with sat.", "NaHCO3, dried and evaporated.", "The residue was redissolved in acetone (20 mL, anhydrous), lithium bromide (excess) added, and the mixture heated at reflux for 30 min.", "The solution was then cooled and the solvent evaporated, and the residue partitioned between EtOAc and water.", "The aqueous layer was extracted twice with EtOAc and the organic layer was dried and evaporated to afford 3-(bromomethyl)-5-isopropoxy-2-methoxypyridine (0.70 g, 78%).", "1H NMR (CDCl3) δ 7.76 (1H, dd, J = 2.6, 3.4 Hz), 7.25 (1H, dd, J = 2.4, 2.4 Hz), 4.5 (2H, d, J = 2.2 Hz), 4.42 (1H, sp, J = 2.6, 6.0 Hz), 3.96 (3H, d, J = 2.9 Hz), 1.32 (6H, dd, J = 3.0, 6.1 Hz).", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (0.80 g, 2.82 mmol), 3-(bromomethyl)-5-isopropoxy-2-methoxypyridine (0.70 g, 2.69 mmol) and cesium carbonate (1.75 g, 5.38 mmol) in toluene (10 mL, anhydrous) and DMF (5 mL, anhydrous) was purged with nitrogen.", "Pd(PPh3)4 (0.12 g, 0.11 mmol) was then added, and the mixture then heated to 80 °C under nitrogen for 4 h.", "The reaction was partitioned between EtOAc and water and the organic fraction was dried and evaporated.", "Column chromatography (19:1 hexanes/EtOAc) gave 6-bromo-3-((5-isopropoxy-2-methoxypyridin-3-yl)methyl)-2-methoxyquinoline (AB-8) (0.57 g, 48%).", "1H NMR (CDCl3) δ 7.77 (1H, d, J = 2.2 Hz), 7.71 (1H, d, J = 2.9 Hz), 7.69 (1H, d, J = 8.9 Hz), 7.62 (1H, dd, J = 2.2, 8.9 Hz), 7.56 (1H, s), 7.02 (1H, d, J = 2.9 Hz), 4.38 (1H, sp, J = 6.0 Hz), 4.08 (3H, s), 3.92 (2H, s), 3.90 (3H, s), 1.29 (6H, d, J = 6.1 Hz).", "To a mixture of 2,6-dimethoxypyridin-3-ol (8.05 g, 51.9 mmol) and imidazole (7.42 g, 108.99 mmol) in DMF (130 mL) at 20 °C was added triisopropylsilyl chloride (13.33 mL, 62.23 mmol), and the resultant mixture stirred at 20 °C for 2 h.", "The solution was then partitioned between EtOAc and water, and the aqueous layer extracted three times.", "The combined organic layers were washed with brine three times, dried and evaporated.", "Column chromatography with 19:1 hexanes/EtOAc afforded the product 2,6-dimethoxy-3-((triisopropylsilyl)oxy)pyridine (15.59 g, 96%).", "1H NMR (CDCl3) δ 7.07 (1H, d, J = 8.2 Hz), 6.13 (1H, d, J = 8.2 Hz), 3.92 (3H, s), 3.86 (3H, s), 1.27–1.18 (3H, m), 1.08 (18H, d, J = 7.1 Hz).", "Found: [M + H] = 312.8.", "To a solution of 2,6-dimethoxy-3-((triisopropylsilyl)oxy)pyridine (8.00 g, 25.69 mmol) and N,N-diisopropylamine (0.18 mL, 1.28 mmol) in THF (100 mL, dist.", "Na) at −40 °C under nitrogen was added n-BuLi (15.41 mL, 30.83 mmol) dropwise.", "The resultant solution was stirred at −40 °C for 5 min, and then warmed to 0 °C and stirred at this temperature for a further 3 h.", "The solution was then again cooled to −40 °C, and formylpiperidine (4.28 mL, 38.54 mmol) was added dropwise, and the mixture stirred at 20 °C for another 1 h. Acetic acid (8 mL) was added and the solvent was removed in vacuo.", "The resultant mixture was partitioned between EtOAc and water, and the organic fraction dried and evaporated.", "Column chromatography with 49:1 hexanes/EtOAc afforded the product 2,6-dimethoxy-5-((triisopropylsilyl)oxy)nicotinaldehyde (7.55 g, 87%).", "1H NMR (CDCl3) δ 10.17 (1H, s), 7.51 (1H, s), 4.02 (3H, s), 4.01 (3H, s), 1.30–1.19 (3H, m), 1.08 (18H, d, J = 7.3 Hz).", "Found: [M−CHO]+= 312.8.", "Tetrabutylammonium fluoride in THF (1 N, 33.36 mL, 33.36 mmol) was added to a solution of 2,6-dimethoxy-5-((triisopropylsilyl)oxy)nicotinaldehyde (7.55 g, 22.24 mmol) in THF (35 mL, dist.", "Na) at 0 °C.", "The reaction was then warmed to 20 °C and stirred for 4 h.", "The solvent was removed and the residue partitioned between EtOAc and water.", "The aqueous layer was extracted with EtOAc three times, and the organic layer dried and evaporated.", "Column chromatography with DCM followed by 3:1 DCM/EtOAc afforded the product 5-hydroxy-2,6-dimethoxynicotinaldehyde (3.15 g, 77%).", "1H NMR (CDCl3) δ 10.20 (1H, s), 7.59 (1H, s), 5.15–4.80 (1H, br s), 4.10 (3H, s), 4.01 (3H, s).", "A mixture of 5-hydroxy-2,6-dimethoxynicotinaldehyde (3.15 g, 17.20 mmol) and potassium carbonate (3.57 g, 25.80 mmol) in DMF (80 mL, anhydrous) was heated at 50 °C for 10 min.", "Methyl iodide (1.29 mL, 20.64 mmol) was then added and the mixture stirred at this temperature for 2 h.", "The resultant solution was diluted with EtOAc and washed with brine three times.", "The organic layer was dried and evaporated to afford the product 2,5,6-trimethoxynicotinaldehyde (3.39 g, 100%).", "1H NMR (CDCl3) δ 10.21 (1H, s), 7.53 (1H, s), 4.10 (3H, s), 4.02 (3H, s), 3.87 (3H, s).", "A solution of N,N,N,N-tetramethylpiperidine (3.59 mL, 21.03 mmol) in THF (40 mL, dist.", "Na) was cooled to −40 °C, n-BuLi (10.52 mL, 21.03 mmol) was added and the solution was stirred at −40 °C for 15 min, then cooled to −78 °C.", "A solution of 6-bromo-2-methoxyquinoline (17.53 mmol) in THF (40 mL, dist.", "Na) was added dropwise, the orange solution was stirred at −78 °C for 1.5 h, then a solution of 2,5,6-trimethoxynicotinaldehyde (3.42 g, 17.53 mmol) in THF (40 mL, dist.", "Na) was added.", "The mixture was stirred at −78 °C for 2 h, then acetic acid (2.5 mL) was added and the solution was allowed to warm to 20 °C.", "The solvent was removed and the residue partitioned between EtOAc and water, and the organic fraction was dried and evaporated.", "Column chromatography with 9:1 hexanes/EtOAc followed by 4:1 hexanes/EtOAc gave the product (6-bromo-2-methoxyquinolin-3-yl)(2,5,6-trimethoxypyridin-3-yl)methanol (5.50 g, 72%) as a white solid.", "1H NMR (CDCl3) δ 7.85 (1H, d, J = 2.1 Hz), 7.79 (1H, s), 7.70 (1H, d, J = 8.9 Hz), 7.65 (1H, dd, J = 2.1, 8.9 Hz), 7.15 (1H, s), 6.14 (1H, d, J = 5.2 Hz), 4.08 (3H, s), 4.02 (3H, s), 3.90 (3H, s), 3.78 (3H, s), 3.48 (1H, d, J = 5.4 Hz).", "Found: [M + H] = 436.1", "Trifluoroacetic acid (11.30 mL, 148.2 mmol) and triethylsilane (17.76 mL, 111.2 mmol) were added sequentially to a solution of (6-bromo-2-methoxyquinolin-3-yl)(2,5,6-trimethoxypyridin-3-yl)methanol (5.35 g, 12.35 mmol) in DCM (125 mL) and the solution was stirred for 1 h at 20 °C, then ice water was added.", "The solution was partitioned between sat.", "aq.", "NaHCO3 and DCM and the aqueous fraction was extracted with DCM.", "The organic fractions were combined, dried and evaporated.", "Column chromatography with 9:1 hexanes/EtOAc gave 6-bromo-2-methoxy-3-((2,4,5-trimethoxypyridin-3-yl)methyl)quinolone (AB-9) (4.45 g, 86%) as a white solid.", "1H NMR (CDCl3) δ 7.76 (1H, d, J = 2.2 Hz), 7.68 (1H, d, J = 8.9 Hz), 7.60 (1H, dd, J = 2.2, 8.4 Hz), 7.49 (1H, s), 7.05 (1H, s), 4.09 (3H, s), 4.01 (3H, s), 3.89 (5H, s), 3.79 (3H, s).", "Found: [M + H] = 419.0", "To a solution of 6-methoxypyridin-3-ol (3.20 g, 25.57 mmol) in E (50 mL, anhydrous) at 0 °C was added sodium hydride (60% in mineral dispersion, 1.23 g, 30.69 mmol) in portions.", "The mixture was warmed to 20 °C and stirred for 1 h. Chloromethyl ethyl ether (2.73 mL, 29.41 mmol) was then added, and the resultant mixture stirred at 20 °C for a further 2 h.", "The reaction was diluted with water and extracted with EtOAc (x3).", "The organic layer was washed with brine (x3), dried and evaporated.", "Column chromatography with 9:1 X4:EtOAc afforded 5-(ethoxymethoxy)-2-methoxypyridine (4.09 g, 87%).", "1H NMR (CDCl3) δ 7.95 (d, J = 3.0 Hz, 1H), 7.34–7.31 (m, 1H), 6.67 (d, J = 9.0 Hz, 1H), 5.13 (s, 2H), 3.88 (s, 3H), 3.72 (q, J = 7.1 Hz, 2H), 1.22 (t, J = 7.0 Hz, 3H).", "Found: [M + H] = 184.4.", "To a solution of 5-(ethoxymethoxy)-2-methoxypyridine (6.20 g, 33.84 mmol) and diisopropylamine (0.24 mL, 1.69 mmol) in THF (100 mL, dist.", "Na) at −40 °C under nitrogen was added n-BuLi (25.4 mL, 50.76 mmol) dropwise.", "The resultant solution was stirred at −40 °C for 5 min, and then warmed to 0 °C and stirred at this temperature for a further 3 h.", "The solution was then again cooled to −40 °C, and n-formylpiperidine (6.76 mL, 60.91 mmol) was added dropwise, and the mixture stirred at 20 °C for another 1 h. Acetic acid (15 mL) was added and the solvent was removed in vacuo.", "The resultant mixture was partitioned between EtOAc and water, and the organic fraction dried and evaporated.", "Column chromatography with 9:1 X4:EtOAc afforded 5-(ethoxymethoxy)-2-methoxyisonicotinaldehyde (4.00 g, 56%).", "1H NMR (CDCl3) δ 10.43 (s, 1H), 8.27 (s, 1H), 7.07 (d, J = 0.4 Hz, 1H), 5.30 (s, 2H), 3.92 (s, 3H), 3.78 (q, J = 7.1 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H).", "A solution of 5-(ethoxymethoxy)-2-methoxyisonicotinaldehyde (4.00 g, 18.85 mmol) and 3 M HCl (60 mL) in THF (40 mL, dist.", "Na) was heated at 40 °C for 3 h.", "The solution was then cooled, diluted with water, and the pH adjusted to 7 using potassium carbonate.", "The aqueous layer was then extracted with EtOAc three times, and the organic layer dried and evaporated.", "Column chromatography with 9:1 X4:EtOAc afforded 5-hydroxy-2-methoxyisonicotinaldehyde (2.50 g, 87%).", "1H NMR (CDCl3) δ 9.97 (d, J = 0.7 Hz, 1H), 9.46 (s, 1H), 8.08 (s, 1H), 6.93 (d, J = 0.6 Hz, 1H), 3.94 (s, 3H).", "A mixture of 5-hydroxy-2-methoxyisonicotinaldehyde (2.50 g, 16.33 mmol) and potassium carbonate (3.39 g, 24.45 mmol) in DMF (80 mL, anhydrous) was heated at 50 °C for 10 min.", "Methyl iodide (1.22 mL, 19.59 mmol) was then added and the mixture stirred at this temperature for 2 h.", "The resultant solution was diluted with EtOAc and washed with brine three times.", "The organic layer was dried and evaporated to afford 2,5-dimethoxyisonicotinaldehyde (2.25 g, 82%).", "1H NMR (CDCl3) δ 10.4 (s, 1H), 8.01 (s, 1H), 7.07 (s, 1H), 3.97 (s, 3H), 3.91 (s, 3H).", "Found [M + MeOH] = 200.4", "A mixture of 2,5-dimethoxyisonicotinaldehyde (2.25 g, 13.46 mmol) and sodium borohydride (1.02 g, 26.92 mmol) in MeOH (50 mL, anhydrous) was stirred at 20 °C for 1 h.", "The solvent was then removed and the residue partitioned between EtOAc and water.", "The organic layer was dried and evaporated to afford (2,5-dimethoxypyridin-4-yl)methanol (2.17 g, 95%).", "1H NMR (CDCl3) δ 7.70 (s, 1H), 6.77 (s, 1H), 4.66 (d, J = 6.0 Hz, 2H), 3.89 (s, 3H), 3.87 (s, 3H), 2.23 (t, J = 6.4 Hz, 1H).", "To a solution of (2,5-dimethoxypyridin-4-yl)methanol (2.05 g, 12.12 mmol) and triehylamine (2.53 mL, 18.18 mmol) in DCM (35 mL, anhydrous) at 20 °C was added methanesulfonyl chloride (1.13 mL, 14.50 mmol) dropwise.", "After 15 min, the reaction was diluted with DCM (20 mL) and the organic layer washed with sat.", "sodium hydrogen carbonate, dried and evaporated.", "The residue was redissolved in acetone (70 mL, anhydrous), Lithium bromide (10 g, excess) added, and the mixture heated at reflux for 30 min.", "The solution was then cooled and the solvent evaporated, and the residue partitioned between EtOAc and water.", "The aqueous layer was extracted twice with EtOAC and the organic layer was dried and evaporated to afford 4-(bromomethyl)-2,5-dimethoxypyridine (2.54 g, 90%).", "1H NMR (CDCl3) δ 7.75 (s, 1H), 6.75 (s, 1H), 4.40 (s, 2H), 3.91 (s, 3H), 3.88 (s, 3H).", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (2.80 g, 9.95 mmol), 4-(bromomethyl)-2,5-dimethoxypyridine (2.54 g, 10.94 mmol) and cesium carbonate (6.50 g, 19.9 mmol) in toluene (50 mL, anhydrous) and DMF (25 mL, anhydrous) was purged with nitrogen.", "Pd(PPh3)4 (0.046 g, 0.40 mmol) was then added, the mixture purged with nitrogen then heated to 80 °C under nitrogen for 4 h.", "The reaction was partitioned between EtOAc and water and the organic fraction was dried and evaporated.", "Column chromatography (19:1 x4:EtOAc) gave 6-bromo-3-((2,5-dimethoxypyridin-4-yl)methyl)-2-methoxyquinoline (AB-10) (2.20 g, 51%).", "1H NMR (CDCl3) δ 7.77 (d, J = 2.2 Hz, 1H), 7.75 (s, 1H), 7.69 (d, J = 8.9 Hz, 1H), 7.62 (dd, J = 8.9, 2.2 Hz, 1H), 7.53 (s, 1H), 6.48 (s, 1H), 4.07 (s, 3H), 3.97 (s, 2H), 3.86 (s, 3H), 3.83 (s, 3H).", "Found: [M + H] = 389.7.", "2,6-Dichloroisonicotinic acid (4.00 g, 20.8 mmol) in DMF (40 mL) was added sodium thiomethoxide (4.38 g, 65.5 mmol) at 0 °C in batches.", "Mixture was stirred at 150 °C for 18 h. Water (40 mL) was added to the resultant solution and pH was adjusted to 3 using 2 M HCl solution.", "Extracted with EtOAc (3x), dried with MgSO4, filtered and the solvent was evaporated to give 2,6-bis(methylthio)isonicotinic acid (4.01 g, 90%) as an orange solid.", "The solid was recrystallized from methanol and was used without further purification for the next step.", "1H NMR (CDCl3) δ 7.44 (s, 2H), 2.61 (s, 6H).", "Found: [M + H] = 216.5.", "Borane dimethyl sulfide complex (8.09 mL, 85.3 mmol) and trimethyl borate (9.68 mL, 85.3 mmol) were added to a solution of 2,6-bis(methylthio)isonicotinic acid (6.12 g, 28.4 mmol) in THF (100 mL, dist.", "Na) at 0 °C.", "The solution was then warmed to room temperature and was stirred for 21 h at 20 °C.", "The reaction mixture was cooled to 0 °C, MeOH (20 mL) was added to quench the reaction and the solvent was evaporated in the fume hood.", "The residue was partitioned between EtOAc and water, extracted with EtOAc (3x), dried with MgSO4, filtered and the solvent was evaporated.", "Column chromatography (4:1 hexanes:EtOAc) gave (2,6-bis(methylthio)pyridin-4-yl)methanol (4.56 g, 80%) as a white solid.", "M.p. 88 – 90 °C.", "1H NMR (CDCl3) δ 6.87 (s, 2H), 4.61 (s, 2H), 2.58 (s, 6H).", "Found: [M + H] = 202.5.", "Phosphorus tribromide (2.97 mL, 31.4 mmol) was added to a solution of (2,6-bis(methylthio)pyridin-4-yl)methanol (5.26 g, 26.1 mmol) in DCM (300 mL) at 0 °C.", "The reaction mixture was stirred at 20 °C for 18 h then solvent was evaporated.", "The residue was diluted with DCM (100 mL) and quenched with ice, the organic layer was washed with sat.", "aq.", "NaHCO3, dried with MgSO4, filtered and the solvent was evaporated.", "Column chromatography (6:1 hexanes:EtOAc) gave 4-(bromomethyl)-2,6-bis(methylthio)pyridine (3.44 g, 50%) as a white solid.", "M.p. 100 – 102 °C.", "1H NMR (CDCl3) δ 6.86 (s, 2H), 4.23 (s, 2H), 2.58 (s, 6H).", "Found: [M + H] = 266.4.", "A mixture of 4-(bromomethyl)-2,6-bis(methylthio)pyridine (3.44 g, 15.0 mmol), (6-bromo-2-methoxyquinolin-3-yl)boronic acid (4.31 g, 15.0 mmol), and Cs2CO3 (11.24 g, 34.5 mmol) in toluene-DMF (60 mL, 2:1) was degassed under N2, then added Pd(PPh3)4 (0.867 g, 0.750 mmol), and heated at 90 °C for 2 h. Reaction mixture was cooled to 20 °C, filtered through a plug of celite, added water and extracted with EtOAc (x4).", "Organic layer washed with brine, dried with Na2SO4, filtered and the solvent was evaporated to give a yellow residue.", "Purification by flash column chromatography with silica using hexane:EtOAc (9:1) to gave 3-((2,6-bis(methylthio)pyridin-4-yl)methyl)-6-bromo-2-methoxyquinoline (AB-11) (2.90 g, 46%) as a white solid.", "M.p. 129 – 131 °C.", "1H NMR (CDCl3) δ 7.80 (d, J = 2.1 Hz, 1H), 7.70 (d, J = 8.9 Hz, 1H), 7.65 (dd, J = 8.9, 2.2 Hz), 7.57 (s, 1H), 6.72 (s, 2H), 4.05 (s, 3H), 3.87 (s, 2H), 2.57 (s, 6H).", "Found: [M + H] = 421.8.", "Sodium hydride (60% w/w, 1.44 g, 36.1 mmol) in DMF (25 mL) at 0 °C was added ethanethiol (2.70 mL, 36.1 mmol) dropwise.", "The mixture foamed up as hydrogen gas was released.", "The mixture was stirred at 0 °C for 15 min, then a solution of 2,6-dichloroisonicotinic acid (2.05 g, 11.6 mmol) in anhydrous DMF (9 mL) was added dropwise at 0 °C.", "The mixture was then stirred at 150 °C for 17 h.", "The mixture was diluted in water, dissolving the white salts.", "The aqueous mixture was purged with air to remove most of the stench.", "2 M hydrochloric acid was added until pH reached ∼ 3, when white solids crashed out.", "The solids were collected by filtration, washed with ice-cold water, dried to yield 2,6-bis(ethylthio)isonicotinic acid (2.82 g, 100%) as light yellow solid which was used directly in the next step.", "1H NMR (CDCl3) δ 7.40 (s, 2H), 3.19 (q, J = 7.3 Hz, 4H), 1.39 (t, J = 7.3 Hz, 6H).", "Found: [M + H] = 244.5.", "Borane dimethyl sulfide complex (3.30 mL, 34.8 mmol) and trimethyl borate (3.90 mL, 34.8 mmol) were added to a solution of 2,6-bis(ethylthio)isonicotinic acid (2.82 g, 11.6 mmol) in THF (100 mL, dist.", "Na) at 0 °C.", "The solution was then warmed to 20 °C and was stirred for 18 h at 20 °C.", "The reaction mixture was cooled to 0 °C, MeOH (20 mL) was added to quench the reaction and the solvent was evaporated in the fume hood.", "The residue was partitioned between EtOAc and water, extracted with EtOAc (3x), dried with MgSO4, filtered and the solvent was evaporated.", "Column chromatography (4:1 hexanes:EtOAc) gave (2,6-bis(ethylthio)pyridin-4-yl)methanol (2.50 g, 94%) as a white solid.", "1H NMR (CDCl3) δ 6.87 (s, 2H), 4.61 (s, 2H), 2.58 (s, 6H).", "Found: [M + H] = 202.5.", "To a solution of (2,6-bis(ethylthio)pyridin-4-yl)methanol (2.48 g, 10.8 mmol) in anhydrous DCM (42 mL) was added triethylamine (2.20 mL, 16.2 mmol) dropwise, followed by methanesulfonyl chloride (1.10 mL, 14.2 mmol).", "The mixture was stirred at 0 °C for 10 min, then allowed to stir at 20 °C for 1 h.", "The mixture was quenched with saturated sodium bicarbonate solution.", "The aqueous mixture was extracted with dichloromethane (2x).", "The combined extract was washed with brine, dried with MgSO4 and concentrated to afford the crude product as a yellow oil.", "The crude intermediate was diluted in acetone (84 mL).", "Lithium chloride (1.85 g, 43.64 mmol) was added.", "The suspension was stirred at room temperature overnight.", "The mixture was concentrated in vacuo and adsorbed onto silica.", "Flash chromatography using a mixture of 98:2 hexane/ethyl acetate gave 4-(chloromethyl)-2,6-bis(ethylthio)pyridine (2.26 g, 84%) as a light yellow oil.", "1H NMR (CDCl3) δ 6.87 (s, 2H), 4.61 (s, 2H), 2.58 (s, 6H).", "Found: [M + H] = 202.5.", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (2.56 g, 9.10 mmol), 4-(chloromethyl)-2,6-bis(ethylthio)pyridine (2.25 g, 9.10 mmol) and Cs2CO3 (5.93 g, 18.19 mmol) in toluene-DMF (60 mL, 2:1) was degassed under N2, then added Pd(PPh3)4 (0.52 g, 0.45 mmol), and heated at 90 °C for 1.5 h. Reaction mixture was cooled to 20 °C, filtered through a plug of celite, added water and extracted with EtOAc (x4).", "Organic layer washed with brine, dried with Na2SO4, filtered and the solvent was evaporated to give a yellow residue.", "Purification by flash column chromatography with silica using hexane:EtOAc (9:1) gave 3-((2,6-bis(ethylthio)pyridin-4-yl)methyl)-6-bromo-2-methoxyquinoline (AB-12) (1.80 g, 47%) as a white solid.", "1H NMR (CDCl3) δ 7.80 (d, J = 2.1 Hz, 1H), 7.70 (d, J = 8.9 Hz, 1H), 7.65 (dd, J = 8.9, 2.2 Hz), 7.57 (s, 1H), 6.72 (s, 2H), 4.05 (s, 3H), 3.87 (s, 2H), 2.57 (s, 6H).", "Found: [M + H] = 421.8.", "Sodium metal (2.04 g, 85.0 mmol) was added to a solution of isopropanol (150 mL) and the mixture stirred at reflux for 3 h.", "The solution was then cooled to room temperature and 5-bromo-2-fluoropyridine (10.0 g, 56.67 mmol) was added, and the reaction heated at 80 °C for 0.5 h.", "The solvent was then removed and the residue partitioned between EtOAc and water, and the organic extract was dried and evaporated to afford 5-bromo-2-isopropoxypyridine (10.9 g, 89%).", "1H NMR (CDCl3) δ 8.16 (d, J = 2.5 Hz, 1H), 7.60 (dd, J = 8.8, 2.6 Hz, 1H), 6.58 (d, J = 8.8 Hz, 1H), 5.23 (sep, J = 6.2 Hz, 1H), 1.34 (s, 3H), 1.32 (s, 3H).", "Found: [M + H] = 215.5.", "To a solution of 5-bromo-2-isopropoxypyridine (9.85 g, 45.6 mmol) in THF (200 mL, dist.", "Na) at −78 °C was added n-BuLi (2.0 M in cyclohexane, 36.48 mL, 72.96 mmol) dropwise over 10 min.", "The reaction was stirred for 20 min and then trimethyl borate (13.24 mL, 72.96 mmol) was added dropwise over 5 min.", "The resulting mixture was stirred for 2 h at −78 °C, then peracetic acid solution (32 wt% in dilute acetic acid, 25.69 mL, 72.96 mmol) was added.", "After 10 min at −78 °C, the reaction was warmed to 0 °C and stirred for 1 h.", "The reaction was then quenched with aqueous sodium bisulfite and stirred for 15 min at 0 °C.", "The solvent was then concentrated, sodium bicarbonate added and the aqueous layer extracted with EtOAc.", "The combined organic extracts were dried and evaporated.", "Column chromatography with 4:1 x4:EtOAc afforded 6-isopropoxypyridin-3-ol (5.40 g, 65%).", "1H NMR (CDCl3) δ 7.76 (dd, J = 3.1, 0.4 Hz, 1H), 7.17 (dd, J = 8.9, 3.1 Hz, 1H), 6.61 (d, J = 8.9 Hz, 1H), 6.16 (br s, 1H), 5.09 (sep, J = 6.1 Hz, 1H), 1.34 (s, 3H), 1.32 (s, 3H).", "To a solution of 6-isopropoxypyridin-3-ol (6.00 g, 32.97 mmol) in DMF (80 mL, anhydrous) at 0 °C was added sodium hydride (60% in mineral dispersion, 1.57 g, 39.54 mmol) in portions.", "The mixture was warmed to 20 °C and stirred for 1 h. Chloromethyl ethyl ether (3.50 mL, 37.71 mmol) was then added, and the resultant mixture stirred at 20 °C for a further 2 h.", "The reaction was diluted with brine (100 mL) and extracted with EtOAc (x3) times.", "The organic layer was washed with brine (x3), dried and evaporated.", "Column chromatography with 19:1 x4:EtOAc afforded 5-(ethoxymethoxy)-2-isopropoxypyridine (6.50 g, 82%).", "1H NMR (CDCl3) δ 7.94 (d, J = 3.0 Hz, 1H), 7.33–7.30 (m, 1H), 6.61 (d, J = 8.9 Hz, 1H), 5.20 (sep, J = 6.2 Hz, 1H), 5.14 (s, 2H), 3.73 (q, J = 7.0 Hz, 2H), 1.33 (s, 3H), 1.32 (s, 3H), 1.23 (t, J = 7.1 Hz, 3H).", "Found: [M + H] = 211.5.", "To a solution of 5-(ethoxymethoxy)-2-isopropoxypyridine (6.50 g, 26.97 mmol) and diisopropylamine (0.19 mL, 1.35 mmol) in THF (80 mL, dist.", "Na) at −40 °C under nitrogen was added n-BuLi (2.0 M in cyclohexane, 16.18 mL, 32.36 mmol) dropwise.", "The resultant solution was stirred at −40 °C for 5 min, and then warmed to 0 °C and stirred at this temperature for a further 3 h.", "The solution was then again cooled to −40 °C, and n-formylpiperidine (4.49 mL, 40.46 mmol) was added dropwise, and the mixture stirred at 20 °C for another 1 h. Acetic acid (12 mL) was added and the solvent was removed in vacuo.", "The resultant mixture was partitioned between EtOAc and water, and the organic fraction dried and evaporated.", "Column chromatography with 19:1 x4:EtOAc afforded 5-(ethoxymethoxy)-2-isopropoxyisonicotinaldehyde (5.51 g, 76%).", "1H NMR (CDCl3) δ 10.41 (s, 1H), 8.25 (s, 1H), 7.01 (d, J = 0.5 Hz, 1H), 5.29 (s, 2H), 5.23–5.16 (m, 1H), 3.79 (q, J = 7.1 Hz, 2H), 1.34 (s, 3H), 1.32 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H).", "A solution of 5-(ethoxymethoxy)-2-isopropoxyisonicotinaldehyde (5.51 g, 20.48 mmol) and 3 M HCl (65 mL) in THF (45 mL, dist.", "Na) was heated at 40 °C for 1.5 h.", "The solution was then cooled, diluted with water, and the pH adjusted to 7 using sodium hydrogen carbonate.", "The aqueous layer was then extracted with EtOAc (x3), and the organic layer dried and evaporated to afford 5-hydroxy-2-isopropoxyisonicotinaldehyde (3.71 g, 100%).", "1H NMR (CDCl3) δ 9.96 (s, 1H), 8.06 (s, 1H), 6.87 (s, 1H), 5.21 (sep, J = 6.2 Hz, 1H), 1.34 (s, 3H), 1.32 (s, 3H).", "A mixture of 5-hydroxy-2-isopropoxyisonicotinaldehyde (3.80 g, 20.99 mmol) and potassium carbonate (4.35 g, 31.49 mmol) in DMF (100 mL, anhydrous) was heated at 50 °C for 10 min.", "Methyl iodide (1.57 mL, 25.19 mmol) was then added and the mixture stirred at this temperature for 2 h.", "The resultant solution was diluted with EtOAc and washed with brine (x3).", "The organic layer was dried and evaporated.", "Column chromatography with 19:1 x4:EtOAc afforded 2-isopropoxy-5-methoxyisonicotinaldehyde (2.60 g, 63%).", "1H NMR (CDCl3) δ 10.42 (s, 1H), 7.99 (s, 1H), 7.02 (d, J = 0.5 Hz, 1H), 5.17 (sep, J = 6.2 Hz, 1H), 1.34 (s, 3H), 1.32 (s, 3H).", "A mixture of 2-isopropoxy-5-methoxyisonicotinaldehyde (2.60 g, 13.32 mmol) and sodium borohydride (1.01 g, 26.65 mmol) in methanol (50 mL, anhydrous) was stirred at 20 °C for 1 h.", "The solvent was then removed and the residue partitioned between EtOAc and water.", "The organic layer was dried and evaporated to afford (2-isopropoxy-5-methoxypyridin-4-yl)methanol (2.59 g, 99%).", "1H NMR (CDCl3) δ 7.68 (s, 1H), 6.71 (s, 1H), 5.17 (sep, J = 6.2 Hz, 1H), 4.64 (d, J = 5.7 Hz, 2H), 3.85 (s, 3H), 2.40 (t, J = 6.1 Hz, 1H), 1.33 (s, 3H), 1.31 (s, 3H).", "To a solution of (2-isopropoxy-5-methoxypyridin-4-yl)methanol (2.59 g, 13.14 mmol) and triethylamine (2.74 mL, 19.71 mmol) in DCM (40 mL, anhydrous) at 20 °C was added methanesulfonyl chloride (1.23 mL, 15.77 mmol) dropwise.", "After 15 min, the reaction was diluted with DCM (40 mL) and the organic layer washed with sat.", "sodium hydrogen carbonate, dried and evaporated.", "The residue was redissolved in acetone (80 mL, anhydrous), Lithium bromide (10 g, excess) added, and the mixture heated at reflux for 30 min.", "The solution was then cooled and the solvent evaporated, and the residue partitioned between EtOAc and water.", "The aqueous layer was extracted with EtOAC (x2) and the organic layer was dried and evaporated to afford 4-(bromomethyl)-2-isopropoxy-5-methoxypyridine (3.42 g, 100%).", "1H NMR (CDCl3) δ 7.73 (s, 1H), 6.69 (s, 1H), 5.17 (sep, J = 6.2 Hz, 1H), 4.38 (s, 2H), 3.90 (s, 3H),1.33 (s, 3H), 1.31 (s, 3H).", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (3.92 g, 13.92 mmmol), 4-(bromomethyl)-2-isopropoxy-5-methoxypyridine (3.45 g, 13.26 mmol) and cesium carbonate (8.64 g, 26.52 mmol) in toluene (50 mL, anhydrous) and DMF (25 mL, anhydrous) was purged with nitrogen.", "Pd(PPh3)4 (0.61 g, 0.53 mmol) was then added, the mixture purged with nitrogen then heated to 80 °C under nitrogen for 4 h.", "The reaction was partitioned between EtOAc and water and the organic fraction was dried and evaporated.", "Column chromatography (19:1 x4:EtOAc) gave 6-bromo-3-((2-isopropoxy-5-methoxypyridin-4-yl)methyl)-2-methoxyquinoline (AB-13) (2.85 g, 49%).", "1H NMR (CDCl3) δ 7.77 (d, J = 2.2 Hz, 1H), 7.72 (s, 1H), 7.70 (d, J = 8.9 Hz, 1H), 7.62 (dd, J = 8.9, 2.2 Hz, 1H), 7.54 (s, 1H), 6.38 (s, 1H), 5.17 (sep, J = 6.2 Hz, 1H), 4.07 (s, 3H), 3.96 (s, 2H), 3.83 (s, 3H), 1.31 (s, 3H), 1.29 (s, 3H).", "A solution of methyl 2-hydroxy-6-methoxyisonicotinate (3.00 g, 16.4 mmol) in DMF (50 mL, anhydrous) was treated with K2CO3 (4.52 g, 32.7 mmol) and bromocyclobutane (2.00 mL, 25.0 mmol).", "The mixture was stirred at 20 °C for 48 h, partitioned between EtOAc and water and the aqueous layer was extracted with EtOAc.", "The combined organic fractions were washed with water, dried and evaporated.", "Column chromatography (DCM) gave methyl 2-cyclobutoxy-6-methoxyisonicotinate (2.215 g, 57%) as a colourless oil.", "1H NMR (CDCl3) δ 6.84 (d, J = 1.0 Hz, 1H), 6.79 (d, J = 1.0 Hz, 1H), 5.08 (pd, J = 7.4, 0.8 Hz, 1H), 3.91 (s, 3H), 3.90 (s, 3H), 2.42–2.52 (m, 2H), 2.12–2.24 (m, 2H), 1.80–1.90 (m, 1H), 1.62–1.75 (m, 1H).", "Found: [M + H] = 238.2.", "A solution of LiOH (0.71 g, 29.6 mmol) in water (20 mL) was added to a solution of methyl 2-cyclobutoxy-6-methoxyisonicotinate (2.205 g, 9.29 mmol) in MeOH (20 mL) and THF (20 mL); the solution was stirred at 20 °C for 18 h and then evaporated.", "The residue was dissolved in water (80 mL) and acidified to pH 3 with 2 M HCl.", "The resulting precipitate was filtered and dried to give 2-cyclobutoxy-6-methoxyisonicotinic acid (2.02 g, 97%) as a white solid.", "M.p. 170–171 °C.", "1H NMR (DMSO‑d6) δ 13.56 (bs, 1H), 6.74 (d, J = 1.0 Hz, 1H), 6.67 (d, J = 1.0 Hz, 1H), 5.07 (pd, J = 7.1, 0.7 Hz, 1H), 3.85 (s, 3H), 2.37–2.46 (m, 2H), 2.14–2.22 (m, 2H), 1.74–1.83 (m, 1H), 1.59–1.72 (m, 1H).", "Found: [M + H] = 224.2.", "Trimethyl borate (1.03 mL, 9.0 mmol) and borane dimethyl sulphide complex were added sequentially to a solution of 2-cyclobutoxy-6-methoxyisonicotinic acid (1.01 g, 4.52 mmol) in anhydrous THF (20 mL) at 0 °C.", "The solution was stirred at 20 °C for 18 h and then quenched with MeOH.", "Removal of the solvent gave an oil, chromatography (3:1 hexanes:EtOAc) of the crude product gave (2-cyclobutoxy-6-methoxypyridin-4-yl)methanol (0.91 g, 96%) as a colourless oil.", "1H NMR (CDCl3) δ 6.29 (d, J = 0.9 Hz, 1H), 6.23 (d, J = 0.9 Hz, 1H), 5.06 (pd, J = 7.2, 0.9 Hz, 1H), 4.62 (d, J = 6.2 Hz, 2H), 3.88 (s, 3H), 2.41–2.49 (m, 2H), 2.11–2.22 (m, 2H), 1.79–1.89 (m, 1H), 1.63–1.75 (m, 2H).", "Found: [M + H] = 210.2", "A solution of (2-cyclobutoxy-6-methoxypyridin-4-yl)methanol 0.842 g, 4.04 mmol) in DCM (25 mL) at 0 °C was treated sequentially with Et3N (2.25 mL, 16.1 mmol) and mesyl chloride (0.47 mL, 6.1 mmol), the solution was stirred for 1 h at 0 °C and then partitioned with water, the organic fraction was dried and evaporated.", "The residue was dissolved in acetone (50 mL), LiBr (3.50 g, 40.3 mmol) was added and the mixture was refluxed for 0.5 h and then evaporated.", "The residue was partitioned between DCM and water and the organic fraction was dried and evaporated.", "Column chromatography (DCM) gave 4-(bromomethyl)-2-cyclobutoxy-6-methoxypyridine (0.994 g, 90%) as a colourless oil.", "1H NMR (CDCl3) δ 6.30 (d, J = 1.0 Hz, 1H), 6.25 (d, J = 1.0 Hz, 1H), 5.07 (pd, J = 7.1, 0.9 Hz, 1H), 4.27 (s, 2H), 3.88 (s, 3H), 2.41–2.49 (m, 2H), 2.11–2.22 (m, 2H), 1.79–1.89 (m, 1H), 1.61–1.75 (m, 1H).", "Found: [M + H] = 272, 274.", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (1.010 g, 3.58 mmol), 4-(bromomethyl)-2-cyclobutoxy-6-methoxypyridine (0.975 g, 3.58 mmol) and Cs2CO3 (2.33 g, 7.2 mmol) in toluene/DMF (2:1, 50 mL) was purged with nitrogen.", "Pd(PPh3)4 (0.083 g, 0.072 mmol) was added and the mixture was heated to 80 °C for 3 h under an atmosphere of nitrogen.", "The mixture was partitioned between EtOAc and water, the organic fraction was dried and evaporated.", "Column chromatography using a gradient of 3:1 hexanes:DCM to DCM gave 6-bromo-3-((2-cyclobutoxy-6-methoxypyridin-4-yl)methyl)-2-methoxyquinoline (AB-14) (1.195 g, 78%) as a white solid.", "M.p. 101–102 °C.", "1H NMR (CDCl3) δ 7.77 (d, J = 2.2 Hz, 1H), 7.69 (d, J = 8.9 Hz, 1H), 7.62 (dd, J = 8.9, 2.2 Hz, 1H), 7.52 (s, 1H), 6.16 (d, J = 0.3 Hz, 1H), 6.10 (d, J = 0.3 Hz, 1H), 5.06 (pd, J = 7.9, 0.9 Hz, 1H), 4.06 (s, 3H), 3.90 (s, 2H), 3.87 (s, 3H), 2.38–2.48 (m, 2H), 2.09–2.21 (m, 2H), 1.77–1.87 (m, 1H), 1.62–1.72 (m, 1H).", "Found: [M + H] = 429.1, 431.1.", "Potassium carbonate (8.65 g, 125 mmol) and 2-iodopropane (12.8 mL, 128 mmol) were added to a solution of ethyl 2-ethoxy-6-hydroxyisonicotinate (10.82 g, 51.2 mmol) in anhydrous DMF (125 mL) and the mixture was stirred at 20 °C for 48 h. 2-Iodopropane (12.8 mL, 128 mmol) and potassium carbonate (8.65 g, 125 mmol) were added and the mixture was stirred for a further 24 h and then partitioned between DCM and water.", "The organic fraction was dried and evaporated, chromatography using DCM as an eluent gave ethyl 2-ethoxy-6-isopropoxyisonicotinate (11.557 g, 89%) as a colourless oil.", "1H NMR (CDCl3) δ 6.81 (s, 2H), 5.23 (sp, J = 6.2 Hz, 1H), 4.36 (t, J = 7.2 Hz, 2H), 4.32 (t, J = 7.1 Hz, 2H), 1.33–1.42 (m, 12H).", "Found: [M + H] = 254.2.", "A solution of LiOH (3.25 g, 136 mmol) in water (60 mL) was added to a solution of ethyl 2-ethoxy-6-isopropoxyisonicotinate (11.424 g, 45.1 mmol) in THF (60 mL) and MeOH (60 mL), the solution was stirred at 20 °C for 60 h then evaporated.", "The residue was dissolved in water (200 mL) and the solution was adjusted to pH 6 with 2 M HCl.", "The oily solid was extracted with EtOAc, the organic fractions were washed with water, dried and evaporated to give 2-ethoxy-6-isopropoxyisonicotinic acid (10.034 g, 99%) as a white solid.", "M.p. > 300 °C.", "1H NMR (DMSO‑d6) δ 13.48 (bs, 1H), 6.66 (d, J = 1.0 Hz, 1H), 6.64 (d, J = 1.0 Hz, 1H), 5.17 (sp, J = 6.2 Hz, 1H), 4.50 (q, J = 7.0 Hz, 2H), 1.28–1.34 (m, 9H).", "No diagnostic peak in the mass spectrum.", "Trimethylborate (3.03 mL, 26.7 mmol) and then borane-dimethylsulfide (2.53 mL, 26.7 mmol) were added to a solution of 2-ethoxy-6-isopropoxyisonicotinic acid (3.00 g, 13.3 mmol) in THF (50 mL, dist.", "Na) at 0 °C and the mixture was stirred at 20 °C for 18 hr.", "The solution was cooled to 0 °C and methanol was cautiously added to quench the reaction.", "Removal of the solvent gave a solid, this was partitioned between EtOAc and water, and the organic fraction was dried and evaporated.", "Column chromatography (3:1 hexanes:EtOAc) gave (2-ethoxy-6-isopropoxypyridin-4-yl)methanol (2.72 g, 97%) as a colourless oil.", "1H NMR (CDCl3) δ 6.24 (s, 1H), 6.23 (s, 1H), 5.21 (sp, J = 6.2 Hz, 1H), 4.60 (d, J = 6.2 Hz, 2H), 4.30 (q, J = 7.0 Hz, 2H), 1.70 (t, J = 6.2 Hz, 1H), 1.38 (t, J = 7.0 Hz, 3H), 1.34 (d, J = 6.2 Hz, 6H).", "Found: [M + H] = 212.2.", "A solution of (2-ethoxy-6-isopropoxypyridin-4-yl)methanol (2.60 g, 12.3 mmol) in DCM (50 mL, anhydrous) at 0 °C was treated sequentially with triethylamine (3.43 mL, 24.6 mmol) then mesyl chloride (1.43 mL, 18.5 mmol), the mixture was stirred at 0 °C for 1 h then partitioned between DCM and water.", "The organic fraction was dried and evaporated and the residue was dissolved in acetone (100 mL), LiBr (10.7 g, 123 mmol) was added and the mixture was refluxed for 1 h and then evaporated.", "The residue was partitioned between DCM and water; the organic fraction was dried and evaporated.", "Column chromatography (DCM) gave 4-(bromomethyl)-2-ethoxy-6-isopropoxypyridine (3.04 g, 100%) as a colourless oil.", "1H NMR (CDCl3) δ 6.26 (s, 1H), 6.25 (s, 1H), 5.20 (sp, J = 6.2 Hz, 1H), 4.26–4.33 (m, 4H), 1.38 (t, J = 7.0 Hz, 3H), 1.34 (d, J = 6.2 Hz, 6H).", "Found: [M + H] = 274.1.", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (3.27 g, 11.6 mmol), (4-(bromomethyl)-2-ethoxy-6-isopropoxypyridine (3.18 g, 11.6 mmol) and Cs2CO3 (7.56 g, 23.2 mmol) in toluene (66 mL) and DMF (33 mL) was purged with nitrogen.", "Pd(PPh3)4 (0.27 g, 0.23 mmol) was added, the mixture was purged with nitrogen then heated to 80 °C under nitrogen for 3 h.", "The reaction was partitioned between EtOAc and water and the organic fraction was dried and evaporated.", "Column chromatography with 3:1 hexanes:DCM eluted impurities, then elution with DCM gave 6-bromo-3-((2-ethoxy-6-isopropoxypyridin-4-yl)methyl)-2-methoxyquinoline (AB-15) (3.42 g, 68%) as white solid.", "M.p. 78–80 °C.", "1H NMR (CDCl3) δ 7.77 (d, J = 2.1 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.62 (dd, J = 8.8, 2.2 Hz, 1H), 7.58 (s, 1H), 6.11 (s, 1H), 6.10 (s, 1H), 5.20 (sp, J = 6.2 Hz, 1H), 4.28 (q, J = 7.1 Hz, 2H), 4.06 (s, 3H), 3.89 (s, 2H), 1.36 (t, J = 7.1 Hz, 3H), 1.32 (d, J = 6.2 Hz, 6H).", "Found: [M + H] = 431.1.", "To a solution of 2,6-dimethoxypyridine (10 g, 71.84 mmol) and N,N-diisopropylamine (0.50 mL, 3.59 mmol) in THF (200 mL, dist.", "Na) at −40 °C under nitrogen was added n-BuLi (43.10 mL, 86.21 mmol) dropwise.", "The resultant solution was stirred at −40 °C for 5 min, and then warmed to 0 °C and stirred at this temperature for a further 3 h.", "The solution was then again cooled to −40 °C, and triisopropylborate (24.87 mL, 107.76 mmol) was added dropwise, and the mixture stirred at 20 °C for another 1 h. Water (50 mL) was added and the solvent was removed in vacuo.", "To the residue was added 1 M NaOH (100 mL) and the aqueous layer washed with EtOAc (2 × 100 mL).", "The aqueous layer was then acidified to pH 3 and a solid precipitated.", "This solid was filtered and dried to afford 2,6-dimethoxypyridin-3-yl)boronic acid (8.10 g, 61%).", "1H NMR (DMSO‑d6) δ 7.87 (1H, d, J = 7.9 Hz), 6.36 (1H, d, J = 7.9 Hz), 3.90 (3H, s), 3.87 (3H, s).", "To a solution of 2,6-dimethoxypyridin-3-yl)boronic acid (8.00 g, 43.49 mmol) in THF (150 mL, dist.", "Na) at 0 °C was added dropwise 32% peracetic acid in acetic acid (21.53 mL, 86.98 mmol) over 10 min.", "The resultant solution was stirred at 20 °C.", "for 2 h.", "A 10% solution of sodium sulfite (75 mL) was then added and the mixture stirred at 20 °C for 0.5 h.", "The solvent was evaporated and the residue partitioned between EtOAc and water.", "The aqueous layer was extracted twice and the organic layer dried and evaporated.", "Column chromatography with 9:1 hexanes/EtOAc afforded 2,6-dimethoxypyridin-3-ol (6.05 g, 90%).", "1H NMR (CDCl3) δ 7.12 (1H, d, J = 8.3 Hz), 6.21 (1H, d, J = 8.2 Hz), 4.90 (1H, s), 7.00 (3H, s), 3.86 (3H, s).", "Found: [M + H] = 156.7.", "To a solution of 2,6-dimethoxypyridin-3-ol (6.45 g, 40.97 mmol) in DMF (70 mL, anhydrous) at 0 °C was added 60% sodium hydride in mineral oil (41.97 g, 9.16 mmol) in portions.", "The mixture was warmed to 20 °C and stirred for 1 h. 1-Chloro-2-methoxyethane (4.37 mL, 47.11 mmol) was then added, and the resultant mixture stirred at 20 °C for a further 2 h.", "The reaction was diluted with brine (100 mL) and extracted with EtOAc three times.", "The organic layer was washed with brine three times, dried and evaporated.", "Column chromatography with 19:1 hexanes/EtOAc afforded 3-(ethoxymethoxy)-2,6-dimethoxypyridine (8.14 g, 93%).", "1H NMR (CDCl3) δ 7.41–7.33 (1H, m), 6.26–6.17 (1H, m), 5.15 (2H, d, J = 1.9 Hz), 3.98 (3H, d, J = 1.8 Hz), 3.87 (3H, d, J = 2.0 Hz), 3.77 (2H, dq, J = 1.8, 7.1 Hz), 1.22 (3H, dt, J = 2.9, 7.0 Hz).", "To a solution of 3-(ethoxymethoxy)-2,6-dimethoxypyridine (4.00 g, 18.78 mmol) and N,N-diisopropylamine (0.13 mL, 0.94 mmol) in THF (60 mL, dist.", "Na) at −40 °C under nitrogen was added n-BuLi (2.0 M in cyclohexane, 14.09 mL, 28.17 mmol) dropwise.", "The resultant solution was stirred at −40 °C for 5 min, and then warmed to 0 °C and stirred at this temperature for a further 3 h.", "The solution was then again cooled to −40 °C, and 1-formylpiperidine (3.75 mL, 33.80 mmol) was added dropwise, and the mixture stirred at 20 °C for another 1 h. Acetic acid (7.5 mL) was added and the solvent was removed in vacuo.", "The resultant mixture was partitioned between EtOAc and water, and the organic fraction dried and evaporated.", "Column chromatography with 19:1 hexanes/EtOAc afforded 3-(ethoxymethoxy)-2,6-dimethoxyisonicotinaldehyde (2.30 g, 51%).", "1H NMR (CDCl3) δ 10.39 (1H, s), 6.61 (1H, s), 6.19 (2H, s), 4.02 (3H, s), 3.88 (3H, s), 3.78 (2H, q, J = 10.1 Hz), 1.21 (3H, t, J = 7.1 Hz).", "A solution of 3-(ethoxymethoxy)-2,6-dimethoxyisonicotinaldehyde (2.30 g, 9.54 mmol) and 3 M hydrochloric acid (60 mL) in THF (30 mL, dist.", "Na) was heated at 40 °C for 1.5 h.", "The solution was then cooled, diluted with water, and the pH adjusted to 7 using NaHCO3.", "The aqueous layer was then extracted with EtOAc three times, and the organic layer dried and evaporated.", "Column chromatography with 19:1 hexanes/EtOAc afforded 3-hydroxy-2,6-dimethoxyisonicotinaldehyde (1.36 g, 78%).", "1H NMR (CDCl3) δ 9.96 (1H, s), 9.61 (1H, s), 6.46 (1H, s), 4.06 (3H, s), 3.91 (3H, s).", "A mixture of 3-hydroxy-2,6-dimethoxyisonicotinaldehyde (1.35 g, 7.38 mmol) and potassium carbonate (1.53 g, 11.07 mmol) in DMF (40 mL, anhydrous) was heated at 50 °C for 10 min.", "Methyl iodide (0.56 mL, 8.86 mmol) was then added and the mixture stirred at this temperature for 2 h.", "The resultant solution was diluted with EtOAc and washed with brine three times.", "The organic layer was dried and evaporated to afford the product 2,3,6-trimethoxyisonicotinaldehyde (1.40 g, 96%).", "1H NMR (CDCl3) δ 10.40 (1H, s), 6.58 (1H, s), 4.04 (3H, s), 3.93 (3H, s), 3.91 (3H, s).", "A mixture of 2,3,6-trimethoxyisonicotinaldehyde (1.40 g, 7.11 mmol) and sodium borohydride (0.54 g, 14.21 mmol) in MeOH (30 mL, anhydrous) was stirred at 20 °C for 1 h.", "The solvent was then removed and the residue partitioned between EtOAc and water.", "The organic layer was dried and evaporated to afford the product (2,3,6-trimethoxypyridin-4-yl)methanol (1.35 g, 95%).", "1H NMR (CDCl3) δ 6.30 (1H, s), 4.68 (2H, d, J = 5.6 Hz), 3.99 (3H, s), 3.88 (3H, s), 3.79 (3H, s), 2.21 (1H, t, J = 5.9 Hz).", "To a solution of (2,3,6-trimethoxypyridin-4-yl)methanol (1.35 g, 6.78 mmol) and triethylamine (1.42 mL, 10.78 mmol) in DCM (20 mL, anhydrous) at 20 °C was added mesyl chloride (0.63 mL, 8.14 mmol) dropwise.", "After 15 min, the reaction was diluted with DCM (20 mL) and the organic layer washed with sat.", "aq.", "NaHCO3, dried and evaporated.", "The residue was dissolved in acetone (40 mL, anhydrous), lithium bromide (excess) added, and the mixture heated at reflux for 30 min.", "The solution was then cooled and the solvent evaporated, and the residue partitioned between EtOAc and water.", "The aqueous layer was extracted twice with EtOAc and the organic layer was dried and evaporated to give the product 4-(bromomethyl)-2,3,6-trimethoxypyridine (1.69 g, 95%).", "1H NMR (CDCl3) δ 6.27 (1H, s), 4.40 (2H, s), 3.98 (3H, s), 3.87 (3H, s), 3.87 (3H, s).", "Found: [M + H] = 262.5", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (1.89 g, 6.69 mmol), 4-(bromomethyl)-2,3,6-trimethoxypyridine (1.67 g, 6.37 mmol) and cesium carbonate (4.15 g, 12.74 mmol) in toluene (40 mL, anhydrous) and DMF (20 mL, anhydrous) was purged with nitrogen.", "Pd(PPh3)4 (0.29 g, 0.26 mmol) was then added, the mixture purged with nitrogen then heated to 80 °C under nitrogen for 4 h.", "The reaction was partitioned between EtOAc and water and the organic fraction was dried and evaporated.", "Column chromatography (19:1 hexanes/EtOAc) gave the product 6-bromo-2-methoxy-3-((2,3,5-trimethoxypyridin-4-yl)methyl)quinoline (AB-16) (1.44 g, 54%).", "1H NMR (CDCl3) δ 7.76 (1H, d, J = 2.2 Hz), 7.68 (1H, d, J = 8.9 Hz), 7.61 (1H, dd, J = 2.2, 8.8 Hz), 7.54 (1H, s), 6.04 (1H, s), 4.07 (3H, s), 4.00 (3H, s), 4.39 (2H, s), 3.85 (3H, s), 3.72 (3H, s).", "Found: [M + H] = 420.0", "To a solution of (2-(dimethylamino)-6-methoxypyridin-4-yl)methanol (3.30 g, 18.1 mmol) in anhydrous DCM (54 mL) was added at 2 °C under nitrogen triethylamine (3.8 mL, 27.2 mmol) dropwise, followed by mesyl chloride (1.7 mL, 21.7 mmol).", "The mixture was stirred from 2 °C for 10 min, then at 20 °C.", "for 2 h.", "The mixture was quenched with sat.", "aq.", "NaHCO3 solution.", "The aqueous mixture was extracted with DCM (2x) and the combined extract was washed with brine, dried (MgSO4) and concentrated to afford the crude product as a beige solid.", "The crude intermediate was dissolved in acetone (60 mL).", "Lithium bromide (1.53 g, 36.2 mmol) was added and the suspension was refluxed for 2 h. Flash chromatography using a mixture of 3–5% Et2O in hexanes as eluent gave 4-(bromomethyl)-6-methoxy-N,N-dimethylpyridin-2-amine (3.78 g, 85%) as a mobile yellow oil.", "1H NMR (CDCl3) δ 6.02 (1H, d, J = 0.8 Hz), 6.00 (1H, d, J = 0.4 Hz), 4.27 (2H, s), 3.88 (3H, s), 3.06 (6H, s).", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (4.34 g, 15.4 mmol), 4-(bromomethyl)-6-methoxy-N,N-dimethylpyridin-2-amine (3.77 g, 115.4 mmol) and cesium carbonate (10.03 g, 30.8 mmol) in a mixture of toluene (40 mL) and DMF (20 mL) was purged with nitrogen.", "Pd(PPh3)4 (0.890 g, 0.774 mmol) was added and the mixture was purged again with nitrogen and heated at 85 °C under nitrogen for 3.5 h.", "The mixture was partitioned between water and EtOAc and the mixture was extracted with EtOAc (2x).", "The extract was washed with water (2x), brine, dried (MgSO4) and concentrated to afford the crude product as a brown solid which was chromatographed using 3–10% Et2O in hexanes as eluent to yield the product 4-((6-bromo-2-methoxyquinolin-3-yl)methyl)-6-methoxy-N,N-dimethylpyridin-2-amine (AB-17) (3.69 g, 60%) as a light yellow solid.", "1H NMR (CDCl3) δ 7.76 (1H, d, J = 2 Hz), 7.68 (1H, d, J = 8.8 Hz), 7.61 (1H, dd, J = 2, 8.8 Hz), 7.55 (1H, s), 5.92 (1H, s), 5.86 (1H, s), 4.07 (3H, s), 3.87 (5H, s), 3.04 (6H, s).", "Found: [M + H] = 402.0", "To a glass tube was charged ethyl 2-chloro-6-ethoxyisonicotinate (WO 2010/080864) (1.00 g, 4.37 mmol), diphenylphosphino-1,1′-binaphthol (0.44 g, 0.70 mmol) and cesium carbonate (1.99 g, 6.12 mmol) under continuous nitrogen flow.", "Anhydrous toluene (24 mL) was added.", "The mixture was purged with nitrogen 5 min.", "Palladium acetate (0.079 g, 0.35 mmol) was added, the mixture was purged again with nitrogen.", "Dimethylamine in THF (2 N, 2.6 mL, 5.246 mmol) was added and the mixture was sealed in the tube and heated at 80 °C overnight.", "The mixture was filtered through Celite, washing with EtOAc and the filtrate was concentrated in vacuo to yield the crude product as a dark red liquid.", "Flash chromatography using 2–4% Et2O in hexanes gave ethyl 2-(dimethylamino)-6-ethoxyisonicotinate (0.85 g, 82%) as a light yellow oil.", "1H NMR (CDCl3) δ 6.60 (1H, d, J = 0.8 Hz), 6.51 (1H, d, J = 0.8 Hz), 4.35 (2H, q, J = 7.0 Hz), 4.33 (2H, q, J = 7.1 Hz), 3.09 (6H, s), 1.38 (3H, t, J = 7.2 Hz), 1.37 (3H, t, J = 7.2 Hz).", "To a solution of ethyl 2-(dimethylamino)-6-ethoxyisonicotinate (4.40 g, 18.50 mmol) in freshly distilled THF (90 mL) was added at −78 °C under nitrogen lithium aluminium hydride (0.91 g, 24.0 mmol) in 3 batches.", "The mixture was stirred at −78 °C for 15 min then at 20 °C for 1 h.", "The mixture was quenched cautiously with water at 2 °C until gas evolution ceased.", "1 M NaOH (32 mL) was added and the mixture was stirred for 1 h, then the aqueous mixture was diluted with water and extracted with EtOAc (3x).", "The combined organic extract was washed with brine, dried (Na2SO4) and concentrated to give the product, which was purified via flash chromatography eluting with mixtures of 6:1 then 4:1 hexanes/EtOAc to give the alcohol intermediate (3.27 g, 90%) as a light yellow oil.", "The material was used directly in the next step without further characterisation.", "To a solution of the alcohol intermediate (3.27 g, 16.7 mmol) in anhydrous DCM (50 mL) was added at 2 °C under nitrogen triethylamine (3.5 mL, 25.0 mmol) dropwise, followed by mesyl chloride (1.6 mL, 20.0 mmol).", "The mixture was stirred from 2 °C for 10 min, then at 20 °C for 0.5 h.", "The mixture was quenched with sat.", "aq.", "NaHCO3 solution.", "The aqueous mixture was extracted with DCM (3x) and the combined extract was washed with brine, dried and concentrated to afford the crude product as a brown oil.", "This was diluted in acetone (60 mL), lithium bromide (1.42 g) was added.", "And the suspension was refluxed for 2 h. Flash chromatography using a mixture of 2–3% Et2O in hexanes as eluent gave 4-(bromomethyl)-6-ethoxy-N,N-dimethylpyridin-2-amine (3.84 g, 89%) as a mobile yellow oil.", "1H NMR (CDCl3) δ 6.011 (1H, s), 5.99 (1H, s), 4.30 (2H, q, J = 6.8 Hz), 4.27 (2H, s), 3.05 (6H, s), 1.37 (3H, t, J = 7.2 Hz).", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (4.13 g, 14.7 mmol), 4-(bromomethyl)-6-ethoxy-N,N-dimethylpyridin-2-amine (3.80 g, 14.7 mmol) and cesium carbonate (9.58 g, 29.4 mmol) in a mixture of toluene (40 mL) and DMF (20 mL) was purged with nitrogen.", "Pd(PPh3)4 (0.68 g) was added.", "The mixture was purged again with nitrogen and heated at 85 °C under nitrogen for 3 h.", "The mixture was partitioned between water and EtOAc and the aqueous mixture was extracted with EtOAc (2x).", "The extract was washed with water (2x), brine, dried and concentrated to afford the crude product as a brown oil.", "This was chromatographed using 3–10% Et2O in hexanes as eluent to give the product as a yellow solid (3.83 g), which was triturated in diethyl ether to afford yield 4-((6-bromo-2-methoxyquinolin-3-yl)methyl)-6-ethoxy-N,N-dimethylpyridin-2-amine (AB-18) (3.51 g, 57%) as a pale yellow solid.", "1H NMR (CDCl3) δ 7.76 (1H, d, J = 2.2 Hz), 7.68 (1H, d, J = 8.8 Hz), 7.61 (1H, dd, J = 2.2, 8.8 Hz), 7.55 (1H, s), 5.91 (1H, s), 5.84 (1H, s), 4.30 (2H, q, J = 7.1 Hz), 4.07 (3H, s), 3.87 (2H, s), 3.03 (6H, s), 1.36 (3H, t, J = 7.1 Hz).", "To a glass tube was charged methyl 2-chloro-6-(dimethylamino)isonicotinate (WO 2010/100475) (2.44 g, 11.40 mmol), rac-bis(diphenylphosphino)-1,1′-binaphthol (0.71 g, 1.140 mmol) and cesium carbonate (4.43 g, 13.60 mmol) under continuous nitrogen flow.", "Anhydrous toluene (30 mL) was added.", "The mixture was purged with nitrogen for 5 min Palladium acetate (0.26 g, 1.158 mmol) was added and the mixture was purged again with nitrogen.", "Ethanethiol (1.0 mL, 13.60 mmol) was added and the mixture was sealed in the tube and heated at 150 °C for 22 h.", "The mixture was filtered through Celite, washing with EtOAc.", "The filtrate was concentrated in the fume hood by heating the solution while purging with air.", "A crude orange solid was obtained.", "Flash chromatography of the product using 2–5% diethyl ether in hexanes provided the product methyl 2-(dimethylamino)-6-(ethylthio)isonicotinate (2.42 g, 88%) as a yellow crystalline solid.", "1H NMR (CDCl3) δ 6.96 (1H, d, J = 1.0 Hz), 6.73 (1H, d, J = 1.0 Hz), 3.89 (3H, s), 3.15 (2H, q, J = 7.3 Hz), 3.12 (6H, s), 1.38 (3H, t, J = 7.3 Hz).", "Found: [M + H] = 241.5.", "To a solution of methyl 2-(dimethylamino)-6-(ethylthio)isonicotinate (1.64 g, 6.824 mmol) in freshly distilled THF (66 mL) was added at 2 °C under nitrogen lithium aluminium hydride (0.31 g, 8.189 mmol) in 3 batches.", "The mixture was stirred at 2 °C for 15 min then at 20 °C for 1 h.", "The mixture was quenched cautiously with water at 2 °C until gas evolution ceased.", "1 M NaOH (20 mL) was added.", "The mixture was stirred for 5 min, then decanted leaving the aluminium salts which were filtered through Celite.", "The aqueous mixture was partitioned between water and EtOAc.", "The aqueous phase was extracted with EtOAc (3x).", "The combined organic extract was washed with brine, dried (Na2SO4) and concentrated to give the crude product as a brown oil.", "The crude product was purified by flash chromatography using a 4:1 mixture of hexanes/EtOAc to afford the product (2-(dimethylamino)-6-(ethylthio)pyridin-4-yl)methanol (1.30 g, 90%) as a brown oil.", "1H NMR (CDCl3) δ 6.43 (1H, s), 6.18 (1H, d, J = 0.8 Hz), 4.56 (2H, d, J = 5.0 Hz), 3.14 (2H, q, J = 7.4 Hz), 3.08 (6H, s), 1.67 (1H, br t, J = 5.8 Hz), 1.57 (3, s), 1.37 (3H, t, J = 7.3 Hz).", "Found: [M + H] = 213.5.", "To a solution of (2-(dimethylamino)-6-(ethylthio)pyridin-4-yl)methanol (0.60 g, 2.84 mmol) in anhydrous DCM (10 mL) was added at 2 °C under nitrogen triethylamine (0.59 mL, 4.26 mmol) dropwise, followed by mesyl chloride (0.26 mL, 3.41 mmol).", "The mixture was stirred from 2 °C to 5 °C over 1 h.", "The mixture was quenched with sat.", "aq.", "NaHCO3 solution.", "The aqueous mixture was extracted with DCM (3x).", "The combined extract was washed with brine, dried (MgSO4) and concentrated to afford the crude product as a light brown oil.", "The crude intermediate was dissolved in acetone (20 mL).", "Lithium bromide (0.99 g, 11.36 mmol) was added and the suspension was stirred at 20 °C for 2.5 h. Flash chromatography using 98:2 hexanes/Et2O furnished the product 4-(bromomethyl)-6-(ethylthio)-N,N-dimethylpyridin-2-amine as a brown solid.", "The reaction was repeated on a 3.29 mmol scale and the products were combined.", "Total yield = 1.39 g, 82%.", "1H NMR (CDCl3) δ 6.46 (1H, d, J = 1.2 Hz), 6.15 (1H, d, J = 0.8 Hz), 4.24 (2H, s), 3.13 (2H, q, J = 7.2 Hz), 3.08 (6H, s), 1.37 (3H, t, J = 7.2 Hz).", "Found: [M + H] = 275.5", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (1.42 g, 5.05 mmol), 4-(bromomethyl)-6-(ethylthio)-N,N-dimethylpyridin-2-amine (1.39 g, 5.05 mmol) and cesium carbonate (3.29 g, 10.09 mmol) in a mixture of toluene (14 mL) and DMF (7 mL) was purged with nitrogen.", "Pd(PPh3)4 (0.29 g) was added.", "The mixture was purged again with nitrogen and heated at 85 °C under nitrogen for 2.5 h.", "The mixture was partitioned between water and EtOAc.", "The aqueous mixture was extracted with EtOAc (2x).", "The extract was washed with water, brine, dried (MgSO4) and concentrated to afford the crude product as an orange oil which was chromatographed using 2–5% Et2O in hexanes as eluent to yield the product as a light yellow solid.", "Recrystallisation from DCM/MeOH provided 4-((6-bromo-2-methoxyquinolin-3-yl)methyl)-6-(ethylthio)-N,N-dimethylpyridin-2-amine (AB-19) (1.25 g, 57%) as a white solid.", "1H NMR (CDCl3) δ 7.79 (1H, d, J = 2.4 Hz), 7.70 (1H, d, J = 8.8 Hz), 7.64 (1H, dd, J = 2.4, 9.2 Hz), 7.56 (1H, s), 6.34 (1H, s), 6.06 (1H, s), 4.09 (3H, s), 3.86 (2H, s), 6.15 (2H, q, J = 7.2 Hz), 3.07 (6H, s), 1.39 (3H, t, J = 7.2 Hz).", "Found [M + H] = 432.1", "To a solution of n-BuLi (26.1 mL, 52.09 mmol) in THF (70 mL, dist.", "Na) at −78 °C was added consecutively N,N-diisopropylamine (7.30 mL, 52.09 mmol) and 2,3-difluoropyridine (5.00 g, 43.41 mmol).", "The resultant mixture was stirred at −78 °C for 1 h, and then poured on crushed dry ice (excess).", "The reaction was allowed to warm to 20 °C for 1 h, and after evaporation of excess dry ice and THF, the residue was taken up into water (100 mL) and washed with EtOAc (2 × 50 mL).", "The aqueous layer was then acidified to pH 1 and extracted with EtOAc (2 × 100 mL).", "The combined organic extracts were dried and evaporated to afford 2,3-difluoroisonicotinic acid (2.39 g, 35%).", "1H NMR (CDCl3) δ 15.3–13.2 (1H, br s), 8.14 (1H, dd, J = 1.2, 5.0 Hz), 7.70 (1H, dd, J = 4.8, 4.8 Hz).", "Found: [M−H] = 158.5.", "Sodium (0.79 g, 33.09 mmol) was added portion wise to MeOH (60 mL) over 0.5 h. 2,3-difluoroisonicotinic acid (2.39 g, 15.04 mmol) was then added and the reaction refluxed for 2 h.", "The solution was cooled and the solvent evaporated.", "The residue was taken up into water (100 mL) and washed with EtOAc (2 × 50 mL).", "The aqueous layer was then acidified to pH 1 and extracted with EtOAc (2 × 100 mL).", "The combined organic extracts were dried and evaporated to afford the product 3-fluoro-2-methoxyisonicotinic acid (2.23 g, 87%).", "1H NMR (DMSO‑d6) δ 14.6–12.8 (1H, br s), 8.05 (1H, d, J = 5.2 Hz), 7.28 (1H, dd, J = 4.7, 4.9 Hz), 3.97 (3H, s).", "Found: [M−H] = 170.5.", "Borane − dimethylsulfide complex (2.47 mL, 26.07 mmol) and trimethyl borate (2.96 mL, 26.07 mmol) were added to a solution of 3-fluoro-2-methoxyisonicotinic acid (13.03 mmol) in THF (80 mL, dist.", "Na) at 0 °C, and the solution warmed to 20 °C and stirred overnight.", "The mixture was then cooled to 0 °C, and quenched with methanol (10 mL).", "The solvent was then evaporated and the residue was partitioned between EtOAc and water.", "The organic layer was then dried and evaporated to afford the product (3-fluoro-2-methoxypyridin-4-yl)methanol (1.89 g, 92%).", "1H NMR (CDCl3) δ 7.92 (1H, d, J = 5.2 Hz), 7.02 (1H, dd, J = 4.5, 5.1 Hz), 4.81 (2H, s), 4.03 (3H, s).", "Found: [M + H] = 158.5.", "To a solution of (3-fluoro-2-methoxypyridin-4-yl)methanol (1.89 g, 12.03 mmol) and triethylamine (2.52 mL, 18.05 mmol) in DCM (30 mL, anhydrous) at 20 °C was added mesyl chloride (1.12 mL, 14.44 mmol) dropwise.", "After 15 min, the reaction was diluted with DCM (20 mL) and the organic layer washed with sat.", "NaHCO3, dried and evaporated.", "The residue was dissolved in acetone (60 mL, anhydrous), lithium bromide (excess) added, and the mixture heated at reflux for 30 min.", "The solution was then cooled and the solvent evaporated, and the residue partitioned between EtOAc and water.", "The aqueous layer was extracted twice with EtOAc and the organic layer was dried and evaporated to give the product 4-(bromomethyl)-3-fluoro-2-methoxypyridine (2.20 g, 83%).", "1H NMR (CDCl3) δ 7.90 (1H, d, J = 5.2 Hz), 6.90 (1H, dd, J = 4.8, 4.9 Hz), 4.43 (2H, s), 4.02 (3H, s).", "Found: [M + H] = 220.2", "A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (2.56 g, 9.09 mmol), 4-(bromomethyl)-3-fluoro-2-methoxypyridine (2.20 g, 10.00 mmol) and cesium carbonate (5.92 g, 18.18 mmol) in toluene (45 mL, anhydrous) and DMF (22.5 mL, anhydrous) was purged with nitrogen.", "Pd(PPh3)4 (0.42 g, 0.363 mmol) was then added, the mixture purged with nitrogen, then heated to 80 °C under nitrogen for 4 h.", "The reaction was partitioned between EtOAc and water and the organic fraction was dried and evaporated.", "Column chromatography (19:1 hexanes/EtOAc) gave the product 6-bromo-3-((3-fluoro-2-methoxypyridin-4-yl)methyl)-2-methoxyquinoline (AB-20) (1.76 g, 51%).", "1H NMR (CDCl3) δ 7.82 (1H, d, J = 5.2 Hz), 7.79 (1H, d, J = 2.1 Hz), 7.69 (1H, d, J = 8.8 Hz), 7.6 4 (1H, dd, J = 2.2, 8.9 Hz), 7.60 (1H, s), 6.69 (1H, dd, J = 4.8, 5.0 Hz), 4.07 (3H, s), 4.06 (2H, s), 4.03 (3H, s).", "Found: [M + H] = 377.2", "Oxalyl chloride (1.34 mL, 15.8 mmol) was added to a suspension of 2,6-dimethoxyisonicotinic acid (2.41 g, 13.2 mmol) in DCM (70 mL) and DMF (0.20 mL, 2.6 mmol) at 20 °C.", "The mixture was stirred for 1 h to give a colourless solution which was cooled to 0 °C.", "N,O-dimethylhydroxylamine hydrochloride (1.42 g, 14.6 mmol) and pyridine (3.51 mL, 28.9 mmol) were added sequentially and the mixture was stirred at 20 °C for 18 h, then partitioned between EtOAc and sat.", "aq.", "NaHCO3.", "Column chromatography with hexanes:EtOAc (2:1) gave N,2,6-trimethoxy-N-methylisonicotinamide (II (Z = Me), 2.49 g, 83%).", "1H NMR (CDCl3) δ 6.47 (s, 2H), 3.93 (s, 6H), 3.58 (br s, 3H), 3.32 (s, 3H).", "Found: [M + H] = 227.2.", "Vinylmagnesium bromide (42.5 mL of a 1 M solution in THF, 42.5 mmol) was added to a solution of N,2,6-trimethoxy-N-methylisonicotinamide (II (Z = Me), 4.81 g, 21.3 mmol) in dry THF (200 mL) at 0 °C.", "The brown solution was warmed to 20 °C for 1 h then dimethylamine (42.5 mL of a 2 M solution in THF, 85 mmol) and water (40 mL) were added.", "The solution was stirred at 20 °C for 1 h, then partitioned between EtOAc and water.", "The solution was dried and evaporated and column chromatography with DCM:MeOH (95:5) eluted impurities while DCM:MeOH (9:1) gave 1-(2,6-dimethoxypyridin-4-yl)-3-(dimethylamino)propan-1-one (IIIa) (2.90 g, 57%) as a brown oil.", "1H NMR (CDCl3) δ 6.74 (s, 2H), 3.95 (s, 6H), 3.06 (t, J = 7.0 Hz, 2H), 2.72 (t, J = 7.0 Hz, 2H), 2.27 (s, 6H).", "Found: [M + H] = 239.1", "Oxalyl chloride (0.73 mL, 8.6 mmol) was added to a suspension of 2,6-diethoxyisonicotinic acid (1.52 g, 7.20 mmol) in DCM (50 mL, anhydrous) and DMF (0.20 mL, 2.6 mmol) at 20 °C.", "The mixture was stirred at 20 °C for 1 h to give a colourless solution, which was then cooled to 0 °C.", "N,O-dimethylhydroxylamine hydrochloride (0.77 g, 17.89 mmol) and pyridine (1.92 mL, 23.7 mmol) were added sequentially and the mixture was stirred at 20 °C for 18 h, then partitioned between EtOAc and sat.", "aq.", "NaHCO3.", "Column chromatography using 3:1 hexanes:EtOAc gave 2,6-diethoxy-N-methoxy-N-methylisonicotinamide (II (Z = Et), 1.26 g, 69%).", "1H NMR (CDCl3) δ 6.43 (s, 2H), 4.33 (q, J = 7.1 Hz, 2H), 3.59 (br s, 3H), 3.32 (s, 3H), 1.39 (t, J = 7.1 Hz, 3H).", "Found: [M + H] = 255.1", "Vinylmagnesium bromide (14.6 mL of a 1 M solution in THF, 14.6 mmol) was added to a solution of 2,6-diethoxy-N-methoxy-N-methylisonicotinamide (II (Z = Et), 1.23 g, 4.85 mmol) in dry THF (50 mL) at 0 °C.", "The brown solution was warmed to 20 °C for 1 h then a solution of 2 M dimethylamine in THF (14.6 mL, 29.2 mmol) and water (10 mL) were added.", "The solution was stirred at 20 °C for 1 h, then partitioned between EtOAc and water.", "The solution was dried and evaporated to give 1-(2,6-diethoxypyridin-4-yl)-3-(dimethylamino)propan-1-one (IIIb) (1.24 g, 96%) as a brown oil.", "1H NMR (CDCl3) δ 6.71 (s, 2H), 4.34 (q, J = 7.1 Hz, 2H), 3.05 (t, J = 7.0 Hz, 2H), 2.72 (t, J = 7.0 Hz, 2H), 2.26 (s, 6H), 1.40 (t, J = 7.0 Hz, 3H).", "Found: [M + H] = 267.2", "Vinylmagnesium bromide (62.7 mL of a 1 M solution in THF, 62.7 mmol) was added to a solution of N,2,6-trimethoxy-N-methylisonicotinamide (II (Z = Me), 6.75 g, 29.8 mmol) in dry THF (150 mL) at 0 °C.", "The yellow/orange solution was warmed to 20 °C for 1 h and water (30 mL) were added.", "The solution was stirred at 20 °C for 24 h, then partitioned between EtOAc and water.", "The solution was dried and evaporated and column chromatography with hexanes:EtOAc (9:1) gave 1-(2,6-dimethoxypyridin-4-yl)-3-(methoxy(methyl)amino)propan-1-one (IV) (5.50 g, 72%) as an oil.", "1H NMR (CDCl3) δ 6.76 (s, 2H), 3.96 (s, 6H), 3.46 (s, 3H), 3.16 (t, J = 6.6 Hz, 2H), 3.04 (t, J = 6.5 Hz, 2H), 2.61 (s, 3H).", "Found: [M + H] = 255.6.", "Vinylmagnesium bromide (18.6 mL of a 1 M solution in THF, 18.6 mmol) was added to a solution of N,2,6-trimethoxy-N-methylisonicotinamide (II (Z = Me), 2.00 g, 8.80 mmol) in dry THF (30 mL) at 0 °C.", "The yellow/orange solution was warmed to20 oC for 1 h then morpholine (3.23 mL, 37.1 mmol) then water (10 mL) were added.", "The solution was stirred at 20 °C for 1 h, the solvent removed in vacuo, and the resultant mixture then partitioned between EtOAc and water.", "The solution was dried and evaporated to afford 1-(2,6-dimethoxypyridin-4-yl)-3-morpholinopropan-1-one (V) (2.40 g, 97%) as an oil.", "1H NMR (CDCl3) δ 6.73 (s, 2H), 3.95 (s, 6H), 3.70 (t, J = 4.4 Hz, 4H), 3.08 (t, J = 7.1 Hz, 2H), 2.79 (t, J = 7.4 Hz, 2H), 2.48 (t, J = 4.4 Hz, 4H).", "Found: [M + H] = 281.6.", "Vinylmagnesium bromide (0.93 mL of a 1 N solution in THF, 0.93 mmol) was added to a solution of N,2,6-trimethoxy-N-methylisonicotinamide (II (Z = Me),0.10 g, 0.44 mmol) in dry THF (3 mL) at 0 °C.", "The yellow/orange solution was warmed to 20 °C for 1 h.", "The solvent was removed in vacuo and the residue partitioned between EtOAc and water, and the pH adjusted to 3 with 1 M HCl.", "The combined organic extracts were dried and the solvent removed in vacuo.", "The crude residue was redissolved in THF (3 mL), cooled to 0 °C, and imidazole (0.18 g, 2.65 mmol) was added followed by water (1 mL).", "The solution was then stirred at 20 °C for 1 h, then partitioned between EtOAc and water.", "The solution was dried and evaporated and column chromatography with hexanes:EtOAc (1:2) gave 1-(2,6-dimethoxypyridin-4-yl)-3-(1H-imidazol-1-yl)propan-1-one (VI) (0.045 g, 39%) as an oil.", "1H NMR (CDCl3) δ 7.54 (s, 1H), 7.04 (s, 1H), 6.95 (s, 1H), 6.68 (s, 2H), 4.41 (t, J = 6.4 Hz, 2H), 3.95 (s, 6H), 3.35 (t, J = 6.4 Hz, 2H).", "Found: [M + H] = 262.6", "Vinylmagnesium bromide (27.9 mL of a 1 N solution in THF, 27.9 mmol) was added to a solution of N,2,6-trimethoxy-N-methylisonicotinamide (II (Z = Me), 3.00 g, 13.3 mmol) in dry THF (30 mL) at 0 °C.", "The yellow/orange solution was warmed to 20 °C for 1.5 h.", "The solvent was removed in vacuo and the residue partitioned between chloroform and water, and the pH adjusted to 1 with 1 M HCl.", "The combined organic extracts were dried and the solvent removed in vacuo.", "The crude residue was redissolved in chloroform (150 mL) and 1H-1,2,4-triazole (2.67 g, 39.8 mmol) and the resultant mixture stirred at 60 °C for 3 h.", "The solution cooled to 20 °C and partitioned between chloroform and water.", "The solution was dried and evaporated and column chromatography with DCM:MeOH (99:1) gave 1-(2,6-dimethoxypyridin-4-yl)-3-(1H-1,2,4-triazol-1-yl)propan-1-one (VII) (1.54 g, 44%) as an oil.", "1H NMR (CDCl3) δ 8.20 (s, 1H), 7.91 (s, 1H), 6.70 (s, 2H), 4.61 (t, J = 6.2 Hz, 2H), 3.94 (s, 6H), 3.51 (t, J = 6.1 Hz, 2H).", "Found: [M + H] = 263.6.", "n-BuLi (2.92 mL of a 2 N solution in cyclohexane, 5.83 mmol) was added at −40 °C under dry nitrogen to a solution of dry diisopropylamine (0.813 mL, 5.83 mmol) in dry THF (6 mL) and the solution was stirred at this temperature for 10 min, then cooled to −78 °C.", "A solution of 6-bromo-3-((2,5-dimethoxypyridin-3-yl)methyl)-2-methoxyquinoline (AB-10) (1.90 g, 4.86 mmol) in dry THF (6 mL) was added dropwise and the mixture was stirred at −78 °C for 90 min, to give a dark, wine-red coloured solution.", "A solution of 1-(2,6-dimethoxypyridin-4-yl)-3-(dimethylamino)propan-1-one (IIIa) (1.15 g, 4.86 mmol) in dry THF (7 mL) was added and the reaction mixture was stirred at this temperature for 4 h. HOAc (0.90 mL) was added and the reaction mixture was warmed to 20 °C.", "Water (100 mL) was added and the mixture was extracted with EtOAc (2x).", "The combined organic extract was washed with sat.", "aq.", "NaHCO3 solution, and brine, then dried (Na2SO4) and the solvent removed under reduced pressure.", "The residue was purified by flash column chromatography.", "Elution with 0–10% MeOH/DCM afforded isomer A of 27 (1.11 g, 36%) followed by isomer B of 27 (1.03 g, 34%) as white solids.", "Isomer A. 1H NMR (CDCl3, 400 MHz) δ 8.12 (d, J = 3.3 Hz, 2H), 7.82 (d, J = 2.2 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.60 (dd, J = 8.9, 2.2 Hz, 1H), 7.48 (d, J = 3.0 Hz, 1H), 6.56 (br s, 2H), 5.31 (s, 1H), 4.19 (s, 3H), 3.88 (s, 6H), 3.74 (s, 3H), 3.63 (s, 3H), 2.30–2.04 (m, 1H), 2.02–1.96 (m, 1H), 1.97 (s, 6H), 1.83–1.68 (m, 2H).", "Found: [M + H] = 627.8.", "Isomer B. 1H NMR (CDCl3, 400 MHz) δ 8.63 (s, 1H), 7.78 (d, J = 1.7 Hz, 1H), 7.68 (d, J = 3.0 Hz, 1H), 7.55–7.47 (m, 3H), 6.56 (br s, 2H), 5.32 (s, 1H), 4.04 (s, 3H), 3.84 (s, 3H), 3.83 (s, 6H), 3.71 (s, 3H), 2.40–2.32 (m, 1H), 2.08 (s, 6H), 2.03–1.98 (m, 1H), 1.87–1.79 (m, 1H), 1.76–1.70 (m, 1H).", "Found: [M + H] = 627.8.", "The mixture was resolved into its four optical isomers using preparative supercritical fluid HPLC at BioDuro LLC (Beijing).", "The data in Table 1 are for the most active R,S-diastereomers.", "The other 6-bromo compounds in Table I were prepared and purified similarly.", "A solution of compound 27 (Table 1) (0.61 g, 0.969 mmol) in DMF (6 mL, anhydrous) was purged with nitrogen and heated to 55 °C for 10 min.", "Tri(o-tolyl)phosphine (0.044 g, 0.145 mmol), zinc dust (0.006 g, 0.097 mmol) and tris(dibenzylideneacetone)dipalladium(0) (0.067 g, 0.073 mmol) were then added, and the reaction was again purged with nitrogen and heated for another 10 min at 55 °C.", "Zinc cyanide (0.063 g, 0.533 mmol) was then added and the reaction mixture was heated to 65 °C for 4 h.", "The reaction was diluted with water and extracted with EtOAc three times.", "The organic layer was washed with brine three times, dried and evaporated.", "Column chromatography with 1:1 hexane/EtOAc followed by 1:3 hexane/EtOAc afforded 28 (0.41 g, 74%); isomer A as a white solid.", "1H NMR (CDCl3, 400 MHz) δ 8.23 (s, 1H), 8.10 (d, J = 3.0 Hz, 1H), 8.05 (d, J = 1.7 Hz, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.71 (dd, J = 8.6, 1.8 Hz, 1H), 7.49 (d, J = 3.0, 1H), 6.56 (br s, 2H), 5.31 (s, 1H), 4.23 (s, 3H), 3.89 (s, 6H), 3.74 (s, 3H), 3.63 (s, 3H), 2.31–2.24 (m, 1H), 2.02–1.96 (m, 1H), 1.97 (s, 6H), 1.78–1.68 (m, 2H).", "Found: [M + H] = 574.6.", "Followed by isomer B, (7%), foamy white solid.", "1H NMR (CDCl3, 400 MHz) δ 8.76 (s, 1H), 8.01 (d, J = 1.7 Hz, 1H), 7.70–7.60 (m, 3H), 7.48 (d, J = 3.0 Hz, 1H), 6.55 (br s, 2H), 5.30 (s, 1H), 4.04 (s, 3H), 3.88 (s, 3H), 3.83 (s, 6H), 3.72 (s, 3H), 2.32–2.25 (m, 1H), 2.05 (s, 6H), 2.04–2.00 (m, 1H), 1.88–1.80 (m, 1H), 1.71–1.64 (m, 1H).", "Found: [M + H] = 574.6.", "The mixtures were resolved into their four optical isomers using preparative supercritical fluid HPLC at BioDuro LLC (Beijing).", "The data in Table 1 are for the most active R,S-diastereomers.", "The other 6-cyano compounds in Table I were prepared and purified similarly." ]
[]
Characterization of the innate stimulatory capacity of plant-derived virus-like particles bearing influenza hemagglutinin
Cell-mediated immunity is an important component of immediate and long-term anti-viral protection. Dendritic cells (DCs) are essential for the induction of cell-mediated immunity by instructing the activation and differentiation of antigen-specific T cell responses. Activated DCs that express co-stimulatory molecules and pro-inflammatory cytokines are necessary to promote the development of type 1 immune responses required for viral control. Here we report that plant-derived virus-like particles (VLPs) bearing influenza hemagglutinins (HA) directly stimulate mouse and human DCs. DCs exposed to H1- and, to a lesser extent, H5-VLPs in vitro rapidly express co-stimulatory molecules and produce pro-inflammatory cytokines including IL-12, IL-6 and TNFα. Furthermore, these VLPs support the activation and differentiation of antigen-specific T cell responses. Mechanistically, H1-VLPs stimulate the activation of kinases typically activated downstream of pattern recognition receptors including AKT, p38, and p42/44 ERK. In vivo, immunization with plant-derived VLPs induce the accumulation of both cDC1s and cDC2 in the draining lymph node and a corresponding increase in T and B cells. VLPs devoid of HA protein activate DCs, suggesting they are intrinsically immunostimulatory. Together, the results demonstrate that these candidate plant-derived VLP vaccines have an inherent and direct stimulatory effect on DCs and can enhance the ability of DCs to promote Type 1 immune responses.
[ "BIOC", "IMMU", "MEDI", "VETE" ]
[ "Dendritic cell", "Influenza", "Innate immunity", "Plant-derived vaccine", "Type 1 response", "Virus-like particle" ]
[ "1313", "2400", "2725", "2739", "3400" ]
[ "Influenza viruses infect the human respiratory tract where they cause a spectrum of pathologies ranging from mild illness to life-threatening disease [1].", "Because these viruses mutate constantly in both humans and reservoir species, yearly vaccination remains the most effective method of prevention and containment [2].", "Traditionally, hemagglutination-inhibition (HI) antibody response was used as a surrogate marker correlating with vaccine efficacy [3].", "However, the cellular response (CD4+ and CD8+ T cells) can control infection in both animal models and humans [4–7] and may be particularly important in elderly subjects who often mount weak or no antibody responses to standard, split virus vaccines [8,9].", "CD4+ T cells contribute to the control of viral infections by sustaining CD8+ T cell responses, supporting B cell functions and supporting long-term T and B cell memory [10,11].", "Cross-reactive CD4+ T cells that recognize conserved epitopes from the HA protein may therefore recognize drifted strains and participate in cross-protection [12].", "Although the currently available vaccines have proved to be safe and to provide some degree of protection (i.e. efficacy averaging 40–60% in young healthy adult populations if vaccine matches circulating strain), it is increasingly clear that new vaccines capable of inducing broader and more durable immune responses (i.e. humoral, cellular, cross-reactive) are needed to improve vaccine efficacy [13,14].", "Virus-like particle (VLP) vaccines for influenza are self-assembling protein structures that contain viral antigens embedded in a lipid bilayer but lack any genetic material [15,16].", "Both pre-clinical (e.g. mouse, ferrets) and clinical studies have demonstrated that plant-derived VLP vaccine candidates bearing influenza hemagglutinin (HA) proteins can generate long-lived, cross-reactive HA-specific humoral responses as well as poly-functional CD4+ T cell responses to both seasonal and avian strains [17–20].", "The mechanisms by which these vaccines induce this broader immune response are not fully understood.", "Because dendritic cells (DCs) are regarded as the principal antigen-presenting cell to both stimulate and direct T cell immunity [21] and the activation of DCs is a necessary step in eliciting effective cellular responses against viruses, we examined whether these plant-derived VLPs had intrinsic immunostimulatory potential.", "We tested two VLP formulations; H1-VLP (Influenza A/California/04/09) and H5-VLP (A/Indonesia/05/2005 strain) [16] with and without adjuvant.", "We observed that unadjuvanted H1-VLPs, and to a lesser extent H5-VLPs, stimulate both murine and human DCs to express co-stimulatory molecules and secrete pro-inflammatory cytokines including the T helper 1 (Th1) promoting cytokine IL-12.", "DCs stimulated with H1-VLP, and to a lesser extent H5-VLP, promoted the proliferation, differentiation and effector function of CD4+ T cells.", "Exposure of murine DCs to H1-VLP also promoted cross-presentation of a simultaneously-administered model antigen ovalbumin (OVA) and CD8+ T cell effector differentiation.", "Mechanistically, we found that the plant-derived VLPs stimulated the activation of kinase cascades that are typically associated with pattern recognition receptor (PRR) ligation and necessary to drive the transcriptional program that supports pro-inflammatory DC function.", "When injected in vivo, plant-derived VLPs stimulated the accumulation of DCs, T cells and B cells in the lymph node.", "These results demonstrate that plant-derived VLPs can directly stimulate DCs to acquire an activation phenotype that supports the development of Type 1 cellular immune responses.", "Female wild-type C57BL/6 or BALB/c mice were purchased from Charles Rivers Laboratories at 6–8 weeks of age (Montreal, QC Canada).", "OTI and OTII transgenic mice were purchased from the Jackson Laboratory (Bar Harbor, ME USA) and bred in house.", "Animals were maintained in a specific pathogen-free environment.", "All experiments were conducted following the guidelines of the Canadian Council of Animal Care, as approved by the animal care committee of McGill University.", "VLP vaccines were obtained from Medicago Inc. (Quebec City, QC) and produced as previously described [15,17].", "H1-VLPs contain HA from A/California/7/09 (H1N1) and H5-VLPs contain HA from A/Indonesia/5/05 (H5N1).", "A detailed characterization of the morphology and biochemical content is reported here [15,22].", "In some experiments, ‘empty’ VLPs (eVLP) were used as an additional control.", "These nanoparticles are generated from plants transiently transfected with ‘null’ A. tumifasciens.", "They are approximately the same size as the HA-bearing VLPs and have very similar lipid composition (data not shown).", "However, since the influenza HA trimers drive budding of the VLPs, the down-stream processes used to isolate and purify the eVLPs are not identical to those used to generate the HA-VLPs.", "Bone marrow was extracted from the femur and tibia of C57BL/6 or BALB/c mice.", "Briefly, bone marrow cells were differentiated to DCs in the presence of GM-CSF (20 ng/ml) in filtered complete DC media (cDCM: RPMI containing 10% FCS, 100 U/ml penicillin, 100 µg/ml of streptomycin, 50 µM β-Mercaptoethanol, 2 mM L-Glutamine (Wisent) and 1% non-essential amino acids) (Life Technologies/Thermofisher) on uncoated TC plates (Falcon) as described [23].", "On day 8–10 of culture, immature DCs were harvested and stimulated in 96-well non-TC plates with either 10 ng/mL Escherichia coli LPS (serotype 0111:B4, Sigma), 5 µg/mL of H1-VLP, 5 µg/mL of H5-VLP (Medicago Inc, Ste Foy, QC: see above), 5 µg/ml of Alhydrogel (alum) (Brenntag/Cedarlane), VLPs with alum at 1:1 ratio, or 8.58 EU/mL of Agrobacterium-LPS (Agro-LPS) (List Biologicals Laboratories USA), 5 µg/ml OK432 (generously provided by Chugai Pharmaceuticals Co.", "INC., Japan) or as otherwise noted.", "After 18 h of incubation at 37 °C, DCs were collected and analyzed by flow cytometry (FACS CANTOII, FORTESSA, Cell Vision Flow Cytometry Facility, McGill University).", "After written informed consent, whole blood was obtained, peripheral blood mononuclear cells (PBMCs) were collected from the venous blood of healthy young adults (18–30 years old) at the MUHC Vaccine Study Center (Pierrefonds, QC).", "PBMCs were separated on a Ficoll density gradient and CD14+ monocytes were positively selected (Stem Cell Technologies, B.C. Canada), resuspended in cDCM and then plated in 6-well tissue culture plates (Falcon) at a concentration of 1 million cells/mL.", "Differentiation into moDCs was completed using human GM-CSF (100 ng/mL) and human IL-4 (50 ng/mL) (Peprotech).", "Human moDCs were fed with 1 ml of fresh media on day 3 and 6 of culture.", "On day 7, moDCs were harvested and activated with either 10 ng/ml LPS, 5 µg/mL of H1-VLP, 5 µg/mL of H5-VLP, 5 µg/mL of Alhydrogel (alum), VLPs with alum at 1:1 ratio, or 8.58 EU/mL of Agro-LPS.", "After 30 min of incubation at 37 °C, moDCs were collected, protein expression was analyzed by Western.", "Otherwise, after 18 h of incubation at 37 °C, moDCs were collected and analyzed by flow cytometry.", "These studies were approved by the Research Ethics Board of the McGill University Health Centre.", "Cell culture supernatants from DCs stimulated with LPS, VLPs or VLPs with alum, were collected after 18 h and quantified for IL-12p70, IL-12p40, IL-6, IL-10, IL-1β and TNFα by enzyme-linked immunosorbent assay (ELISA) kits (eBioscience/Affymetrix) according to the manufacturer’s instructions.", "On day 10 of culture, BMDCs from C57BL/6 mice were harvested and cultured for 6 h with whole ovalbumin (OVA) protein (in house) (0.001 mg, 0.01 mg or 0.1 mg per well) with either Agro-LPS (0.5 ng/mL), H1-VLP (3 µg/mL) or H5-VLP (3 µg/mL).", "T cells from OTII or OTI transgenic mice were isolated from the spleen and lymph nodes using CD4+ and CD8+ positive selection kits (Stem cell), respectively.", "Sorted T cells were then co-cultured with the activated DCs at a 1:10 ratio of DCs to T cells.", "Prior to co-culture, half of the isolated T cells were additionally stained with a violet proliferation dye (eBioscience/Affymetrix) to examine proliferation.", "On day 3–5, T cells were collected and analyzed by flow cytometry.", "Surface staining: Following activation, live DCs or T cells were stained with fluorochrome-conjugated antibodies and acquired on a FACSCANTO II or FACSFortessa flow cytometer (BD Pharmingen).", "Analysis was performed using FlowJo software (Tree Star, Oregon, USA).", "See figure legends for analysis gates.", "Intracellular staining: T cells were stimulated with 3 µg/mL Brefeldin A (eBioscience/Affymetrix), 0.5 µg/ml ionomycin (Sigma) and 0.05 µg/ml PMA (Sigma) for 4 h prior to their harvest.", "T cells were stained with fluorochrome-conjugated antibodies against surface markers, for 60 min at 4 °C in the dark.", "T cells were then fixed and permeabilized with Foxp3 fix/perm buffer from eBioscience/Affymetrix for 40 min.", "Intracellular proteins were stained overnight at 4 °C.", "See figure legends for analysis gates.", "All conjugated antibodies used in flow cytometry analyses were purchased from eBioscience/Affymetrix: Anti-human CD11c-Percp710 3.9; anti-human CD86-PeCy7 IT2.2; anti-human CD83-PE HB15e; anti-human CD14-FITC 61D3; anti-mouse CD11c-e450 N418; anti-mouse CD11c-APC-780N418; anti-mouse CD86-FITC GL1; anti-mouse CD86-PeCy7 GL1; anti-mouse CD40-APC 1C10; anti-mouse MHCII-AF700 M5/114.15.2; anti-mouse MHCII-APC-780 M5/114.15.2; anti-mouse MHCII-PE M5/114.5.2; anti-mouse Flt3 (CD135)-PE A2F10; anti-mouse CD115-AlexaFluor488 AFS98; anti-mouse CD62L-APC MEL-14; anti-mouse CD4-APC-780 RM4-5; anti-mouse CD4-e450 RM4-5; anti-mouse CD8-APC-780 53–6.7; anti-mouse CD8-e450 53–6.7; anti-mouse CD44-e450 IM7 anti-mouse CD44-PercpCy5.5 IM7; anti-mouse CD44-PeCy7 IM7; anti-mouse CD25-PE PC61.5; anti-mouse CD25-PeCy7 PC61.5; anti-mouse XCR1-BV650 ZET; anti-mouse CD3- FITC 17A2; anti-mouse CD3- Biotin 17A2; streptavidin V500 (BDHorizon 561419); anti-mouse B220-APC RA3-6B2; anti-mouse B220-FITC RA3-6B2; anti-mouse Ly6G- FITC 1A8; anti-mouse NK1.1-FITC PK136; anti-mouse CD69 FITC H1.2F3; anti-mouse CD172a-PeCy7 P84; anti-mouse T-bet-PeCy7 eBio4B10; anti-mouse IFNγ-FITC XMG1.2; anti-mouse IFNγ-APC XMG1.2; anti-mouse Granzyme B-FITC NGZB; anti-mouse IL-2-PE JES6-5H4; anti-mouse CD16/CD32 FcR Block 93; 7AAD Viability Staining Solution; Fixable Viability APC-780; Cell Proliferation dye efluor 450; 123count eBeads Counting Beads.", "RNA from BMDCs was isolated using Trizol (Life Technologies, ThermoFisher), cDNA were synthesized using random primers and multiscribe reverse transcriptase (Applied Biosystems).", "Real-time qRT-PCR analysis was performed using SYBR green (FroggaBio).", "Relative expression of IL-12p40 and pro-IL-1β to HPRT was calculated using the 2-ΔΔCt method.", "Real-time primers for HPRT, IL-12p40 and pro-IL-1β were designed by Primer3 and PrimerBlast.", "The primer sequences used are listed below: Hprt Fwd: CTC CGC CGG CTT CCT CCT CA Rev:ACC TGG TTC ATC ATC GCT AAT C IL-12p40 Fwd: CTG GAG CAC TCC CCA TTC CT Rev: CGC CTT TGC ATT GGA CTT CG Il-1β Fwd: GCC ACC TTT TGA CAG TGA TGA G Rev:AAG GTC CAC GGG AAA GAC AC.", "5 μg of VLPs, 0.1 μg of LPS, or PBS were injected into each footpad of female wild-type C57BL/6 mice at 6–8 weeks of age.", "After 24–36 h, the draining popliteal lymph nodes were digested and stained with antibodies for flow cytometry.", "Statistical analysis on murine data was performed using one-way ANOVA using GraphPad Prism.", "Statistical analysis on human data was performed using the Friedman test (nonparametric test).", "Single stars above bars indicate statistically significant difference relative to control.", "Lines indicate where direct comparisons were made between groups.", "Differences were considered significant at p < 0.05.", "To examine whether plant-derived VLPs bearing influenza HA proteins could directly activate DCs, BM-derived DCs from both C57BL/6 and BALB/c mouse strains were stimulated with H1-VLPs or H5-VLPs and examined for expression of co-stimulatory molecules and cytokine production.", "LPS from E. coli was used as a control in all experiments.", "DCs exposed to H1-VLPs increased the expression of co-stimulatory molecules CD86 and CD40, markers of proinflammatory DC activation which were further increased by alum (Fig. 1A and B).", "H5-VLPs were less stimulatory than H1-VLPs although their activity was also enhanced by alum (Fig. 1A and B).", "H1-VLPs induced the secretion of pro-inflammatory cytokines IL-12p40, IL-6, and TNFα and levels were enhanced with the addition of alum (Fig. 1C).", "In contrast, pro-inflammatory cytokine secretion in response to H5-VLPs was generally weak and was not significantly enhanced by alum (Fig. 1C).", "IL-10 secretion, which can be induced in murine DCs by LPS, also increased in response to H1-VLP stimulation, but not H5-VLPs.", "Increasing the concentration of H5-VLPs improved their immunostimulatory capacity, but cytokine levels were never comparable to those stimulated by H1-VLPs (Fig. S1).", "Together, these findings demonstrate that H1-containing VLPs, and to a lesser extent H5-VLPs, can directly stimulate murine DCs to acquire a pro-inflammatory phenotype.", "Since these VLPs are produced in plants using a Gram-negative bacterial vector (Agrobacterium tumefaciens), small amounts of Agro-LPS are found in VLP preparations (range ∼312–1610 EU/mL in Drug Substance).", "Thus, the possible effect of Agro-LPS on murine DC activation was evaluated.", "DCs from young mice were stimulated for 18 h with 1.72 ng/ml (8.58 EU/ml) of Agro-LPS (amount found in 5 µg/ml of H1-VLP) and the induction of activation marker expression and cytokine production was compared to 1.72 ng/ml of E. coli LPS (serotype 0111:B4).", "DCs stimulated with Agro-LPS alone expressed CD86 and CD40 levels almost identical to the unstimulated control (Fig. S2).", "The addition of alum with Agro-LPS did not increase CD86 or CD40 expression (data not shown).", "We also examined whether the VLPs could similarly stimulate human DCs.", "Human monocyte-derived DCs (moDCs) were stimulated with either 5 µg/mL of H1- or H5-VLP for 18 h with or without alum.", "Following stimulation, the activation status was evaluated using CD83 and CD86 expression as well as cytokine secretion.", "We observed that both H1- and H5-VLPs significantly induced the expression of CD83 and CD86 compared to the negative control and expression was not increased by alum (Fig. 1D).", "H1-VLP (with or without alum) significantly increased the secretion of IL-6, TNFα and IL-10 (Fig. 1E).", "Although there was a trend observed in some experiments for increased cytokine secretion with H5-VLP stimulation, significance was not reached as compared to the control cultures (Fig. 1E).", "Neither alum nor Agro-LPS on their own induced up-regulation of activation marker expression (Fig. 1D) and did not stimulate any significant cytokine secretion (Fig. 1E).", "With respect to IL-6, IL-10, and TNFα production and CD83 expression, H1-VLPs were generally more stimulatory than H5-VLPs.", "However, differences between H1- and H5-VLPs were not observed for CD86 upregulation and IL-12 production.", "The promotion of effective Type 1 immunity (including both Th1 CD4+ and CD8+ cytotoxic T lymphocyte responses (CTL) is necessary for optimal protection from most viruses and is thus an important criterion for evaluating novel influenza vaccine candidates.", "Because the plant-derived HA-VLPs stimulated the production of Type 1 immunity promoting cytokines by both murine and human DCs (IL-12), we examined whether the VLP-stimulated DCs could promote the development of Th1 responses.", "DCs from C57BL/6 mice were exposed to H1- and H5-VLPs for 6 h in the presence of whole ovalbumin (OVA) and subsequently co-cultured with OVA-specific TCR transgenic CD4+ T cells from OTII transgenic mice.", "CD4+ T cell responses were evaluated by flow cytometry based on the extent of proliferation, activation marker expression (CD44 and CD25), Th1 transcription factor (T-bet) up-regulation and effector cytokine secretion (IFNγ).", "We observed that both H1- and H5-VLP-stimulated DCs promoted the activation (measured by CD44 expression) and proliferation of CD4+ T cells (Fig. 2A and B).", "Both H1- and H5-VLP-stimulated DCs promoted a significant increase in T-bet expression compared to OVA-stimulated DCs, highlighting the promotion of a Th1 program (Fig. 2C).", "Consistently, the percentage of activated CD4+ T cells producing IFNγ (CD44+IFNγ+) was also increased by H1- and H5-stimulated DCs, indicating that both VLPs can induce Th1 effector function (Fig. 2D).", "Although the increase in Th1 differentiation by H5-stimulated DCs was routinely observed, statistical significance was not achieved in every experiment.", "Overall, the CD4+ T cell responses promoted by DCs exposed to H1-VLPs were stronger and more consistent than those observed following H5-VLP stimulation, similar to the differences observed in murine DC activation.", "VLPs are extracellular particles therefore antigen must be cross-presented on MHCI in order to stimulate antigen-specific CD8+ T cell responses.", "Thus, the ability of VLP-stimulated DCs to cross-present antigen and stimulate CD8+ T cells was also evaluated.", "DCs from C57BL/6 mice were stimulated for 6 h with H1- or H5-VLPs in the presence of OVA and subsequently co-cultured with OVA-specific TCR transgenic CD8+ T cells from OTI transgenic mice.", "Activation marker expression (CD44), T-bet upregulation and effector molecule secretion (IL-2, IFNγ and granzyme B) were examined.", "The percentage of activated CD8+ T cells producing IL-2 (CD44+IL-2+) was significantly increased with H1-, but not H5-stimulated DCs compared to OVA alone (Fig. 3A).", "A trend for H1- and H5-VLP stimulated DCs to activate CD8+ T cells to produce IFNγ (CD44+IFNγ+) was also observed (Fig. 3A).", "The expression of T-bet and granzyme B, as well as the percentage of activated CD8+ T cells producing T-bet and granzyme B, were significantly increased with H1-VLP-stimulated DCs compared to negative controls (Fig. 3B).", "Again, the changes induced with H5-VLP-activated DCs trended in the same direction but did not reach significance compared to the OVA control (Fig. 3B).", "Together, these findings suggest that H1-VLP, and to some extent H5-VLP, can stimulate DCs to cross-present antigen to CD8+ T cells and promote antigen-specific CD8+ T cell responses.", "To begin to understand the mechanisms by which the plant-derived VLPs mediate DC activation, we first determined whether they stimulate a priming signal (much like pattern recognition receptor ligation), or whether their particulate nature activated mechanisms associated with inflammasome activation.", "In murine, but not human DCs, IL-1β secretion is dependent on two signals; a priming signal that leads to Il1b gene transcription and then a second signal mediated by the inflammasome that results in IL-1β protein cleavage and secretion.", "We found that exposure of DCs to the H1-VLP increased both Il1b and Il12b transcription.", "(Fig. 4A).", "Although H5-VLP stimulated significant transcription of pro-IL-1β compared to the control, the levels were similar to Agro-LPS and were less than H1-VLP (Fig. 4A).", "However, VLPs on their own could not induce the secretion of active IL-1β and required the addition of alum, which stimulates inflammasome activation, for significant secretion of IL-1β (Fig. 4B).", "Together these results indicate that the plant-derived VLPs, in particular H1-VLP, acts as signal 1 to prime the activation of DCs and that, as expected, alum then acts as signal 2 to trigger inflammasome assembly, resulting in pro-IL-1β cleavage.", "Human moDCs stimulated with H1-VLP or LPS were able to produce significant amounts of IL-1β in the absence of alum (Fig. 4C).", "This result is consistent with previous reports that human monocytes have a path to IL-1β production distinct from mouse cells that does not require a second signal [24–26].", "Since H1-VLP was shown to act as a strong priming signal for DC activation, we determined which intracellular signaling pathways H1-VLP might activate to induce the expression of inflammatory cytokines and co-stimulatory molecules.", "DCs were exposed to H1- or H5-VLPs and the activation of signaling pathways known to be important for DC activation were examined.", "As expected, LPS strongly activated many kinases known to be downstream of PRRs (Fig. 4D).", "In DCs, H1-VLP induced the phosphorylation and thus activation of AKT, p42/44 and p38, but only minimally activated JNK (Fig. 4D).", "H5-VLP induced activation of AKT, p38 and to a lesser extent p42/44 but all were less strongly induced than with H1-VLP exposure.", "Similar results were obtained from human monocyte-derived DCs (Fig. 4E).", "Together, these results demonstrate that H1-VLP, and to a lesser extent H5-VLPs, directly activate kinase cascades that lead to the activation of DCs with their production of pro-inflammatory cytokines and ability to stimulate T cell responses.", "To determine whether H1 and H5-VLPs stimulated DCs in vivo, the footpads of mice were injected with H1-VLP or H5-VLP.", "Injection of the VLPs led to a trend for increased numbers of total DCs, conventional DC1s (cDC1s; XCR1+) and cDC2s (CD172a+) in the draining lymph nodes 36 h following immunization (Fig. 5A).", "Consistent with DC activation and lymphocyte recruitment, we found a significant increase in the total number of B cells and T cells (Fig. 5B), some of which expressed markers of activation (CD69+ and CD44+ respectively).", "These experiments demonstrate that H1- and H5-containing VLPs have inherent immunostimulatory activity in vivo.", "Influenza HA proteins are not thought to have any intrinsic immunostimulatory properties on their own.", "Therefore, we assessed the ability of the VLPs devoid of HA proteins to stimulate murine and human DCs.", "Biochemical analyses of the VLPs reveal the presence of low levels of residual host cell proteins and glucosylceramides associated with plant lipid rafts [15].", "Empty VLPs (eVLP), the VLP without the HA proteins, stimulated co-stimulatory molecule expression and cytokine production by DCs (Figs. 6A, B and S3).", "LPS and OK432 bacterial vaccine were used as controls.", "DCs stimulated with eVLPs also induced in vitro T cell responses (Fig. 6C), demonstrating enhanced function of the stimulated DCs.", "As in Fig. 5, eVLPs were injected into the footpads of mice and compared to PBS controls.", "eVLPs induced substantial DC accumulation in the draining LN indicating that the eVLPs were stimulatory in vivo.", "The proportion of cDC subsets were not significantly different upon eVLP immunization, with an increase in both cDC1s and cDC2s (Fig. 6D).", "To determine whether eVLPs also stimulate human DCs, moDCs were stimulated with eVLP.", "eVLPs induced the activation of p38 and p44/42 but minimal activation of AKT (Fig. 6E).", "High serum antibody titers can protect against many viral infections, including influenza.", "Although antibodies that mediate hemagglutination inhibition (HI) have long been used as surrogates to license influenza vaccines [3], there is increasing interest in formulations that can induce more effective and broader immunity.", "Plant-derived VLP vaccines bearing the influenza virus HA protein have recently been shown to generate broad and cross-reactive cellular and humoral immune responses in both animal models [16] and clinical trials [17].", "In animal challenge studies with highly pathogenic avian strains, some of the VLP-vaccinated animals are protected despite very low or even undetectable antibody titers [16,18].", "To better understand the mechanism(s) that underlie the broader pattern of immunity induced by the plant-derived vaccines, we evaluated the innate stimulatory potential of H1- and H5-containing VLPs and eVLPs devoid of any HA protein, in DCs isolated from mice and humans.", "We found that the plant-derived VLPs can directly activate DCs and stimulate subsequent CD4+ and CD8+ T cell responses.", "In vitro, H1-VLPs were generally superior to H5-VLPs in stimulating these responses.", "However in vivo, H1 and H5-VLPs showed similar ability to stimulate DC migration to the draining LN and lymphocyte recruitment.", "Activated, or mature, DCs play a pivotal role in the induction of anti-viral immunity.", "Activated DCs express increased levels of co-stimulatory molecules including CD86 and CD40, antigen presenting machinery (MHC molecules), and cytokines such as Type 1 interferons and IL-12.", "The latter two are necessary for the development and function of CD4+ Th1 cells, CD8+ cytolytic cells and NK cells.", "Our data demonstrate that plant-derived VLPs, with and without HA protein, can intrinsically stimulate DCs.", "In vitro, plant-based VLPs alone were able to stimulate DCs to induce the activation and proliferation of CD4+ T cells and promote Th1 differentiation and effector function.", "Even though the H5-VLPs were less effective than their H1 counterparts in activating murine DCs, they still induced readily detectable and significant antigen-specific Th1 CD4+ T cell responses.", "H1-VLP- and eVLP-stimulated DCs also promoted cross presentation of the model antigen OVA to stimulate antigen-specific CD8+ T cell activation, proliferation and effector function.", "H5-VLP-stimulated DCs showed a similar trend for increased CD8+ T cell responses but these changes did not reach statistical significance.", "Overall, these animal data are consistent with early studies of T cell responses in humans with the same H1- and H5-VLPs, in which strong CD4+ and more limited CD8+ T cell responses to the H1 antigen were detectable up to 6 months after H1-VLP vaccination while only CD4+ T cell responses were seen in the H5-VLP vaccine recipients [17].", "Furthermore, the H5-VLP vaccine candidate needed to be adjuvanted in order to see significant CD4+ T cell responses, while the H1-VLP vaccine did not [17].", "Although our mouse data suggest that H1- and H5-VLPs that differ in only their HA cargo have different capacity to stimulate DCs, the differences in apparent T cell immunogenicity of these two formulations in humans may be attributable, at least in part, to prior experience of most human study subjects with H1 but not H5 antigens.", "However, prior exposure would not affect the studies reported here.", "Although our principal interest was in the intrinsic stimulatory capacity of the plant-derived VLPs, alum was included in many of our experiments because of its widespread use.", "Alum remains the most commonly used adjuvant in human vaccines, even though aluminum-containing adjuvants have long been thought of as promoting Th2 responses [27,28].", "In pre-clinical studies (ferret) and early clinical trials with the plant-derived VLP vaccines, alum had shown unexpected promise in eliciting T cell responses and protection from challenge [17,29].", "In the current work, we focused on testing the combined effects of alum and VLPs on DC activation.", "Similar to previous findings [30,31], we observed that alum by itself does not activate murine or human DCs to any significant degree.", "However, when alum was combined with H1-VLP, there was a marked increase in activation marker expression and cytokine secretion from murine DCs compared to H1-VLP alone.", "Alum was less effective at promoting enhanced responses to H5-VLPs.", "In combination with H1-VLPs, alum drove increased secretion of Th1-promoting cytokines such as IL-12p70 and IL-12p40.", "This finding suggests an unexpected feature of combined VLP plus alum stimulation and demonstrates that alum can play a role in promoting Th1 responses in certain contexts.", "Because particles are known to activate the inflammasome, a known target of alum [32], we examined whether the VLPs had any impact on inflammasome activation.", "Although IL-1β secretion by DCs was only detected when H1-VLP and alum were used together, Il1b transcription occurred following exposure of DCs to H1-VLP alone.", "These findings suggest that the plant-derived VLPs serve as a priming rather than an activating signal for the inflammasome.", "When primed by the plant-derived VLP, alum can then support greater Th1 responses.", "This observation raises the possibility that aluminum-containing adjuvants, in some circumstances, may stimulate immune responses beyond promoting antibody production.", "In in vitro assays used to evaluate the innate immune-stimulatory potential of H1- and H5-VLPs on murine DCs, we found that H1-VLPs were more stimulatory than H5-VLPs.", "For human cells, even though there were fewer differences in the ability of H1-VLPs and H5-VLPs to activate DCs, H1-VLPs still stimulated greater cytokine production.", "Interestingly, in vivo, the ability of H1- and H5-VLPs to stimulate in vivo the recruitment of DCs, B cells, and T cells to the draining lymph node was equivalent.", "The observations that the H5-VLP effectively elicits an immune response in vivo are in accordance with the findings obtained during clinical trials [17].", "Considering that both these VLPs are produced using the same expression platform, it was unexpected to observe such a striking difference between their innate stimulatory capacities.", "Detailed morphological analyses of H1- and H5- containing VLPs by cryostatic electron microscopy did not reveal differences between the two types of VLPs [22].", "Even when much higher doses of H5-VLPs were used to stimulate murine DCs, the expression of activation markers never reached the same level as with H1-VLP in vitro.", "These differences were also evident in the activation of signaling pathways that are essential for the development of a pro-inflammatory phenotype capable of promoting Type 1 immune responses.", "Interestingly DCs activated by eVLPs resemble those activated by H1-VLPs.", "Further work is needed to determine the mechanism behind the differential activation of DCs by empty VLPs, H1 and H5-containing VLPs.", "Such intrinsic stimulatory capacity could be the result of the non-HA components of the VLPs (ie: plant-origin lipids/glycolipids) or to the physical structure of the VLPs themselves.", "We also cannot definitively rule out a role for residual Agrobacterium- or plant-derived proteins, particularly in response to eVLP.", "Although >98.5% of the protein in the VLP vaccine is viral HA, the eVLPs cannot be produced in the same way and have slightly higher residual protein content.", "None of the proteins identified to date in either the VLP vaccine or the eVLPs is known to have immunostimulatory activity.", "Recent work suggests that the initial interactions of plant-derived VLPs with antigen presenting cells recapitulate those of intact virions [33].", "However, the different binding specificities of the hemagglutinin trimers themselves (α2,6 and α2,3 sialic acid respectively) may result in distinct VLP interactions with immune cells [34].", "Injection in vivo of eVLPs, H1- and H5-VLPs resulted in an increase in cDC1 and cDC2 populations in the draining lymph node.", "Both cDC1s and cDC2s are capable of stimulating antigen-specific CD4+ T cell responses.", "While XCR1+ cDC1s are more widely known to be able to cross-present antigen and stimulate CD8+ T cells, cDC2s can also stimulate cross-presentation in some contexts, in particular for human DCs [35].", "These findings suggest that plant-derived VLPs may stimulate the activation of multiple DC types to induce protective immunity.", "Future studies will assess the contribution of cDC1s and cDC2s to the induction of CD4+ and CD8+ T cell immune responses in vivo and the generation of protective immunity.", "In this study, we demonstrate the intrinsic stimulatory potential of plant-derived VLPs.", "These VLP vaccine candidates effectively activate murine and human DCs in vitro.", "The combination of alum and plant-derived VLPs had the unexpected effect of enhancing the production of Th1-promoting cytokines by DCs.", "The cooperation between alum and plant-derived VLPs raises the possibility that this well-established adjuvant could be re-purposed to drive broader immune responses in certain circumstances.", "Overall, the unusual innate stimulatory activities of the plant-derived VLPs may prove to be useful in generating more effective vaccines for influenza as well as other targets for which both humoral and cellular responses are important.", "K.H., C.K., N.L., N.C. and B.W. conceived and designed the study.", "K.H., C.K. and SY.W.", "prepared the manuscript.", "K.H., SY.W.", "H.G. and B.H. performed experiments." ]
[ "Plant-derived VLPs stimulate murine and human DCs.", "VLPs promote CD4+ and CD8+ T cell effector function and cross-presentation of antigen.", "VLPs activate signaling cascades in murine and human DCs.", "VLPs stimulate the accumulation of DCs, B cells, and T cells in lymph nodes in vivo." ]
Exploring social preferences for ecosystem services of multifunctional agriculture across policy scenarios
Multifunctional agroecosystems are the result of complex adaptive interactions between humans and nature where trade-offs between food production and other ecosystem services are key. Our objective is to explore the social preferences for ecosystem services, and the associated willingness to pay, in three multifunctional agroecosystem in Europe (Mediterranean, Atlantic, Alpine) under alternative agrienvironmental policy scenarios. We use the same methodology (a choice experiment including equivalent attributes and levels) to rank and estimate the economic value of provisioning, regulating, supporting and cultural ecosystem services. We define the scenarios (current situation, abandonment and enhanced management) in biophysical terms to elucidate changing relations between social perception and level of delivery of ecosystem services. We derive some lessons. i) Value of ES: biodiversity and regulating ecosystem services always produce welfare gains; people, however, perceive trade-offs between delivery of agricultural landscapes and quality food products. Nevertheless, preferences are heterogeneous and vary across regions, scenarios and ES. ii) Policymaking: society's willingness to pay for the delivery of ecosystem service exceeds largely the current level of public support. Moreover, further abandonment and intensification of agriculture is clearly rejected by the public. iii) Methodological: monetary valuation is context dependent and extrapolation of economic values can be misleading.
[ "AGRI", "ENVI", "SOCI" ]
[ "Abandonment", "Agrienvironmental policy", "Economic values", "Intensification", "Social-ecological systems", "Trade-offs" ]
[ "1101", "2303", "2306", "2308", "2309", "3305" ]
[ "Agriculture in marginal lands, such as mountains or arid areas, constitutes a paradigmatic social-ecological system.", "These landscapes are the result of complex adaptive interactions between humans and nature that have occurred across spatial and temporal scales (Liu et al., 2007).", "For example, through the practice of the transhumance of livestock across seasons and regions to optimize the utilization of natural resources (Oteros-Rozas et al., 2013).", "Agriculture is multifunctional in these landscapes, delivering multiple ecosystem services (ES) to humans, both provisioning ES such as food products, and regulating, supporting and cultural ES, which normally constitute public goods.", "In Europe, the geographical distribution of multifunctional agroecosystems occupy 41.2% of the total utilized agricultural area (Schwaiger et al., 2012).", "However, these agroecosystems are in a continuous, often concurrent, process of abandonment and intensification, due to general socio-economic trends driven by the increasing opportunity cost of labor and changes in the relative prices of inputs and outputs (Strijker, 2005).", "Agricultural policies, specifically the European Common Agricultural Policy (CAP), recognizes the multifunctionality of particular agricultural systems and aim, at least in its spirit, at increasing the provision of public goods.", "However, the CAP has so far failed to do so (Erjavec and Erjavec, 2015; Pe’er et al., 2019), due to distribution of budgetary resources, the choice of instruments and the design and implementation of policy measures (Cooper et al., 2009).", "Scenario analysis can help exploring ex-ante the outcomes of alternative policy settings in terms of provision of public goods by defining potential but realistic trends in agricultural land use at European scale.", "The existence of trade-offs between the provision of private and public goods (typically between food production and non-marketable ES) is a key feature in multifunctional agroecosystems (Kovács et al., 2015; Ripoll-Bosch et al., 2012).", "They are traditionally considered as low-input/low-output, implying a reduced use of off-farm resources and productivity.", "In contrast, these agroecosystems can provide a wide range of benefits and services to society.", "Nevertheless, since they have no market price, farmers have little or no incentives to produce them regardless of their high societal demand (Cooper et al., 2009).", "To correct this situation, equitable and effective agrienvironmental policies need to be designed.", "On the production side, there is a need to measure in biophysical terms the range of services that farmers provide, for example by analyzing the effect that particular agricultural practices have on biodiversity (Rodríguez-Ortega et al., 2018).", "On the demand side, it is necessary to understand how the public perceive the relationships between agriculture and the environment (Bernués et al., 2016), identify the most demanded ES that should guide policy targets, and pay famers for the delivery of these ES.", "Despite economic valuation of ES is controversial (Gómez-Baggethun and Ruiz-Pérez, 2011; Laurans and Mermet, 2014), when combined with socio-cultural and biophysical approaches, it can help in prioritizing conservation objectives, quantifying trade-offs among different management options, or estimating the amount of money that society is willing to pay for ES.", "Despite the literature on economic valuation is rapidly increasing, a number of challenges remain.", "First, location-specific ecological and social-economic factors determine the ES delivery and value (Randall, 2002).", "Therefore, comparing economic value between places or aggregating this value to larger scales may become problematic.", "Benefit transfer methods aim at transposing the value estimates across locations, adjusting for differences in ecological and economic contexts (Pascual et al., 2010), however there are major sources of uncertainty that question the validity of transferred values (Huber et al., 2018; Schmidt et al., 2016).", "Second, there is a substantial gap between research and the informational needs of decision makers and practitioners (Olander et al., 2017).", "Decision processes are normally taking place at local or regional scales, where tangible problems relevant to stakeholders occur (Fischer et al., 2015; Tammi et al., 2017).", "Yet, valuation models tend to focus on biophysical outcomes (ES) that do not always matter to people, or do not represent the interplay of biophysical, ecological and social components at relevant temporal and spatial scales (Costanza et al., 2017).", "In addition, valuation methods are rarely comparable as they are developed to address specific problems and repeatability is rarely feasible (Small et al., 2017).", "Third, most studies are not capable of estimating the changes in value arising from changes in management or policies (Olander et al., 2017).", "Nature-human relationships are dynamic, often nonlinear and can reveal trade-offs among ES.", "Defining alternative policy scenarios, representing complex but plausible futures, can help investigating how value evolves by linking landscape changes to ES delivery (Plieninger et al., 2013).", "Finally, the disaggregation of the beneficiaries of ES is key to analyze trade-offs and inform policy to resolve potential conflicts among them (Martín-López et al., 2012).", "Moreover, few studies analyze the heterogeneity of preferences within social groups, despite a greater knowledge of the underlying attitudes and motivations of lay people can increase the public support for agrienvironmental and conservation policies (Rodríguez-Ortega et al., 2016).", "The main objective of this study was to explore the social preferences for ecosystem services, and the associated willingness to pay, in three multifunctional agroecosystem in Europe (Mediterranean, Atlantic, Alpine) under alternative agrienvironmental policy scenarios.", "To do so, we first analyze how the preferred level of delivery of ecosystem services (provisioning and non-provisioning) differ from the current level; then we analyze how these preferences, and their heterogeneity, change across policies and regions; and finally we calculate the total willingness to pay of society for the delivery of the ES, as a proxy to estimate the total economic value of multifunctional agroecosystems.", "This study covers the three largest terrestrial biogeographical regions in western Europe holding multifunctional agroecosystems: Mediterranean, Atlantic and Alpine.", "Table 1 summarizes the main characteristics of the agroecosystems; further biogeographical information on the study areas can be obtained from Bernués et al. (2014), Bernués et al. (2015) and Faccioni et al. (2019), for Mediterranean, Atlantic and Alpine agroecosystems, respectively.", "The ES under consideration in each agroecosystem were derived from previous socio-cultural analyses (focus groups in Mediterranean and in-depth interviews in the Atlantic and Alpine agroecosystems), as described by Bernués et al. (2014), Bernués et al. (2015) and Faccioni et al. (2019).", "These ES, which were identified as the most important by the public, were (i) the preservation of agricultural landscape (cultural ES); ii) the conservation of biodiversity (supporting ES); (iii) the prevention of forest fires in Mediterranean conditions, the maintenance of soil fertility in Atlantic conditions and the preservation of water quality in Alpine conditions (regulating ES); and iv) the provision of specific quality food products linked to the agroecosystems (provisioning ES).", "We used choice modeling to assess the relative importance of individual ES and the willingness to pay (WTP) of the populations under study.", "The ES corresponded to the four types of the Economics of Ecosystems and Biodiversity (TEEB) taxonomy (Kumar, 2010) (provisioning, regulating, supporting and cultural).", "We assumed that these ES embodied the most important use and non-use values of the Total Economic Value (TEV) (Pearce and Pretty, 1993).", "Choice experiments are one of the few methodologies able to estimate all use and non-use values of the TEV taxonomy (Rodríguez-Ortega et al., 2014).", "In choice modeling, we used individuals’ stated behavior in a hypothetical choice setting to derive the value of nonmarket goods (Hensher et al., 2005).", "Each respondent was asked to choose his most preferred alternative of five choice situations defined in terms of policy scenarios described by their ES biophysical attributes (Table 2).", "For each choice situation, one of the scenarios was fixed (the current policy) and represented the current situation in the respective study areas.", "The other two scenarios in each choice situation were referred to as policy A and B and were represented with different combinations of attribute levels (Table 2).", "These were contrasting policy scenarios that we call here ‘liberalization’ and ’targeted support’ representing two consistent macroeconomic framework conditions.", "The general rationale for the scenarios was as follows.", "The liberalization of policy assumed a reduction in support for agrienvironmental schemes and a subsequent acceleration of the processes of abandonment of the most marginal lands and the intensification and homogenization of production systems in the most favorable lands.", "Consequently, a process of landscape closure and homogenization, loss of biodiversity, erosion of regulating ES and lower availability of quality products linked to the territory was hypothesised.", "The targeted support scenario represents an alternative policy that leads to additional funding for agrienvironmental schemes, specifically designed to deliver higher levels of public goods.", "Hence, higher levels for the abovementioned ES was expected.", "Full details of scenario definition, attributes and levels are described in detail in Appendix A.", "The survey was designed to collect responses from the general populations (above the age of 18) in the regions nearby the agroecosystems under consideration.", "The regions were Aragon administrative region in Spain (N > 18 = 1,103,864 inhabitants), Bergen municipality in Norway (N > 18 = 271,949) and the Provinces of Belluno, Bolzano, Brescia, Sondrio, Verona, and Vicenza in Italy (N > 18 = 3,265,736).", "Sample sizes collected were 402, 240 and 402, respectively.", "Date of data collection was July 2013, June 2014 and July 2016, respectively.", "The questionnaires were implemented through three professional online panels that were representative of the adult population (above 18 years old) in the respective areas according to gender, age and place of residence.", "The panellists were selected randomly and recruited by invitation only to ensure representativeness.", "All responses provided by the professional panel were complete and used for analysis.", "The questionnaire was formulated equally for all study areas and translated to the native languages.", "Before the choice options, basic information on the agroecosystems that are present, the ES attributes, the existing agrienvironmental policies, and the societal cost of policy was supplied.", "We made it explicitly clear that the societal cost of each choice corresponded to the amount of money that each member of a family who was above the age of 18 would have to pay as an annual tax.", "The original questionnaires (block 1) can be seen in Appendix B.", "Given the large number of combinations of attributes and levels (34 * 51 = 405) we used the software Ngene (Choice Metrics, Ltd.) to develop an efficient experiment design that included a fraction of these combinations.", "Thirty choice sets divided in six blocks were obtained, i.e., each respondent made five choices.", "The design used prior parameter estimates obtained in a previous test survey.", "We carefully checked all the choice options for illogical or counterintuitive patters.", "In addition, the questionnaire was tested face-to-face to detect problems with particular choice settings.", "Policy scenarios were unlabelled to ensure that respondents focussed on the attribute combinations without their choices being affected by policy names.", "When individuals made their choice, they had to make a trade-off between the levels of the attributes and the costs that were associated with the different policies in the choice set.", "To estimate the effect of the different attributes on the choices the respondent made, we used a mixed logit model.", "Mixed logit models explicitly assume that there is a distribution of preference weights across the sample reflecting differences in preferences among respondents, and it models the parameters of that distribution for each attribute level (Hauber et al., 2016), which allows for unobserved preference heterogeneity among the respondents to be revealed (Revelt and Train, 1998).", "Parameter estimates indicated the effect of the attributes on the probability of choice of an alternative.", "Positive parameter values indicated that the presence of the attribute in a policy scenario increase the choice probability of that scenario, and negative values indicated a reduction in choice probability.", "The relative size of the parameters within one sample can be used to calculate the respondents’ marginal rate of substitution between the attribute levels, and the marginal WTP for the attribute levels.", "We used these marginal WTP estimates in our calculations of TEV of the policy scenarios, taking as reference the targeted support scenario.", "We calculated the WTP for each of the attributes included in the choice experiment by dividing the absolute value of the estimates of the highest levels of the attributes by the absolute value of the estimate for the annual cost.", "Then, we calculated the TEV by summing the partial WTP for the individual ES, as proxies to the different values of the TEV taxonomy (Table 1).", "Table 3 shows the results of the mixed logit model used to analyze the choice experiment data from the participants in Mediterranean, Atlantic and Alpine agroecosystems.", "The parameter estimates indicate preference for changes relative to the current situation.", "All parameter estimates show the expected sign.", "Positive estimates means that respondents preferred (obtained welfare gains from) the level of delivery of a particular ES (i.e. rich landscape mosaic, biodiversity increase, forest fires decrease, soil fertility increase, water quality increase and higher number of quality products).", "Negative estimates, in contrast, means that respondents disapproved (obtained welfare loses from) the level of delivery of a particular ES (i.e. abandonment of agricultural landscapes, biodiversity decrease, forest fires increase, soil fertility decrease, water quality decrease and lower number of quality products).", "The estimates for ES are significant (between 1 and 10% significance level) except for the high level of provision of quality products in the Alpine agroecosystem (P = 0.187).", "The annual cost has as expected a negative sign, meaning that respondents preferred to pay lower taxes, all else being equal.", "The evolution of estimates across scenarios is plotted in the last column of Table 3.", "In general, the estimates for the liberalization scenario are greater in absolute value than the estimates for the targeted support scenario.", "In other words, the welfare gain that accrued from preventing abandonment was greater than the welfare gain that accrued from enhancing ES from agricultural activity (when compared with the current scenario).", "This happened in all agroecosystems and ES, except for landscape and biodiversity in the Alpine agroecosystem.", "However, the pattern of evolution of ES estimates across scenarios varies.", "For some, the pattern from the liberalization to the targeted support scenarios is close to linear (e.g. forest fires in the Mediterranean agroecosystem and water quality and biodiversity in the Alpine agroecosystems).", "For others, the evolution of estimates shows a nonlinear pattern (i.e. discontinuity in the current scenario), particularly intense in the case of conservation of landscape and provision of quality products.", "We selected mixed logit models because they yield both the mean and the standard deviation of effects across the samples.", "The heterogeneity of preferences calculated by the mixed logit model among respondents is represented in Fig. 1.", "In general, the targeted support scenario shows greater heterogeneity of responses (people differed more in their responses) than the liberalization scenario (people concurred more in their responses).", "The Mediterranean region shows the greatest heterogeneity of responses among respondents and the Alpine region the lowest.", "Exceptions are the provisioning of quality products linked to the territory in the targeted support scenario (highest heterogeneity in the Alpine region) and in the liberalization scenario (intermediate heterogeneity in the Alpine region), and the agricultural landscape in the liberalization scenario (intermediate heterogeneity in the Alpine region).", "Within regions, Mediterranean people showed the greatest heterogeneity for a rich mosaic agricultural landscape, and the lowest for reduction of forest fires and for the abandonment of agricultural landscape.", "Atlantic people showed very similar level of heterogeneity of responses for all ES and scenarios.", "Alpine people showed the greatest discrepancy of preferences for the delivery of quality products, as mentioned before, but agreed about the importance of water quality in the two scenarios.", "WTP and TEV results are expressed as € per person per year, as cost was included in the model in this manner.", "Table 4 shows ES in order of importance (regarding WTP) in each agroecosystem.", "In the Mediterranean agroecosystem, the prevention of forest fires was the most important ES (more than 50% of total WTP), followed by the provision of quality foods, the conservation of biodiversity and cultural landscapes.", "In the Atlantic agroecosystem, all ES had similar importance, with the provision of quality foods and maintenance of soil fertility having slightly more WTP.", "In the Alpine agroecosystem, the preservation of water quality was the most important ES (50% of total WTP), followed by biodiversity, landscape and, with much lower WTP, the provision of quality foods.", "TEV was 121.2€ (2.7 times the tax reference in choice experiment) for the Mediterranean agroecosystem, 715.0€ (7.1 times) for the Atlantic agroecosystem, and 159.3€ (5.3 times) for the Alpine agroecosystem.", "WTP was recalculated according to the average wage in each country, taking as reference the average wage in Spain (see Table 4 footnotes for details).", "In this case, the wage equivalent TEV was 121.2€, 510.1€ and 169.5€, respectively.", "The evolution of the value of particular ESs varied greatly, as did the level of heterogeneity of responses among individuals.", "Regarding landscape, by definition its appreciation is subjective and therefore difficult to include in choice models or other valuation exercises (Daniel et al., 2012).", "Socio-cultural background and the former trajectory in terms of land use and land cover seemed key factors in landscape appreciation.", "In the Mediterranean and Atlantic agroecosystems, the landscape of the current and targeted support scenarios received similar appreciation, which implied that further agricultural development was not a priority, or even slightly rejected (Mediterranean agroecosystem).", "In the Alpine agroecosystem, the landscape that keeps mountain farming and a moderate intensification of agriculture in valleys was clearly preferred, probably because abandonment and intensification in the current landscape has already gone too far in the eyes of respondents (Faccioni et al., 2019).", "Biodiversity conservation was perceived similarly across agroecosystems.", "This ES had moderate estimates in absolute value (i.e. its relative importance was intermediate: 18.4–25.3% of total WTP).", "The comparatively low importance assigned to biodiversity could be because the links with human well-being are not immediate or not easily perceived.", "People tend to value more those ES that have direct effects and satisfy more tangible needs (Hartter, 2010; Olander et al., 2017).", "However, biodiversity followed a linear pattern across scenarios, so people always obtained moderate welfare gains from increasing biodiversity through policy scenarios.", "Regulating services also showed a linear evaluation across scenarios.", "These ES were very important in all cases (around 50% of total WTP for forest fires and water quality and 27% for soil fertility) for a majority of people (lower level of heterogeneity of responses in the two policy scenarios).", "However, regulating ES were rated differently across agroecosystems, which confirmed the need of previous socio-cultural studies to choose relevant ES -for people- before analyzing their economic value (Martín-López et al., 2014).", "The prevention of forest fires in the Mediterranean (located in a sparsely populated area with abandonment of large marginal areas) and the maintenance of water quality in the Alpine agroecosystem (located close to a very populated region with high agriculture development) have a perceived direct impact on people wellbeing.", "Consequently, the importance attached by the participants was large and homogeneous.", "Moreover, people were not willing to compromise on these two ES and welfare gain was linear across scenarios.", "Differentiated quality products are often neglected in ES valuation exercises, as food is normally considered in terms of food security (bulk quantity).", "However, food quality has demonstrated to be a distinctive feature of multifunctional agroecosystems (Bernués et al., 2016).", "An intriguing outcome of our analyses is the changing public perception of this provisioning service across scenarios, which suggest a non-linear pattern and strong contextual and individual differences.", "In places where the current provision of quality products is high (in Mediterranean and more intensely in Alpine agroecosystems) many people are willing to compromise on the number or quality products in the target support scenario, but not on biodiversity and regulating services as previously mentioned.", "In Atlantic conditions, where quality products are not so abundant, the importance attached to them is the highest (with the lowest heterogeneity of responses), and the public always obtain welfare gains across scenarios.", "Both abandonment and intensification of agroecosystems were rejected by the public.", "In general, the loss obtained from reduced levels of ES in the liberalization policy was larger than the gains from increased levels in the targeted support scenario.", "This asymmetry of welfare variation around the status quo scenario was observed in the Mediterranean and Atlantic agroecosystems, where the main problem is abandonment.", "This finding is consistent with the large literature on loss aversion in behavioral economics, which shows that losses have greater impacts on preferences than gains (Tversky and Kahneman, 1991).", "Psychological explanations for this asymmetry include ethical dimensions and the sense of moral responsibility or affection towards other people or the environment (Brown and Gregory, 1999; Peters et al., 2003).", "However, in the Alpine agroecosystem, where intensification and abandonment coexist and are visible in the same area, the gains of the targeted support scenario (which means a de-intensification of agriculture) had similar relevance to the losses due to the liberalization scenario that produced an increment of ecosystem disservices, water pollution in particular.", "More generically, these results give empirical evidence to the theory of a displacement of the modernization paradigm in European agriculture by a sustainability/rural development paradigm (van der Ploeg et al., 2000).", "Today, urban citizens expect the countryside to provide much more than cheap food products and new needs and expectations must be satisfied by agriculture, which is therefore considered multifunctional (Renting et al., 2009).", "Respondents preferred a multifunctional configuration of agricultural systems oriented towards a mix of quality products, landscape management, biodiversity conservation and improvement of regulating services.", "This suggests a “land-sharing” model of agriculture as the desirable pathway for sustainable rural development (Pedroli et al., 2016), at least in the European context.", "For effective design and implementation of agrienvironmental policies, we need to be aware of the potential changes in the value of ES arising from the different levels of provisioning as consequence of changes in management regimes or policies (Olander et al., 2017), as well as trade-offs between ES that also can change (e.g. provision of quality food products and regulating services).", "We believe it is wise to focus on particular ES that matter to people at the right local/regional spatial scales.", "In the three case studies, regulating ES and biodiversity showed consistent increments of WTP across scenarios; however, the WTP for regulating ES (specially prevention of forest fires and maintenance of water quality) was much higher.", "Regulating ES are perceived as having direct effects on human wellbeing and therefore may constitute better “entry points” to convince people of the need to preserve (agro-) ecosystems.", "Eventually, this strategy could contribute to increase policy acceptance and effectiveness.", "It derives that the optimal scale for policy implementation might not be political, but one in which social and environmental systems share their boundaries (Small et al., 2017).", "Agrienvironmental schemes with targeted objectives, and the corresponding indicators that demonstrate the societal gains or losses, should contribute to reverse the abandonment/intensification trend that is widely rejected by the public.", "There is room for manoeuvre, as the WTP of the population is three to seven times larger than the current social cost in the respective locations.", "However, better targeted agrienvironmental policy should not necessarily mean a higher social cost (i.e. higher taxes).", "Economic resources of agricultural policies (e.g. the Common Agricultural Policy of the European Union) could be reallocated following a “provider gets” principle (i.e. moving from subsidies to increase farmers’ rents to payments for their services to society).", "Our approach aimed at addressing some of the challenges of economic valuation of ecosystem services as described in the introduction, such as: the dynamic relations between humans and nature, the gap between research results and information relevant for decision-making at a definite scale, the lack of repeatability of many valuation studies, and the heterogeneity of value perception in the population.", "To address the changes of value arising from different levels of delivery to ecosystems services, we defined realistic contrasting policy scenarios with biophysical effects tailored to the different multifunctional agroecosystems.", "We made some assumptions to link the policies to the level of delivery of ES (detailed in Appendix A).", "These assumptions were specific for the different indicators representing the ES in each location.", "To make results relevant for management, we chose the ES that matter to people, according to previous socio-cultural studies; moreover, in the considered agroecosystems the study areas were delimited on the basis of administrative units (different types of governances: natural park, municipality or province).", "A different representation of these ES (in particular the manipulation of pictures to represent land use intensities and the indicators of biodiversity) might have altered the final WTP values, which need to be used with caution.", "We also assumed that respondents understood the rationale of the experiment and further that they understood complex environmental phenomena and made rational choices.", "Yet, the results showed consistent patterns; some were general, whereas others were influenced by the socio-cultural contexts in which the agroecosystems were located.", "Repeatability in valuation exercises is impaired due to high cost of time and resources, and the heterogeneity of local conditions (e.g. in this study, different regulating ES were important across populations as detected by previous socio-cultural studies).", "The replication of the analysis in the three agroecosystems showed that the economic value of ES depended on model specification (especially tax reference which depends on number of taxpayers in the study area) (Tammi et al., 2017), but also on economic and socio-cultural context (Randall, 2002).", "There were differences due to income level, but even when wealth of the population average was considered, the differences between countries were still large.", "It is possible that in the Atlantic case, where WTP was substantially higher, a number of people might not attend to the cost attribute relative to the ecosystem services attributes (Bernués et al., 2015).", "Consequently, the TEV varied greatly across agroecosystems, although it was always much larger than the current cost for the taxpayers.", "Taking into account that we used the same methodological framework and a similar model specification in terms of ES and change in provision levels, this finding opens up questions around the usability of monetary values estimated with stated-preference methods and the benefit transfer approach, even if adjusted to income level, to enlarge the scale of economic assessments (Plummer, 2009).", "Historical and cultural differences between countries can play a major role when explaining the differences of current and future use values in very specific local contexts (Brouwer, 2000).", "As mentioned above, current values need to be compared with caution.", "In addition, other studies have demonstrated that, within countries, WTP varies enormously among geographical location of the population (rural versus urban) and psychographic (conservationists vs. productivists) profiles (Rodríguez-Ortega et al., 2016).", "The different degree of heterogeneity of responses across ecosystems services underlines the variation within the population and indicates an area for further research.", "Our approach allowed visualizing the changing relationships between (agro-) ecological and social subsystems by quantifying the societal demand of ES across diverse policy scenarios defined in levels of biophysical delivery.", "A number of lessons could be derived.", "1 – The WTP for the provision of ES exceeded the current level of public support so there is room for further investment or reallocation of resources in agrienvironmental policies.", "2 – Further abandonment and intensification of agriculture were clearly rejected by the public.", "Respondents preferred a multifunctional configuration of agricultural systems oriented towards a mix of quality products, landscape management, biodiversity conservation and improvement of regulating services.", "3 – Monetary valuation with stated preference methods is highly dependent on context, therefore it can help in ES prioritization but extrapolation of economic values can be misleading.", "4 – Increasing the provision of biodiversity and specially regulating ES always produced welfare gains, however, the optimal level of delivery of agricultural landscapes and quality products was context dependent and people perceived trade-offs between ES across policy scenarios." ]
[ "We value ecosystem services in three agroecosystems and three policy scenarios.", "Higher levels of regulating services and biodiversity always produce welfare gains.", "People perceive trade-offs between agricultural landscapes and quality products.", "The WTP for the provision of ES exceeded the current level of public support.", "Further abandonment and intensification of agriculture is rejected by the public." ]
Cardiac Remote Conditioning and Clinical Relevance: All Together Now!
Acute myocardial infarction (AMI) is the leading cause of death and disability worldwide. Timely reperfusion is the standard of care and results in decreased infarct size, improving patient survival and prognosis. However, 25% of patients proceed to develop heart failure (HF) after myocardial infarction (MI) and 50% of these will die within five years. Since the size of the infarct is the major predictor of the outcome, including the development of HF, therapies to improve myocardial salvage have great potential. Over the past three decades, a number of stimuli have been discovered that activate endogenous cardioprotective pathways. In ischemic preconditioning (IPC) and ischemic postconditioning, ischemia within the heart initiates the protection. Brief reversible episodes of ischemia in vascular beds remote from the heart can also trigger cardioprotection when applied before, during, or immediately after myocardial ischemia—known as remote ischemic pre-, per-, and post-conditioning, respectively. Although the mechanism of remote ischemic preconditioning (RIPC) has not yet been fully elucidated, many mechanistic components are shared with IPC. The discovery of RIPC led to research into the use of remote non-ischemic stimuli including nerve stimulation (spinal and vagal), and electroacupuncture (EA). We discovered and, with others, have elucidated mechanistic aspects of a non-ischemic phenomenon we termed remote preconditioning of trauma (RPCT). RPCT operates via neural stimulation of skin sensory nerves and has similarities and differences to nerve stimulation and EA conducted at acupoints. We show herein that RPCT can be mimicked using electrical stimulation of the abdominal midline (EA-like treatment) and that this modality of activating cardioprotection is powerful as both a preconditioning and a postconditioning stimulus (when applied at reperfusion). Investigations of these cardioprotective phenomena have led to a more integrative understanding of mechanisms related to cardioprotection, and in the last five to ten years, it has become clear that the mechanisms are similar, whether induced by ischemic or non-ischemic stimuli. Taking together much of the data in the literature, we propose that all of these cardioprotective “conditioning” phenomena represent activation from different entry points of a cardiac conditioning network that converges upon specific mediators and effectors of myocardial cell survival, including NF-кB, Stat3/5, protein kinase C, bradykinin, and the mitoKATP channel. Nervous system pathways may represent a novel mechanism for initiating conditioning of the heart and other organs. IPC and RIPC have proven difficult to translate clinically, as they have associated risks and cannot be used in some patients. Because of this, the use of neural and nociceptive stimuli is emerging as a potential non-ischemic and non-traumatic means to initiate cardiac conditioning. Clinical relevance is underscored by the demonstration of postconditioning with one of these modalities, supporting the conclusion that the development of pharmaceuticals and electroceuticals for this purpose is an area ripe for clinical development.
[ "CENG", "COMP", "ENER", "ENGI", "ENVI", "MATE" ]
[ "cardiac conditioning", "clinical feasibility", "electroceuticals", "neural and molecular mechanism", "non-ischemic conditioning", "peripheral nociceptive stimulus", "remote cardioprotection" ]
[ "1500", "1700", "2102", "2200", "2305", "2501" ]
[ "CardioCeption, LLC holds licenses relevant to this field of study.", "W. Keith Jones owns equity in CardioCeption, but has received no income or financial benefit from any entity related to this work.", "The University of Cincinnati, W. Keith Jones, and Xiaoping Ren filed the initial patents but have received no royalties.", "Kristin Luther, Yang Song, Yang Wang, Xiaoping Ren, and W. Keith Jones declare that they have no conflict of interest or financial conflicts to disclose.", "In 1986, Murry et al. [1] first described ischemic preconditioning (IPC), in which brief episodes of ischemia/reperfusion (I/R) preceded an injurious I/R resulting in reduced myocardial infarct size in a canine model.", "The phenomenon of IPC has since been successfully demonstrated in multiple animal species including dogs, rats, pigs, rabbits, and mice, as well as confirmed in human patients [2–7]; reviewed in Ref. [8].", "Marber et al. [9] reported a second window of protection (late preconditioning), which develops 12−24 h after the initial preconditioning stimulus.", "Unlike early IPC, which is powerful but short-lived, late IPC remains protective for 24−72 h, although the magnitude of the protection is less over time [3].", "Repeated preconditioning episodes of the same type are neither additive nor cumulative [10].", "In 1993, Przyklenk et al. [11] demonstrated that in the canine heart, a preconditioning stimulus applied to the vascular bed supplied by the circumflex branch reduced infarct size in the area of the myocardium supplied by the left anterior descending coronary artery.", "Subsequently, it was demonstrated by McClanahan et al. [12] that cardioprotection could even be achieved by preconditioning ischemic stimuli in distant organ sites, such as the kidney.", "Remote ischemic preconditioning (RIPC) has been demonstrated to elicit cardioprotection following ischemia of kidneys, intestine, limbs, liver, skeletal muscle, and brain (reviewed in Ref. [13]).", "While the release of diffusible factors from ischemic preconditioned tissues/organs is a key aspect of RIPC [14–18], it is also evident that neural connections to the preconditioned limb or tissue are required.", "For instance, it has been shown in most [19] but not all [20] animal models that blocking sympathetic transmission using hexamethonium abrogates RIPC's protective effect.", "In a mouse hindlimb model, it has been demonstrated that either occlusion of the femoral vein or transection of the femoral and sciatic nerves likewise abolishes the cardioprotection observed after RIPC, indicating the requirement for both humoral and neural pathways [21].", "RIPC has been shown to improve short- and long-term clinical outcomes when applied prior to emergency or elective percutaneous intervention [22], coronary artery bypass grafting [23], and valve replacement [24].", "Many of the detailed mechanistic aspects of RIPC are shared with IPC (see below).", "Although IPC and RIPC have emerged as powerful methods of ameliorating I/R injury to the myocardium, their use as clinical cardioprotective strategies to attenuate the pathophysiological consequences of I/R injury (i.e., infarction and ventricular dysfunction) is limited by the inability to predict the onset of clinical ischemia.", "As a direct result of considering this limitation, Zhao et al. [25] demonstrated that rapid sequential intermittent interruption of coronary blood flow during the early moments of reperfusion after ischemia attenuates I/R injury.", "This phenomenon, termed postconditioning, is extremely valuable in that it supports the efficacious use of repetitive ischemia at a clinically relevant time point (e.g., at reperfusion) [26].", "The more recent findings in this field [27–30] have combined postconditioning and RIPC concepts by demonstrating that ischemia of a remote site (such as a limb) can elicit cardioprotection at reperfusion.", "A related phenomenon, perconditioning, results from administering a cardioprotective stimulus during the I/R injury [31, 32].", "Remote perconditioning involves using a conditioning stimulus at a remote site during myocardial infarction (MI), and has been shown to be protective and to have clinical value [24].", "However, all of these cardioprotective strategies have a limitation in that they require the use of an ischemic stimulus to an organ; this is often not clinically desirable.", "Furthermore, clinical trials of such approaches have been disappointing [33], and/or come with significant limitations and risks that prevent their use [34, 35].", "The clinical trials of remote ischemic conditioning have some limitations, mainly as a result of including too few subjects, and of confounding variables inherent within the patient population.", "The patients involved are of heterogeneous age and overall health; many have co-morbidities that are known to reduce the efficacy of cardioprotection, and are administered drugs that mask or block cardioprotection [8].", "Furthermore, factors such as collateral circulation and spontaneous or very early clinical reperfusion can lead to small infarcts that do not benefit from adjunctive therapy [13].", "Even though recent results with limb ischemia have supported cardioprotection in humans [36], this approach can be difficult to tolerate and cannot be employed in some patients, including the morbidly obese, or patients who have had axial lymphadenectomy.", "An alternative would be to achieve cardioprotection using a non- or minimally-invasive technique, such as a remote non-ischemic stimulus.", "Cardioprotection has been shown to result from non-ischemic stimuli including nerve stimulation (spinal, vagal, femoral), acupuncture and electroacupuncture, skin incision, and the chemical or electrical treatment of skin [37–43].", "To the extent that some of these modalities are less traumatic yet efficacious, they are being studied as potential therapies.", "Over the past 25 years, we have learned a great deal about the detailed mechanisms that underlie cardioprotection in general, and IPC, RIPC, and related phenomena specifically.", "Mechanistic studies usually focus upon initiators, mediators, and effectors of protection, though over the years, our insights have blurred some of these distinctions.", "Clearly, understanding the mechanism of cardioprotection after IPC, RIPC, and postconditioning has the potential to elucidate potential therapeutic targets that could be used to initiate or intensify cardioprotection.", "These targets could be useful in preconditioning in certain situations, including preventing/reducing perisurgical MI and treating patients at risk for MI [5, 44, 45].", "Alternatively, understanding the mechanism may be the key to discovering more clinically applicable ways to initiate perconditioning and/or postconditioning in the clinical setting.", "Generally speaking, similar—yet not identical—mechanisms underlie the mediator and effector aspects of diverse protective stimuli including IPC, RIPC, preconditioning, postconditioning, and perconditioning [46].", "The most diverse aspect of cardioprotective phenomena seems to be in the initiating phase, but this may simply represent the point of stimulation in a more general convergent mechanism.", "There are some distinct mechanistic differences and specific pathways that have been shown not to be necessary for certain types of cardioprotective stimuli.", "Combinatorial studies (use of multiple stimuli) have sometimes shown additive effects supporting mechanistic differences [47, 48].", "However, other combinations have not shown such effects, supporting a similarity or at least an overlap of mechanism.", "In this paper, we compare and contrast some of these data, extract some meaning, and add to what is known in a way that may point to more efficacious and practical types of cardioprotection to invoke in a clinical setting.", "Some studies of combinatorial stimuli are shown as follows.", "In recent years, there have been several studies combining conditioning stimuli in order to determine if there is overlapping, additive, or synergistic protection.", "Several groups have studied the combinatorial effects of postconditioning and perconditioning.", "The rationale for such studies is that the reduction in infarct size by either stimulus is often less than that of preconditioning.", "Given the greater clinical relevance of these stimuli, any additivity would be of interest.", "Xin et al. [47] first studied combinatorial effects in 2010.", "They found that postconditioning and perconditioning, while each less protective than preconditioning, additively mimicked the extent of protection afforded by preconditioning.", "Furthermore, they found that combining the effects of preconditioning with those of the postconditioning plus perconditioning combination resulted in no further protection, suggesting that preconditioning and the postconditioning/perconditioning combination have similar overlapping mechanisms.", "Studies of kinase activity showed the activation of Akt and ERK1/2 phosphorylation by all stimuli, but that the extent of activation was highest in IPC and was mimicked by the postconditioning plus perconditioning combination.", "In each instance, protection was blocked by PI3K or ERK1/2 inhibitors.", "Xin et al. concluded that the stimuli, which differ in the timing of initiation, work through the same general mechanisms and that it is the extent of kinase activation that confers the extent of protection in mice.", "This conclusion is consistent with mechanistic studies of these phenomena, and with a study by the same group showing that repeated remote postconditioning stimuli increase the extent of cardioprotection [49].", "Interestingly, Tamareille et al. [30] found that IPC and RIPC act additively to reduce infarct size, but that, in addition to the activation of reperfusion injury salvage kinase (RISK) pathways, the addition of RIPC resulted in increased levels of phospho-Stat3.", "They concluded that the mechanisms of IPC and RIPC were overlapping but that they were somewhat different.", "However, it must be noted that a requirement for Stat3 has independently been established for IPC [50, 51].", "A study by Kloner's group showed that perconditioning and remote postconditioning were not effective in the rat model used, obviating the question of the combination in that case [52].", "The recent remote ischemic postconditioning (RIPOST) clinical trial showed that remote perconditioning and postconditioning were both effective, but that the combination did not produce a significant additive effect (reduction in creatinine kinase-MB (CK-MB) AUC from 31% to 29%, P>0.05) [48].", "Thus, the evidence of combinatorial studies in animal models and in humans supports the concept that overall, protective pathways work by overlapping if not similar mechanisms.", "Because of the growing recognition of this concept in the field, the term “conditioning” is coming into vogue as a general term to refer to these types of effects.", "In addition to cardiac ischemia, which initiates IPC, and ischemia elsewhere, including limb ischemia, which initiates RIPC to the heart, several non-ischemic stimuli have been studied as initiators of cardiac conditioning, notably electroacupuncture (EA), skin incision, and the pharmacological activation of skin nociception.", "We review these stimuli here due to their potential clinical relevance, and for what they can tell us about the mechanism of cardiac conditioning.", "We primarily discuss the effects of EA, spinal stimulation, vagal stimulation, and skin nociceptor activation, all of which are able to activate cardiac conditioning.", "This work was presaged by three papers from the late 1980s and early 1990s that showed the adaptive effects of EA and vagal stimulation upon the heart [53–55].", "In particular, the paper published in 1991 by Meerson et al. showed that EA of an isolated heart reduced necrosis.", "Subsequently, several investigators observed a role for nerves and neuropeptides in conditioning phenomena.", "The peptide bradykinin is well known to be required for ischemic pre- and post-conditioning [56, 57], and Erşahin et al. and Shoemaker et al. first showed its involvement in RIPC [58, 59].", "Bradykinin is involved in sensory nerve signaling, and cardiomyocytes have the BKR2 receptor for bradykinin.", "In the rat model, bradykinin in the coronary circulation is degraded quickly by angiotensin converting enzyme (ACE) and by aminopeptidase P. Erşahin et al. [58] showed that apstatin, an aminopeptidase P inhibitor, is cardioprotective against I/R injury in an isolated rat heart model, supporting the idea that the local action of bradykinin is protective, but not ruling out effects of systemic bradykinin.", "The Vatners's group later showed that innervation and specifically the α1-AR is required for the late phase of IPC in a porcine model, but that the early phase of IPC is independent of this pathway [60].", "RIPC is known to require intact innervation from the ischemic bed; while there is evidence for hormonal mediators, it appears that neural signals are critical as well [61].", "In addition to neural involvement in ischemic stimuli, all known forms of non-traumatic/non-ischemic remote conditioning involve the neural system at some level.", "EA is now thought to work through the stimulation of skin sensory or other innervation.", "Vagal and spinal stimulation invoke the direct stimulation of nerves.", "We have shown that skin nociception-induced conditioning (NIC), whether by skin incision (remote preconditioning of trauma, or RPCT), or chemical nociceptor stimulation, involves the stimulation of skin sensory nerves [42, 62].", "This phenomenon likely has both neural and humoral components, but the relative contribution of these to the protection observed is unknown.", "Most of these non-ischemic cardiac conditioning stimuli are capable of initiating both an immediate protection (i.e., early phase) and a longer lasting protection (i.e., late phase).", "As will be seen, all of the non-ischemic remote initiators of conditioning seem to involve neural and hormonal mechanistic aspects.", "In this, they are similar to RIPC.", "However, remote perconditioning and postconditioning are more similar to the early phase in their timing of action (immediate onset of protection).", "Activation of the spinal dorsal horn neurons by spinal cord stimulation (SCS) is known to be effective in treating chronic intractable angina pectoris.", "In addition to pain relief, clinical studies using SCS for the treatment of chronic refractory angina demonstrate increases in exercise tolerance, improvements in ischemia-related EKG changes, and improvements in the quality of life [37, 63, 64].", "Animal studies also indicate that SCS improves the function of the heart.", "It has been demonstrated that SCS reduces infarct size in a rabbit model with transient coronary artery occlusion, an effect that can be prevented by adrenergic blockers [65].", "However, this effect was not observed when performed as a postconditioning stimulus.", "This result, along with the invasive nature of the procedure, limits its clinical applicability, although SCS is being studied and has other potential uses, such as in heart failure (HF) and arrhythmia.", "It has long been recognized that balance of the autonomic nervous system is critical to many aspects of homeostasis and functional regulation in the heart.", "After myocardial ischemia, there is an imbalance between the sympathetic and parasympathetic systems, and increased chronic sympathetic activity is thought to contribute to mortality after MI and in HF [66–68].", "Vagal stimulation has been studied for its potential to slow left ventricle (LV) remodeling and improve heart function and survival in post-MI HF.", "Based on this potential, Kong et al. [38] showed that vagal stimulation reduces infarct size and improves function in a rat MI model.", "The mechanism they describe includes the activation of protein kinase C (PKC), nitric oxide (NO) production, and anti-inflammatory effects that are not dissimilar to those that have been described after IPC and RIPC [38].", "It has been demonstrated that transection of the femoral nerve likewise abolishes the cardioprotection of RIPC [61] and that stimulation of the femoral nerve can mimic RIPC of limb ischemia as well [39], supporting the concept that neural pathways are common to many forms of cardiac conditioning and that direct stimulation of some nerves can elicit cardioprotection.", "In 2003, Tsou et al. [69] showed that EA at the Neiguan acupoints (corresponding to a part of the human wrist area, innervated by the thoracic vertebral region) reduces I/R injury, measured by reduced cardiac enzymes in a rat model.", "They also showed that this effect was blocked by the severing of the bilateral median nerve, the bilateral severing of the vagus, or by IV naloxone, implicating neural pathways, including the vagus, whose stimulation is already known to be capable of conditioning.", "Wang et al. [70] demonstrated that EA induces cardiac conditioning against MI and that this effect adds to the protective effect of hypothermia, suggesting complementary mechanisms.", "In 2006, Gao et al. [40] showed that EA at bilateral Neiguan acupoints is cardioprotective, reducing infarct size in a rat model by nearly 50%.", "This effect was blocked by propranolol, revealing dependency upon the adrenergic signaling.", "In 2012, Zhou et al. [41] showed in a rabbit model that perconditioning by EA (EA during coronary occlusion) reduced infarct size concomitant with the release of cardiac norepinephrine (NE), and that this protection was reduced by the pharmacologic blockade of PKC and opioid receptors in an additive manner.", "This work implicates many of the same signaling pathways involved in IPC/RIPC, as well as the neural signaling in EA-induced conditioning.", "Redington's group, which had previously shown the involvement of a dialyzable circulating factor in RIPC [61], showed that such is also produced in the rabbit after bilateral EA at the Neiguan points [71].", "Despite more than 15 years of research, the humoral factor(s) have never been identified.", "Other work has implicated roles for Hsp70 (also critical for IPC and RIPC [72, 73]) and miR-214 (also involved in ischemic postconditioning [74, 75]), supporting the idea that in addition to common signaling and neural pathways, gene regulatory pathways may be common to these different forms of cardiac conditioning.", "To date, EA has not been shown to be effective as a postconditioning stimulus, but the perconditioning protection may have clinical relevance [41].", "In 2004, we published a paper describing a cardioprotective effect of skin surgical incision and coined the term remote preconditioning of trauma (RPCT) for the effect.", "Briefly, we found that a surgical incision in the abdominal peri-umbilical region elicited cardioprotection against a subsequent ischemic insult, resulting in an 80% to 85% reduction of cardiac infarct size.", "Unlike either IPC or RIPC, RPCT is ischemia-independent and yet is highly efficacious.", "Like RIPC, RPCT requires intact neural connections [42].", "RPCT has both an early and late phase, like IPC; however, unlike IPC, both the early and late phases of remote preconditioning of trauma are TNF-α and iNOS independent [42, 62, 76].", "This shows that RPCT is not mechanistically identical to IPC; however, as we will see, there is mechanistic overlap.", "An unusual aspect of RPCT is that the initiation of the protective phenomenon is associated with peripheral nerve stimulation of a particular site—the skin bilaterally located around the umbilicus [42, 62].", "We showed that this stimulus acts though skin sensory nerves, as the effect is blocked by topical lidocaine and mimicked by topical capsaicin cream, with the effect of topical capsaicin being completely blocked in TRPV1 knockout mice [42].", "This result implicates neural pathways, and indeed, we showed that transection of the spine at the T7 vertebral level blocks protection, while C7 transection does not, demonstrating a lack of central nervous system (CNS) regulation, but the necessity of spinal integrity between the umbilicus and the heart.", "Based upon the fact that skin nociception is the initiator of this protection, and that chemical and other pain initiators can activate it, we have coined the term nociception-induced conditioning (NIC) to refer to the broader phenomenon (activated by incision, chemical, or as we shall see, electrical means).", "Further work showed that the neural pathway could be traced via injection of a fluorescent dye, 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine (DiI) at the skin incision site.", "The DiI signal was shown to stain the dorsal horns at both the T1−T5 (abdomen) and T9−T10 (cardiac) vertebral levels.", "This result suggests a neural pathway from the skin sensory nerves to the spinal dorsal root ganglia (DRG) at the level of the skin incision to the level of the cardiac innervation [42].", "The literature supports the idea that a dorsal root reflex induced by peripheral nociceptive stimulation may form the basis of the interactions between the somatosensory and cardiac sympathetic systems at different spinal levels [77, 78].", "The dorsal root reflex is a volley of action potentials that can be recorded emerging antidromically from the spinal cord in the dorsal roots following the electrical stimulation of an adjacent dorsal root or sensory nerve.", "The dorsal root reflex is thought to arise on the rising phase of the primary afferent depolarization (PAD) generated within the terminals of afferent fibers in the cord by a synchronous afferent volley arriving in the cord after electrical stimulation of sensory nerves and roots [79, 80].", "This causes depolarization of the terminals of the afferent fibers within the cord beyond their threshold, producing action potentials that travel antidromically out of the cord along the dorsal roots.", "Spreading of the dorsal root reflex both along and across the cord has been noted by several groups [81, 82].", "In an isolated hamster spinal cord, the dorsal root reflex has been shown to spread over at least fifteen segments from the lower lumbar to upper thoracic roots, in both rostro-caudal and caudo-rostral directions [82].", "This suggests the existence of a pathway within the mammalian cord for the transmission of PAD between segments.", "The involvement of this pathway is supported by our spinal transection results [42].", "The fact that both Hoe 140 and propranolol block cardioprotection, as does the genetic ablation of BKR2, supports the involvement of bradykinin and adrenergic signaling, as was found for IPC and RIPC [59, 60].", "We also showed a requirement for PKC activation and activity of the mitoKATP channel, supporting the hypothesis that common mediators and end-effectors are involved in NIC [42] and IPC [83].", "The blockade of calcitonin gene-related peptide (CGRP) and substance P signaling supports a pathway whereby the antidromal activation of cardiac sensory nerves activates cardiac sympathetic nerves.", "NE and bradykinin likely then act upon cardiomyocytes and are the activation signals for PKC.", "The Gross's lab and others [84, 85] have confirmed that RPCT (which we now see as one sort of stimulus for NIC) operates in dogs and rats, and have confirmed the requirement of bradykinin signaling.", "While the pharmacologic blockade of adenosine and opioid signaling had no effect upon cardioprotection, unlike in IPC.", "Gross et al. showed that signaling by epoxyeicosatrienoic acids (EETs) are important downstream of bradykinin signaling in NIC (induced by either RPCT or topical capsaicin) and have previously shown EETs to be involved in IPC and ischemic postconditioning [85–87].", "Although EETs are thought to act locally in an autocrine or paracrine manner, longer-distance actions cannot yet be ruled out in NIC.", "Finally, using a topical inhibitor of PKCγ [88], which is a neuron-specific PKC isoform, they blocked NIC, confirming the role of skin nerve fibers and providing a new pharmacologic target for inducing NIC [86].", "We believe that the published evidence supports a mechanism for NIC whereby the activation of skin nociceptors at the anterior abdominal location (near T9−T10) sends a signal from the DRG at T9−10 to the DRG at T1−5, and that this initiates an antidromic signal via cardiac sensory nerves to the heart.", "In the heart, the sensory nerves would release CGRP and substance P, which would trigger the release of NE from sympathetic nerves and bradykinin from either the sensory or sympathetic nerves [89].", "NE and/or bradykinin would act in part upon cardiomyocytes, triggering PKC activation that would mediate the signaling and activation of end-effectors, including the mitochondrial KATP channels [42, 85].", "This sympathetic system-mediated cardiac conditioning is triggered in response to “warning signals” relayed by the cord from the remote skin nerves.", "Although there is a great deal of overlap between the mechanisms of IPC, RPCT, and NIC, it is notable, based on our limited data at present, that NIC does not employ TNF-α, adenosine, or opioid signaling [62, 85], while IPC requires each of these.", "Interestingly, sympathetic activation, bradykinin, and β-adrenergic signaling have been implicated in NIC and IPC, PKC seems to be required for all of these conditioning modalities, and CGRP has been shown to be cardioprotective, and to be involved in RIPC, postconditioning, and cardioprotection after heat stress [90–93].", "Overall, the available evidence supports the hypothesis that cardiac conditioning phenomena share aspects of mechanism while not requiring completely identical molecules.", "We propose that this is indeed the case and that different cardioprotective phenomena essentially represent different entry points into a large signaling network consisting of receptors, kinase and metabolic signaling cascades, gene networks, and effector pathways (Figure 1).", "Each type of conditioning signal activates this network at a distinct “edge” of the network, with the consequence that specific portions of it are the focus of detectable signaling pathways that converge upon more common protective effector pathways.", "The common mediator and effector pathways seem to include bradykinin (discussed above), PKC, and mitoKATP.", "PKC plays an integral role in many of the signaling cascades that govern cellular behavior [94].", "The current paradigm of PKC signaling in cardioprotection has been primarily formulated on the basis of information acquired through the pharmacologic inhibition of various signaling elements.", "In support of this linear paradigm of PKC signaling, the activation of multiple proteins in a PKC-dependent fashion has been demonstrated in response to IPC [95] and RIPC [96], indicating that these proteins may be signaling elements in the PKC pathway.", "As it is one of the most highly expressed myocardial PKC isoforms [95], PKC-α is the least studied because, unlike the novel isoforms PKC-δ and-ε, it is not regulated in acute myocardial ischemia.", "Translocation modification has confirmed that PKC-δ is a critical mediator of post-ischemic cardiomyocyte necrosis and contractile dysfunction in mice, rats, and pigs [97, 98], although the activity in the porcine model is disputed by one study [99].", "In the normal myocardium, PKC-ε forms signaling complexes with at least 36 different proteins that can be classified as structural elements, signaling molecules, and stress-responsive proteins [100].", "PKC-ε dependent cardioprotection induces dynamic modulation of these complexes, suggesting a functional role for these complexes in the genesis of protection against ischemic injury [94, 101].", "Mitochondria play an important part in myocyte energy production through oxidative phosphorylation.", "They are also involved in key aspects of cardiomyocyte survival, through regulating intracellular reactive oxygen species (ROS) generation, calcium homeostasis, and the release of apoptosis-inducing factors [102–104].", "Both mitoKATP and sarcKATP channels are present in the mammalian heart [105] and appear to be involved in IPC and cardioprotection [102].", "Gaudette et al. [106] demonstrated in a sheep model that protection provided by direct mitoKATP openers could be abolished by PKC antagonists (chelerythrine); and that protection mediated via the activation of PKC could be abrogated by administration of the mitoKATP channel antagonist 5-hydroxydecanoate (5-HD), a selective blocker of the mitoKATP channels, implying that the activation of PKC and of mitoKATP channels are both codependent and necessary for protection in the heart.", "Nozawa et al. [107] showed that IPC in rats produced a translocation of both PKC-ε and PKC-δ; however, 5-HD blocked the effect of IPC without blocking the translocation of the two PKC isoforms.", "These results suggest that the mitoKATP channel or the site of action of 5-HD is distal to PKC.", "The mitoKATP is one of the major and likely common effector pathways for improving cell survival after I/R injury.", "However, as for other pharmacologic inducers of cardiac conditioning, severe limitations exist on the clinical utility of drugs targeting this channel.", "As mentioned earlier in this paper, EA has not been shown to be useful for cardiac postconditioning; however, one positive study supports its use as a perconditioning stimulus [41].", "Because of mechanistic similarities, and because of the limited clinical utility of RPCT (i.e., involving surgical skin incision), we sought an easier, non-traumatic way to initiate NIC by abdominal sensory nerve activation using electrical stimulation—EA of the skin in this instance.", "We tested the hypothesis that remote electrical stimulation of the same skin field stimulated using RPCT and NIC (with capsaicin [42]) in our previous work could be used to activate cardiac conditioning, and herein report our results, for the first time.", "We performed electrical skin stimulation (ES) using electroacupuncture needles and current (for technical details, see the legend of Figure 2) 15 min prior to I/R or at the beginning of reperfusion (for 15 min thereafter).", "For ES, an EA needle was not inserted deep into the tissue as it is for acupuncture, but was inserted and only hooked through the skin (thus, the term ES).", "Infarct size was used to evaluate the results of I/R injury with or without prior ES treatments.", "The results of this study demonstrate that ES of specific points spaced equally along the line lateral to the umbilicus (the same region used for RPCT) induces a powerful 80%−85% (P≤0.05) reduction in infarct size when applied either as a preconditioning or as a postconditioning stimulus (Figure 2).", "This result supports the conclusion that NIC by ES at the anterior abdominal site is efficacious against I/R injury in the mouse, similar to RPCT.", "Since this intervention works as a postconditioning stimulus, is non-traumatic, non-ischemic, and does not require surgical intervention, we believe it has great clinical relevance.", "Nearly three decades of studies have taught us much about the mechanisms that underlie the endogenous cardioprotection afforded by IPC.", "The discovery of RPCT has offered significant therapeutic strategies for protecting the heart against myocardial I/R injury, and opened the door to the exploration of new modalities for triggering cardioprotection.", "The fact that some of these modalities work in multiple model species, and in humans [108], supports that these models are worthwhile laboratories to further our understanding and for preclinical development.", "Mechanistic and combinatorial studies support the hypothesis that preconditioning, perconditioning and postconditioning, as well as IPC, RIPC, nerve stimulation, EA, and NIC have many common mechanistic components.", "Yet mechanistic differences remain as well.", "For example, both IPC and RIPC involve the action of TNF-α, adenosine, and opioids, and yet these are not involved in the mechanism underlying RPCT [62] and NIC [85].", "EA has been reported to be effective as a preconditioning but not as a postconditioning stimulus [41].", "Yet all of these modalities require and employ the activity of NF-κB, PKC, bradykinin, and the mitoKATP channel [42, 62].", "We propose that all of these cardioprotective phenomena are in fact a reflection of a broader cardiac conditioning network, and that each stimulus works through slightly different initiating points in that network (Figure 1).", "IPC seems to invoke multiple initiating points, including vascular, humoral, and neural, while NIC and EA work primarily through the neural initiating portions of the network.", "Differences in initiation may relate to differential activation of components of the network; thus, their detectable activation, necessity, or dispensability in the particular phenomena under study are different.", "Our view of these phenomena as discrete derives primarily from the timing over the last 29 years of our experimental discovery of the different modalities and the mechanistic pieces.", "The proposed conditioning network includes: humoral factors; neural and vascular signaling; kinase, oxidative, and metabolic cascades in cardiac cells (and perhaps between cells of different types); gene expression; and related miRNA networks.", "As far as we can tell, these network components converge at several points, including PKC, bradykinin, and the mitochondria.", "As our work progresses, we predict that we will find additional components of the network that are more critical for NIC, ES, and RIPC, for example, but that are dispensable for IPC.", "We recognize that the existence of such a conditioning network is really a human construct that designates parts of the overall cellular and intercellular informational network as having a specific function related to myocardial salvage.", "However, for the purposes of research and translation, such constructs are helpful to delineate functions for molecules that may eventually be considered as therapeutic targets.", "There remains a great clinical need for non-ischemic and non-traumatic methods to initiate cardiac conditioning.", "At the moment, the discovery of NIC and EA/ES-induced neural pathways as remote non-ischemic initiators holds great promise for the development of pharmacologic and electroceutical approaches [109, 110] to elicit cardioprotection without causing ischemia or requiring surgical intervention.", "We demonstrate herein for the first time that NIC is protective as a postconditioning stimulus, establishing the clinical relevance of studying the underlying mechanism and further preclinical development." ]
[]
First reports of nasal and traumatic myiasis infection in endangered Przewalski's horses (Equus ferus przewalskii)
Myiasis has great economic and medical importance. However, myiasis in wildlife that is caused by oestroid flies is relatively rarely recorded compared with that in humans and domestic animals. Recently, during our research on the conservation of Przewalski's horse (PH), we observed two new records of oestroid flies parasitizing wildlife in China. The first is the horse nasal bot fly, Rhinoestrus sp. (Diptera: Oestridae), found in a dead PH from Kalamaili Nature Reserve. One morphotype (R. purpureus-like) was identified. The second is the Wohlfahrt's wound myiasis fly, Wohlfahrtia magnifica (Schiner, 1862) (Diptera: Sarcophagidae), which was collected from an open wound of a PH in the Wild Horse Breeding Research Centre. These observations extend the records of known hosts of these two oestroid myiasis agents. To the knowledge of the authors, infestation by Rhinoestrus and Wohlfahrtia larvae causing myiasis in wildlife has not been reported in China previously.
[ "AGRI", "IMMU", "MEDI" ]
[ "China", "Myiasis", "Przewalski's horse", "Rhinoestrus", "Wildlife", "Wohlfahrtia magnifica" ]
[ "1103", "2405", "2725" ]
[ "Myiasis is the infestation of live vertebrates, including humans, with dipterous larvae, which at least briefly feed on dead or living tissue, liquid body substances, or ingested food of the host (Zumpt, 1965).", "The Oestridae, Calliphoridae, and Sarcophagidae represent the three major families of myiasis-producing flies (Hall and Wall, 1995).", "They all belong to the superfamily Oestroidea, making oestroid flies the primary myiasis agents.", "There are records worldwide of myiasis in people and domestic animals caused by oestroid flies (Smith et al., 2005; Gaglio et al., 2011; Singh and Singh, 2015; Hall et al., 2016), suggesting that they have considerable global economic and medical importance.", "Although myiasis is one of the most widespread parasitological problems in veterinary practices, the distribution and impacts of even the important agents are poorly recorded in wildlife compared with records of domestic animals, probably because of the high technical difficulty of obtaining such records for wildlife (Hall and Wall, 1995; Hall et al., 2016).", "Our conservation research on Przewalski's horse (Equus ferus przewalskii, PH), the only extant wild horse species (Orlando et al., 2013), in Xinjiang over the past decade includes epidemiological data on stomach myiasis in equids and detailed biological information of horse stomach bot flies (Diptera: Oestridae, Gasterophilus) (Yang et al., 2013; Huang et al., 2016; Liu et al., 2016).", "Gastric myiasis caused by oestrids is very common in the released population from Kalamaili Nature Reserve (KNR) and the captive population from Xinjiang Research Centre for Breeding Przewalski's Horse (XRCBPH), especially in KNR, where almost all PHs are infested with Gasterophilus spp., resulting in gastric myiasis (Liu et al., 2016).", "The Gasterophilus parasite burden of deceased PHs in KNR can be more than 1000 individuals on average (Yang et al., 2013), which is much higher than the levels found in the Mongolian wild ass (Equus hemionus hemionus) and the local domestic horse (Equus ferus caballus) from the same area (Yang et al., 2013).", "A heavy parasitic load would risk the health condition of host and could even result in the death of the host (Czosnek, 1988; Getac hew et al., 2012).", "Therefore, we pay close attention to the myiasis in PH in order to conserve this iconic endangered species.", "Unlike gastric myiasis, very little information is available regarding the nasal or traumatic myiasis in PH or other wild animals in China.", "The infection of traumatic myiasis occurs when fly larvae infest open wounds of a vertebrate host, accompanied by significant pain, swelling, irritation, or inflammation (Zumpt, 1965), leading to severe health problems (Hall et al., 2016).", "The nasal bots infest the nasopharyngeal cavities and internal organs of mammal hosts, inducing rhinitis, sinusitis, and even death of the host (Angulo-Valadez et al., 2010).", "Here, we present two new records of oestroid flies causing nasal and traumatic myiasis in PH, the horse nasal bot fly, Rhinoestrus sp. (Diptera: Oestridae), and the Wohlfahrt's wound myiasis fly, Wohlfahrtia magnifica (Schiner, 1862) (Diptera: Sarcophagidae).", "There are two re-introduced populations of endangered PHs in Xinjiang of China: the captive population in the XRCBPH located in Jimsar County (44°12′12″N, 88°44′26″E), with 89 individuals, and the free-ranging population in the KNR located in the southeast corner of the northeast Junggar Basin, with up to 130 individuals.", "The PHs described in the present study are under the general surveillance program of the Wildlife Conservation Office of Altay Prefecture, Forestry Department of Xinjiang.", "During May to June in 2014, one injured PH from XRCBPH and one dead PH from KNR were examined for myiasis infection (see section 2.2).", "All of the larvae recovered were either placed in a plastic cup to pupate or stored in 70% alcohol for subsequent identification in the lab.", "Species identification was carried out following morphological traits described by Zumpt (1965), Grunin (1966) and Colwell et al. (2006).", "Specimens were deposited in the Museum of Beijing Forestry University (MBFU), Beijing (Accession number: larvae of Rhinoestrus sp., BFU138-139 & BFU208-209; larvae of W. magnifica, BFU70-72; adults of W. magnifica, BFU205, BFU8710-8715).", "This study was carried out in accordance with the guideline of the Institution of Animal Care and the Ethics Committee of Beijing Forestry University.", "All sample procedures in this study were performed with the help of a local veterinarian with approval of Wildlife Conservation Office of Altay Prefecture (Forestry Department of Xinjiang) and School of Nature Conservation (Beijing Forestry University).", "A 10-year-old male PH at KNR was observed ill in the morning of May 2, 2014 and died about 4 h later.", "Two living third-instar dipteran larvae were found on the ground near its head.", "The larvae were assumed to be oestrids, so the horse's nasal cavity was probed with tweezers and another four living third-instar larvae were collected.", "Two of the larvae were brought to the lab to pupate, and the rest were stored in alcohol for morphological study.", "An adult male PH raised at XRCBPH was hurt when fighting with another stallion on June 22, 2014, exposing a 10 cm × 10 cm wound on the left stifle (Fig. 1A).", "The PH was kept in a stall and could move freely.", "Three days later, it was immobilized to clean the wound, and dipteran larvae were found parasitizing the wound (Fig. 1B).", "A total of 52 larvae were collected.", "The wound was cleaned using normal saline before the PH was released.", "The PH has since recovered.", "The larvae isolated from the nasal cavity of the PH in case I failed to pupate.", "One larva preserved in ethanol was identified as (R. purpureus-like) on the basis of the shape of the posterior spiracular plates (i.e., higher than broad) and the distribution of spines on the dorsal surface of the third segment (i.e., two complete rows of spines) (Fig. 2) (see Zumpt, 1965: Fig. 222; Otranto et al., 2005: Fig. 1B and C).", "Morphological characteristics used to identify Rhinoestrus spp. were mainly limited to the descriptions by Zumpt (1965) and Grunin (1966).", "However, morphological variability of Rhinoestrus spp. has been documented previously (Otranto et al., 2005; Mula et al., 2013; Hilali et al., 2015): these studies demonstrate that the morphological features used by Zumpt (1965) to separate R. purpureus and R. usbekistanicus affecting horses in Italy and Egypt were inadequate.", "Further studies are required to identify which Rhinoestrus species exist in China.", "All larvae in case II (Fig. 1) were identified as W. magnifica according to the diagnosis of Zumpt (1965:109, Fig. 125), with 6, 25, and 21 individuals recognized to be first-, second-, and third-instar larvae, respectively.", "Fifteen of the third-instar larvae were brought to the lab, where they pupated on June 27th, and all emerged after 12 days.", "These two oestroid myiasis agents have not been previously reported in PHs in China.", "Wohlfahrtia magnifica, one of the most important obligatory traumatic myiasis agents worldwide (Hall et al., 2016), is often found in domesticated animals, e.g., in wounds of dogs and horses (Diakakis et al., 2006; Farkas et al., 2009) and inside the vulva of goats (Gaglio et al., 2011).", "More than 200 larvae of W. magnifica can be found in a single host (Gaglio et al., 2011).", "Cases of humans infested with this species are also reported from time to time (Singh and Singh, 2015).", "In Xinjiang, the Wohlfahrt's wound myiasis fly was first recorded infesting humans in 1981 (Yao, 1981), and the existence of this species has been confirmed by occasional reports of infesting humans or domesticated animals and in subsequent investigations of dipteran diversity (Han et al., 1998; Tang et al., 2003).", "However, this is the first documented case of Wohlfahrt's wound myiasis fly parasitizing wild animals in China.", "Rhinoestrus purpureus is an obligatory parasite of equids (Zumpt, 1965), and is rarely reported compared with Wohlfahrt's wound myiasis fly.", "Nevertheless, attacks on humans causing ophthalmomyasis and conjunctivitis by this species have been reported occasionally (Peyresblanques, 1964; Rastegaev, 1980).", "Myiasis caused by Rhinoestrus spp. in equids has rarely been reported in China and was confined to domestic Mongolian horses (Wang and Shi, 1963).", "Although there is little information available concerning the impact of nasal and wound myiasis on wildlife in China, it is well known that myiasis is an important animal welfare problem, even if it rarely causes severe pathology (Hall and Wall, 1995; Colwell et al., 2006).", "Current control strategies, including insect light traps and applying regular chemical control, may be effective in decreasing populations of pest flies for captive host populations.", "However, long term strategies should pay close attention to predicting the distribution of pest species based on present distributions, since this may help determine where these two species occur currently and highlight where they may occur following dispersal or climate change (Hall et al., 2016).", "This study was supported by the Fundamental Research Funds for the Central Universities [No. JC2015-04], and the National Science Foundation of China [No. 31572305]." ]
[ "First report of Rhinoestrus sp. and Wohlfahrtia magnifica parasitizing Przewalski's horses.", "Infestation by Rhinoestrus and Wohlfahrtia larvae causing myiasis in wildlife has not been reported in China previously.", "This study will enrich the epidemiological data of myiasis infection of wildlife in China." ]
Soft X-ray detection and photon counting spectroscopy with commercial 4H-SiC Schottky photodiodes
The results of electrical characterisation and X-ray detection measurements of two different active area (0.06 mm2 and 0.5 mm2) commercial 4H-SiC Schottky photodiodes at room temperature are reported. The devices exhibited low dark currents (less than 10 pA) even at a high electric field strengths (403 kV/cm for 0.06 mm2 diodes; 227 kV/cm for 0.5 mm2 diodes). The results of the X-ray measurements indicate that the diodes can be used as photon counting spectroscopic X-ray detectors with modest energy resolutions: FWHM at 5.9 keV of 1.8 keV and 3.3 keV, for the 0.06 mm2 and 0.5 mm2 devices, respectively. Noise analysis of the photodiodes coupled to a custom low noise charge sensitive preamplifier is also presented.
[ "PHYS" ]
[ "COTS", "Commercial off the shelf", "Photodiode", "Silicon carbide", "X-ray detector" ]
[ "3105", "3106" ]
[ "Wide bandgap semiconductors such as AlGaAs [1,2], GaN [3,4], GaAs [5,6] and SiC [7–10], are good potential candidate materials for radiation detectors required to operate in harsh environments and as such have received significant attention in recent years.", "SiC has been subject to particularly intensive study [11,12].", "The wide bandgap of SiC (4H-SiC=3.27 eV; 6H-SiC=2.86 eV), brings the benefits of a good visible−blind performance and low leakage currents even when operating at high temperature [9].", "Furthermore, SiC detectors are radiation hard [13,14].", "SiC has shown particular promise for use as an X-ray detector.", "The first experimental report of X-ray spectroscopy with SiC was in 2001; Np L X-rays and the 59.5 keV γ-ray emissions from an 241Am radioisotope γ-ray source were detected using a heavily N-doped 4H-SiC device with a Schottky contact [15].", "More recently, SiC detectors coupled to ultra-low-noise preamplifier electronics have been demonstrated to have excellent energy resolution (233 eV FWHM at 5.9 keV) even at high temperatures (100 °C) [10].", "Whilst SiC UV photodiodes have been readily commercially available from standard electronic component retailers for some years, there has so far been no report of the potential utility of these readily available devices as X-ray detectors.", "If such relatively cheap and easily available devices can be used to detect X-rays, it opens possibilities for low cost, radiation hard and high temperature tolerant X-ray detectors to be used for applications such as university-led cubesat missions and industrial applications.", "In this paper, results are reported charactering four commercially available 4H-SiC UV photodiodes of two different active areas, as current mode and photon counting spectroscopic detectors of soft X-rays from a 55Fe radioisotope X-ray source (Mn Kα=5.9 keV; Mn Kβ=6.5 keV).", "Four SiC UV Schottky photodiodes of two different active areas (0.06 mm2 and 0.5 mm2) manufactured by sglux SolGel Technologies GmbH, Berlin, Germany [16,17], were purchased from a standard electronics retailer.", "The photodiodes were supplied mounted in TO-18 packages.", "The UV windows of the TO-18 packages were removed as shown in Fig. 1 so that the devices could be directly illuminated with X-rays.", "The capacitances of all four photodiodes were measured as functions of applied reverse bias at room temperature (24 °C) up to 150 V in 1 V steps using an HP 4275 A Multi Frequency LCR meter and a Keithley 6487 Picoammeter/Voltage Source as the external voltage supply.", "The AC test voltage signal magnitude and frequency of the LCR meter were set at 60 mV r.m.s. and 1 MHz, respectively.", "National Instruments Labview software was used to automate the capacitance measurements.", "In order to extract the capacitances of the photodiodes themselves, the bond wires of sacrificial devices of the same type were removed to measure packaging capacitances.", "The capacitance of each photodiode was calculated by subtracting the capacitances of packages (0.67 pF for the 0.06 mm2 photodiodes and 0.65 pF for 0.5 mm2 photodiodes) from the total capacitances of the packaged devices.", "The measured capacitances and the calculated depletion width of each diode as functions of applied reverse bias are shown in Figs. 2 and 3, respectively.", "The photodiodes were fully depleted at an applied reverse bias of 120 V and 150 V for the 0.06 mm2 and 0.5 mm2 devices, respectively.", "The thickness of the depletion widths of the 0.06 mm2 and 0.5 mm2 photodiodes were determined to be 2.5 µm and 4.5 µm at 150 V reverse bias, respectively.", "In comparison to other previously reported wide bandgap X-ray detectors, they are thinner than previously reported GaAs X-ray detectors (7 µm) [5] and SiC X-ray detectors (25 µm) [10] but comparable to AlGaAs X-ray detectors (1.7 µm) [1].", "The leakage currents of the four photodiodes were measured as functions of applied reverse bias from 0 V to 100 V at room temperature (24 °C) with 1 V per step using a Keithley 6487 Picoammeter/Voltage Source.", "The results are presented in Fig. 5.", "National Instruments Labview software was used to automate the measurements.", "The photodiodes have low leakage currents (less than 10 pA) at high reverse bias (corresponding to leakage current densities of 937 pA/cm2 and 347 pA/cm2 at electric fields of 403 kV/cm for the 0.06 mm2 diodes; and1.4 nA/cm2 and 1.18 nA/cm2 at electric fields of 227 kV/cm for the 0.5 mm2 diodes).", "These are greater than some previously reported SiC X-ray detectors (1 pA/cm2 with mean electric fields of 53 and 103 kV/cm at room temperature) [10], but better than semi-insulating SiC X-ray photodiodes (from 2.6 to 65 nA/cm2 with internal electric fields from 7 to 28 kV/cm) [8] and GaAs X-ray photodiodes (e.g. 17.4 nA/cm2 with internal electric field 22 kV/cm) [5].", "In order to characterize the response of the diodes to illumination with soft X-rays, an 55Fe radioisotope X-ray source (Mn Kɑ=5.9 keV; Mn Kβ=6.5 keV; activity=260 MBq; active area=28.27 mm2) was placed 2 mm above the diodes.", "The reverse bias was increased from 0 V to 100 V in 1 V steps.", "The measured photocurrent (the current measured with the devices illuminated with the previously measured leakage current subtracted) are presented for the 0.06 mm2 and 0.5 mm2 devices in Fig. 6.", "Given the quantum efficiencies of the devices computed from the calculated depletion widths at 100 V reverse bias (2.48 µm and 4.4 µm, for the 0.06 mm2 and 0.5 mm2 devices, respectively), the activity geometry of the 55Fe radioisotope X-ray source, and the relative emission probabilities (Mn Kα 5.9 keV=0.245; Mn Kβ=6.49 keV=0.0338 [23]), the calculated expected photocurrents at 100 V reverse bias of 0.06 mm2 photodiodes (0.44 pA) and 0.5 mm2 photodiodes (6.3 pA), assuming only charge created in the depletion width contributed to the signal and 100% charge collection efficiency [19], were much smaller than the measured photocurrents of the 0.06 mm2 photodiodes (4.6 pA) and 0.5 mm2 photodiodes (18 pA) at 100 V reverse bias.", "The greater than expected photocurrent suggests that there were significant contributions to the signal from charge created outside of the depletion region by photons absorbed there.", "Furthermore, one 0.5 mm2 photodiode showed an exponential trend in photocurrent at reverse biases >60 V indicative of avalanche multiplication even though the electric field strength, assuming a uniform electric field across the depletion layer, was lower than that at which significant impact ionization has been previously observed to occur [20].", "This may have been due to defects in the material of the detector leading to localised regions of high electric field strength.", "The diodes were each in turn connected to a custom-made low-noise charge-sensitive preamplifier with a 2N4416A silicon input JFET (capacitance=2 pF).", "An ORTEC 572 A shaping amplifier and a multi-channel analyser (MCA) were connected to the preamplifier.", "Spectra were accumulated using an 55Fe radioisotope X-ray source.", "The 55Fe radioisotope X-ray source was placed 2 mm above the diodes.", "Spectra were accumulated at reverse biases from 0 V to 100 V in 10 V steps.", "The temperature of the detector and preamplifier was 22 °C.", "The live time limit for each spectrum was 60 s.", "Because of the large capacitances and low leakage currents of these devices, the longest shaping time available (10 µs) was selected [21,22].", "The resulting spectra were calibrated in energy terms by using the position of the zero energy noise peak and the position of the fitted Mn Kɑ at 5.9 keV for each spectrum as points of known energy on MCA's charge scale and assuming a linear variation of detected charge with energy.", "The energy resolution of the devices as quantified by the full width half maximum (FWHM) of the 5.9 keV peak is presented in Fig. 8 as a function of applied reverse bias.", "A photopeak could not be resolved with the 0.5 mm2 photodiodes at reverse biases below 50 V due to the large capacitance of the devices.", "For both sizes of device (0.06 mm2 and 0.5 mm2) the best energy resolutions, 1.8 keV FWHM and 3.3 keV FWHM, were obtained at the maximum investigated reverse bias (100 V).", "The 55Fe X-ray spectrum obtained at 100 V with one of the 0.06 mm2 photodiodes is presented in Fig. 7; two Gaussians were fitted to the observed photopeak to represent the Mn Kα (5.9 keV) and Mn Kβ (6.5 keV) emissions from the radioisotope X–ray source in the accepted ratio [23].", "The MCA's low energy threshold was set at 2.85 keV as a compromise between minimising the number of noise counts from the tails of the zero energy noise peak and maintaining the low energy response of the spectrometer.", "The energy resolutions at 5.9 keV were not as good as some wide bandgap devices specifically designed for X-ray detection e.g. GaAs mesa photodiodes (840 eV, area of 0.03 mm2; and 1.07 keV, area of 0.13 mm2) [24], AlGaAs X-ray detectors (1.1 keV, area of 0.03 mm2 [25]; and 1.27 keV, area of 0.13 mm2 [1]), and the best result of SiC X-ray detectors (196 eV at 30 °C, area of 0.4 mm2) [10].", "However, the FWHM of the 0.06 mm2 area diode was better than some larger area AlGaAs photodiodes (0.13 mm2) (1.95 keV) [26].", "The electronic noise of this system consists of five contributors: series white noise, parallel white noise, 1/ƒ series noise, dielectric noise and the induced gate drain current noise [28].", "A detailed explanation of the origin of each electronic contributors can be found in Ref. [1].", "The total electronic noise contribution was measured for the system with the 0.06 mm2 devices by injecting a delta test pulse signal into the preamplifier with the detector connected but unilluminated [21,22,29].", "The dielectric noise (and series white noise from unknown stray capacitances [1]) can be calculated by the subtraction of the calculated series white noise including induced gate drain current noise, parallel white noise, and 1/f noise in quadrature from the measured total electronic noise [5].", "The other noise components were calculated as per Ref. [1].", "The calculated electronic noise components are presented as a function of reverse bias in Fig. 9.", "As is the case for other detectors connected to similar charge-sensitive preamplifiers, the dielectric noise (and noise from stray capacitances) is the most significant noise component [1,5].", "Such noise is thought to arise from dielectric materials of the capacitances and packages [21].", "The presently reported preamplifier used a wire-end input JFET for convenience; a bare die JFET directly wire-bonded to the detector would be expected to significantly reduce the noise.", "If this noise could be eliminated entirely, an energy resolution (FWHM at 5.9 keV) of 1.1 keV could be achieved with the 0.06 mm2 photodiode.", "Incomplete charge collection noise is related to the detector's charge diffusion and collection properties as well as to the trap density distribution in the detector [28].", "In this system, the incomplete charge collection noise can be calculated by subtracting in quadrature the predicted Fano noise and the total electronic noise from the measured energy resolution at 5.9 keV.", "The calculated contributions from incomplete charge collection for the 0.06 mm2 photodiode are shown in Fig. 10.", "Interestingly, the incomplete charge collection noise was independent of applied electric field.", "This supports the hypothesis – introduced in Section 3.2 to explain the greater than expected photocurrent – that significant amounts of charge created outside of the depletion region (by photons absorbed in those positions) contributed to the detected signal.", "Since the applied bias mainly resulted in an electric field across the epi-layer/depletion region, the field strength in the volume below the depletion region remained low, regardless of applied bias.", "Thus the incomplete charge collection noise, which probably arose from recombination of electrons and holes in the low field region remained constant.", "The data and explanation are consistent high quality material close to top Schottky contact exhibiting comparatively little charge trapping and a volume of material with consistently low electric field strength below it showing moderate amounts (≈58 e− rms equivalent noise charge) of incomplete charge collection noise.", "Measurements characterizing four commercial 4H-SiC Schottky photodiodes of two different areas (0.06 mm2 and 0.5 mm2) at room temperature have been presented.", "The measurements of capacitance as functions of applied reverse bias showed consistent characteristics between photodiodes of the same active area.", "The depletion widths (2.5 µm and 4.5 µm, respectively) of the devices were calculated based on the capacitance measurements.", "The diodes were found to be fully depleted at ≥120 V reverse bias.", "Measurements of leakage current as a function of applied reverse bias showed low leakage currents among these diodes (less than 10 pA) at 100 V reverse bias; the current densities were found between 300 and 900 pA/cm2 with the 0.06 mm2 diodes (at electric field strengths of 403 kV/cm) and 1.2 to 1.4 nA/cm2 with 0.5 mm2 diodes (at electric field strengths of 227 kV/cm).", "The performance of these devices as X-ray detectors was investigated by the measuring the photocurrent generated when the devices were illuminated by an 55Fe radioisotope X-ray source, and by connecting the photodiodes to a custom low-noise charge-sensitive preamplifier and investigating their performance as detectors for photon counting X-ray spectroscopy.", "The photocurrents were measured to be 5 pA with the 0.06 mm2 photodiodes and 7–19 pA with 0.5 mm2 devices.", "The detectors functioned as photon counting spectroscopic X-rays detectors with modest energy resolution (the FWHM at 5.9 keV of the 0.06 mm2 and 0.5 mm2 diodes were 1.8 keV and 3.3 keV at 100 V reverse bias, respectively).", "Although the energy resolutions were not as good as many custom X-ray detectors, the results indicated these photodiodes can be used for photon counting X-ray spectroscopy at room temperature albeit with modest energy resolution.", "Noise analysis of this system was also presented.", "It showed that dielectric noise and series white noise from stray capacitances were the dominant noise sources in the system.", "If the noise, which was a consequence of the non-optimal design of the connection of the detectors to the preamplifier, could be entirely eliminated the energy resolution would be expected to improve to 1.1 keV.", "In future publications, the performance of these devices at high temperature will be reported, as will characterisation of the detectors when illuminated with X-ray photons of different energies." ]
[]
Extensions of the mTPI and TEQR designs to include non-monotone efficacy in addition to toxicity for optimal dose determination for early phase immunotherapy oncology trials
With the emergence of immunotherapy and other novel therapies, the traditional assumption that the efficacy of the study drug increases monotonically with dose levels is not always true. Therefore, dose-finding methods evaluating only toxicity data may not be adequate. In this paper, we have first compared the Modified Toxicity Probability Interval (mTPI) and Toxicity Equivalence Range (TEQR) dose-finding oncology designs for safety with identical stopping rules; we have then extended both designs to include efficacy in addition to safety – we determine the optimal dose for safety and efficacy using these designs by applying isotonic regression to the observed toxicity and efficacy rates, once the early phase trial is completed. We consider multiple types of underlying dose response curves, i.e., monotonically increasing, plateau, or umbrella-shaped. We conduct simulation studies to investigate the operating characteristics of the two proposed designs and compare them to existing designs. We found that the extended mTPI design selects the optimal dose for safety and efficacy more accurately than the other designs for most of the scenarios considered.
[ "PHAR" ]
[ "Early phase immunooncology design considering efficacy and safety", "Eff-Tox design", "Extended TEQR design", "Extended mTPI design", "Optimal biological dose isotonic design", "Umbrella-shaped dose-response curve" ]
[ "3004" ]
[ "Several dose finding oncology designs have been developed that are improvements over the 3 + 3 design in terms of accuracy of maximum tolerated dose (MTD) selection as well as other operating characteristics such as the percentage of patients under-dosed [1–6].", "There are also designs that incorporate efficacy in dose selection, in addition to safety.", "These include the seamless Phase 1/2 SEARS design [7,8], a seamless 2-step Phase 1/2 design [9,10], designs to find the optimal biological dose [11,12], the Eff-Tox design [13,14] and the Toxicity and Efficacy Probability Interval (TEPI) design [15] among others [16–19].", "In this paper, we focus on two relatively recent dose-finding designs that have been proposed to determine the MTD, namely the mTPI and the TEQR designs [4,5], and then extend them to choose the optimal dose for both safety and efficacy.", "Our aim is to identify the best (optimal) dose using a practical design, and not specifically to optimize our proposed design(s).", "We define the optimal dose to be the dose with the highest efficacy below or at the MTD.", "The mTPI design is a Bayesian dose finding design, where the dose finding decisions are based on whether a statistic called the Unit Probability Mass (UPM) has its highest value in the target dose limiting toxicity (DLT) interval or in the interval above or below it.", "The TEQR design uses a similar concept for dosing decisions but provides a frequentist counterpart to the Bayesian mTPI design, since the dosing decisions in the TEQR design are based on the empirical DLT rates.", "Phase I trials are generally very small and the accuracy of MTD selection is low with such a small sample size.", "Hence, we first compare the frequentist TEQR and the Bayesian mTPI dose-finding designs for accuracy of MTD selection for various sample sizes while requiring identical stopping rules.", "We then extend the mTPI and TEQR designs with a moderately large sample size to choose an optimal dose based on both safety and efficacy by considering safety and efficacy outcomes using Bernoulli distributions.", "A key part of our evaluation of these designs is to determine their performance when the efficacy response rate does not necessarily increase monotonically with increasing dose.", "With immunotherapy and other novel therapeutics, the traditional assumption of increasing efficacy with increasing dose may no longer hold [11,20].", "Thus, in our simulations to evaluate these designs, we assume that the true DLT rates increase monotonically with an increase in dose but we do not assume that this is true for the efficacy response rates.", "We allow multiple types of curves for dose-response in the simulations: monotonically increasing, plateau, or umbrella-shaped curves.", "In this context of potentially non-monotone efficacy, we apply isotonic regression to the differences in observed response rates between adjacent dose levels and investigate its use in selecting an optimal dose for safety and efficacy.", "The work by Li et al. [15] proposes using a statistic called the joint unit probability mass (JUPM) to incorporate both toxicity and efficacy, to extend the mTPI design.", "This Toxicity and Efficacy Probability Interval (TEPI) design, as well as other designs such as the Eff-Tox design and the Optimal Biological Dose (OBD) Isotonic design [12], requires that efficacy or a surrogate of efficacy be available in a similar time frame as the DLT observation period, for dosing decisions.", "Our extended mTPI and TEQR designs do not require this, since we use the efficacy information for optimal dose selection only at the end of the trial.", "Thus, we propose a simple way of extending the mTPI and TEQR designs to include efficacy in dose selection, using isotonic regression.", "We finally compare the accuracy of dose selection of the extended mTPI and TEQR designs to that of the Eff-Tox design, the OBD Isotonic design and the TEPI design.", "The mTPI design is a Bayesian design that uses the unit probability mass (UPM) statistic, defined as the ratio of the probability mass of the interval and the length of the interval [4], for the dose finding decisions.", "The toxicity probability scale is divided into three intervals, namely (0, pT-ε1), [pT-ε1, pT+ε2] and (pT+ε2, 1), where pT is the target probability of DLT and ε1 and ε2 are used to define the interval for the target DLT rate.", "These three intervals correspond to under-dosing, correct dosing and over-dosing respectively.", "The rules for escalating, staying at the same dose or de-escalating depend on which of these intervals has the highest UPM for that dose level, based on a beta-binomial posterior distribution formed from the likelihood of the observed DLT data and a beta (1,1) prior.", "For example, the next cohort of patients will be treated at the same dose if the UPM is the largest for the correct dosing interval.", "The trial stops if dose level 1 is too toxic or if the pre-specified maximum sample size is reached or exceeded.", "The TEQR design is a frequentist design based on the empirical DLT rate [5].", "As in the mTPI design, the toxicity probability scale is divided into three intervals, namely (0, pT-ε1), [pT-ε1, pT+ε2] and (pT+ε2,1).", "The rules for escalating, staying at the same dose or de-escalating depend on which of these intervals contains the empirical DLT rate for that dose level – for example, if the empirical DLT rate lies in the interval [pT-ε1, pT+ε2], the next cohort of patients will be treated at the same dose.", "The trial stops if dose level 1 is too toxic or when a dose level achieves the pre-specified MTD sample size.", "In both the mTPI and TEQR design, we stay at the current dose if the current dose is safe but the DLT data indicate that the next higher dose is too toxic.", "When the true underlying DLT rate (or response rate) increases with an increase in dose, the observed DLT (or response) rate is also expected to be a monotonically non-decreasing function of dose.", "However, this may not always be what is observed due to the small sample size in each dose level in dose-finding oncology trials.", "Isotonic regression is a weighted regression and a smoothing procedure that can be used to provide estimates of the DLT (or response) rate that are monotonically non-decreasing functions of dose [21].", "This then enables us to determine the highest dose level that is acceptable for safety and the lowest dose level that is acceptable for efficacy.", "In a trial using a standard mTPI or TEQR design, the dose chosen for safety is the highest dose level with a DLT rate that is closest to (and below) the pre-specified DLT threshold rate (say 0.33) after applying isotonic regression at the end of the trial to the observed DLT rates.", "In our extensions of the mTPI and TEQR designs, isotonic regression is also applied independently to the observed efficacy response rates at the end of each trial, when the true underlying response rates are thought to be monotonically increasing or monotonically non-decreasing with an increase in dose.", "Since the estimated response rates will be monotonically non-decreasing with an increase in dose after applying isotonic regression, we choose as the optimal dose for safety and efficacy the highest dose level where the DLT rate is less than or equal to 0.33 after isotonic regression, only if the smoothed response rate at that dose level is equal to or above the efficacy threshold (say response rate of 0.4).", "For example, if dose level 4 is chosen after isotonic regression as the highest dose level with a DLT rate <= 0.33 and dose level 3 or lower is chosen after isotonic regression as the lowest dose level with a response rate >= 0.4, then dose level 4 is the optimal dose for safety and efficacy since the response rate at dose level 4 will be >= 0.4 in this monotone case.", "However, if dose level 3 is chosen for safety after isotonic regression and dose level 4 is chosen for efficacy after isotonic regression, then no dose level is optimal for safety and efficacy because the efficacy threshold of a response rate of 0.4 is not crossed at dose level 3, but only at dose level 4.", "If dose level 3 is chosen for both safety and efficacy after isotonic regression, then dose level 3 is the optimal dose for safety and efficacy (Figs. 1 and 3).", "The OBD Isotonic design by Zang et al. [12] uses a double-sided isotonic approach to determine the peak of an umbrella-shaped dose response curve.", "We use a simpler method to determine the peak.", "When there is a peak in the dose-response curve, an umbrella-shaped dose-response curve, we apply isotonic regression to the differences in observed response rates between adjacent dose levels obtained at the end of each simulated trial.", "These differences provide the change between discrete dose levels and function like a derivative, or rate of change.", "For a convex curve, the derivative is 0 at the peak, and the sign of the derivative changes from positive before the peak to negative after the peak.", "This provides the approach we use to determine the peak of an umbrella shaped dose-response curve – we apply isotonic regression at the end of each simulated trial to the differences in observed response rates between adjacent dose levels.", "As the version of isotonic regression we used allows only monotonically non-decreasing estimates, the differences were constructed to be negative when the curve increases and positive when the curve decreases.", "Applying isotonic regression to the differences, we observe where the sign of the differences switches from negative to positive, to determine the peak of the curve (Fig. 1).", "This method to determine the peak of an umbrella-shaped dose response curve is demonstrated to work well with examples in the Results Section and Appendix Section 5.", "Once the peak of the dose response curve is found, the optimal dose for efficacy and safety can be determined, as explained in Figs. 2 and 3.", "We compare the results for accuracy of optimal dose selection of the extended mTPI and TEQR designs to those of the Eff-Tox, OBD Isotonic and TEPI designs for various scenarios of true toxicity and efficacy rates.", "The Eff-Tox design is a Bayesian design that considers the trade-off between the probabilities of drug toxicity and efficacy to determine the optimal dose for each new cohort of patients.", "The stopping point of the trial is usually at a pre-specified sample size.", "Further details are provided in the references by Thall et al. [13,14].", "The details of the OBD Isotonic design are provided in the reference by Zang et al. [12].", "To determine the OBD, an admissible set of doses satisfying a safety criterion similar to that used in the Eff-Tox design, is first defined.", "The OBD is then the lowest dose with the highest response rate within the admissible set of doses, while still being safe.", "The stopping point of the trial is usually at a pre-specified sample size.", "As mentioned earlier, the TEPI design is an extension of the mTPI design that includes efficacy and safety in dose selection.", "The stopping point of the trial is usually at a pre-specified sample size.", "Further details are provided in the reference by Li et al. [15].", "We generate two Bernoulli distributed binary random variables for the toxicity and efficacy outcomes of simulated patients – these random variables can be generated as either uncorrelated or correlated.", "In most of the simulations presented in this paper, we generate the DLT occurrence for patients at each dose level from values based on a logistic dose toxicity curve, whose two coefficients are calculated using the following parameters: true DLT rate at starting dose (dose level 1, 100 units) of 0.01 and true DLT rate of 0.2 at the MTD (dose level 4, 501 units).", "However, the dose response curve for the efficacy of simulated patients at each dose level varies by the simulation scenario, with 3 possibilities: it can monotonically increase, increase until reaching a plateau and then remain at the same level, or follow an umbrella-shape where it increases until reaching a peak after which it decreases (Table 1).", "We have created SAS codes, available on request, to simulate both the extended mTPI and TEQR designs.", "To obtain the statistical operating characteristics for each design, we perform 1000 simulated trials for each scenario.", "The rules for escalation, de-escalation or remaining at the same dose for each simulated trial are based on the number of observed DLTs.", "Two different stopping rules are considered in our simulations, which are the usual stopping rules for the mTPI and TEQR designs respectively; a simulated trial stops when a) the total planned sample size is reached or b) the planned MTD sample size is reached.", "For both stopping rules, the simulated trial would also stop if dose level 1 is determined to be too toxic.1", "In our simulations of these designs, we also track the efficacy response of each patient and the resultant efficacy response rate at each dose level.", "Although the dose escalation/staying/de-escalation decisions during the trial are determined only by the number of observed DLTs, at the end of each simulated trial we choose a dose that is optimal for both safety and efficacy based on the observed DLT and response rates at each dose level (Figs. 1–3).", "The input parameters used in our SAS code for the mTPI and TEQR designs are provided in Appendix Table 1.", "The coefficient of correlation r between efficacy and toxicity is set to 0 (independent true toxicity and efficacy rates) for the simulation results presented in the main text.", "This is because Cai and co-authors [22] showed that joint modeling of efficacy and safety does not necessarily improve the performance of the dose finding, especially when efficacy is weakly correlated with toxicity.", "However, the results can be investigated for correlation coefficients other than zero (Appendix Section 2) within the valid range of values that the correlation coefficient can assume.", "The simulations presented in this paper consider the following scenarios: 1) as a reference, we consider only toxicity rates and ignore efficacy; 2) both toxicity and response rates increase with increasing dose; 3) toxicity rates increase with increasing dose, and response rates are monotonically increasing but reach a plateau after a certain dose; 4) toxicity rates increase with increasing dose, but the response rate has an umbrella-shape with a peak at an intermediate dose.", "We then compared the accuracy of dose selection of the extended mTPI and TEQR designs with that of the Eff-Tox, OBD Isotonic and TEPI design.", "These simulations comparing the various designs include the scenarios above (monotonically increasing, plateauing and umbrella-shaped dose-response curves).", "Only the monotonically increasing DLT rates with increasing dose shown in Table 1 are used in the simulations for Table 2 with no efficacy considered; isotonic regression is applied to the observed DLT rates at the end of each simulated trial to determine the MTD.", "We use the same stopping rules for the mTPI and TEQR designs and compare them for accuracy of MTD selection.", "We use the usual stopping rules of the mTPI design (stopping rule a)), namely stop the trial when the total planned sample size is reached or when dose level 1 is too toxic, for both the mTPI and TEQR designs and compare their performance for the accuracy of MTD selection (Table 2); dose level 4 with a true DLT rate of 0.2 is the true MTD in this scenario.", "Results for the accuracy of MTD selection for the mTPI and TEQR designs when the stopping rules of the TEQR design (stopping rule b)) are used are not shown here.", "However, in general, when identical stopping rules are used for both the designs, the Bayesian mTPI design is more accurate than the frequentist TEQR design in selecting the true MTD, with the same (Table 2) or a similar number of subjects.", "Using the UPM statistic for dose finding as in the mTPI design, rather than the empirical DLT rate as in the TEQR design, appears to estimate the MTD more accurately, put a larger percentage of patients at the MTD as well as under-dose a smaller percentage of patients.", "Although the association between accuracy of MTD selection and cohort size given the same sample size may not be very clear from Table 2, very small or very large cohort sizes would not be optimal.", "However, it is clear that given the same cohort size, the accuracy of MTD selection increases when the total sample size is increased.", "Thus, in the following sets of simulations, we use a moderately large sample size of 50 subjects to evaluate efficacy and safety.", "We show results in the following sections for a cohort size of 5, that is moderate, with a sample size of 50 but our codes can be used to obtain results for other cohort sizes (for e.g. cohort size of 3 with a total sample size of 51).", "We compare in Table 4 the accuracy of optimal dose selection of our extended mTPI and TEQR designs to that of the Eff-Tox design, the OBD Isotonic design and the TEPI design, for some scenarios of true DLT and response rates.", "All the input parameters used in the Eff-Tox design, OBD Isotonic design and TEPI design simulations are provided in Appendix Section 6.", "For scenarios 1 and 2, where the DLT rate increases substantially between dose levels 3 and 4, while the response rate increases only slightly, the proposed extended mTPI and TEQR designs outperform the other designs with a higher percent of selecting dose level 4 as the optimal dose.", "The TEPI design has a higher percentage of selecting dose level 3 as the optimal dose, while the other designs pick dose level 4.", "This may be justified as clinicians may have different judgements on which of these two dose levels is optimal in terms of a trade-off between efficacy and toxicity.", "The TEPI design selects the dose based on a utility function with a safety and efficacy trade off.", "In these two scenarios, the utility values for dose levels 2, 3 and 4 are similar (refer to Appendix Section 6 for the TEPI utility function).", "For scenario 3, the TEPI design does very well in selecting the optimal dose, and all designs consistently select dose level 3 as the optimal dose most frequently.", "For Scenario 4, the extended mTPI and TEQR designs choose dose level 1 as the optimal dose most frequently.", "The Eff-Tox design also chooses dose level 1, which has the highest trade-off value calculated per the Eff-Tox method, as the optimal dose most frequently.", "The TEPI and OBD Isotonic designs choose dose level 2 as the optimal dose more frequently than dose level 1.", "For the TEPI design, this may be due to the fact that the design allows enrollment of the next cohort at the current dose level 2 even if the toxicity rate is between 0.2 and 0.33 provided the efficacy rate is high enough (refer to TEPI dosing decision table in Appendix Section 6).", "For the TEPI and OBD Isotonic designs, when we start at dose level 2, they may take a longer time or more subjects to de-escalate given the acceptable efficacy at dose 2.", "However, both designs eventually select dose level 1 as the optimal dose with higher probability than dose level 2, as the sample sizes increase in our simulations (results not shown here).", "For scenario 5, all designs pick dose level 3 as the optimal dose most frequently.", "The OBD Isotonic design performs very well for this scenario (umbrella-shaped dose response curve), while the Eff-Tox and TEPI designs have a lower probability of selecting the optimal dose for such a dose-response curve.", "In summary, among the designs considered, the extended mTPI design selects the optimal dose more accurately than the other designs for most of the scenarios.", "The extended TEQR design performs as well as or better than the Eff-Tox design in terms of accuracy of optimal dose selection in most of the scenarios considered.", "We have first compared the frequentist TEQR design with the Bayesian mTPI design for accuracy of MTD selection, when using the same stopping rules for both designs.", "In the scenarios considered, the Bayesian mTPI design is generally more accurate in selecting the true MTD than the frequentist TEQR design, when identical stopping rules and the same or similar sample sizes are used for both the designs.", "The mTPI design also puts a larger percentage of patients at the MTD and under-doses a smaller percentage of patients compared to the TEQR design.", "For both designs, given the same cohort size, the accuracy of MTD selection increases when the total sample size is increased.", "We then extended the mTPI and TEQR designs to also consider efficacy in addition to safety in dose selection, in a moderately sized trial.", "In our extended mTPI or TEQR trial designs, isotonic regression is always applied to the observed DLT rates at the end of the trial, since the true DLT rate is always assumed to increase with an increase in dose.", "The technique that is most appropriate to apply to the observed response rates depends on the drug's properties (Fig. 1).", "For this, clinical knowledge or judgement about the true underlying response rates of the study drug is required to have a good initial guess at the shape of the true dose-response curve.", "When the true underlying response rates are thought to increase monotonically with an increase in dose or are thought to first increase monotonically and then plateau after a certain dose level, isotonic regression can also be applied to the observed response rates at the end of the extended mTPI or TEQR trial.", "The optimal dose level for safety and efficacy is chosen to be the highest dose level for which the DLT rate after applying isotonic regression is below or at the chosen toxicity threshold (e.g. DLT rate <= 0.33), only if the threshold for response rate is crossed at that dose.", "If the threshold for response rate is not reached at the highest dose level at which the smoothed DLT rate is below or at the toxicity threshold, then no dose level is chosen as optimal for safety and efficacy (Fig. 3).", "When the underlying true response rates are thought to possess a clear peak (e.g. umbrella shaped dose-response curve), isotonic regression on the differences in observed response rates between adjacent dose levels, along with the sign of these differences, can be used to reveal or identify this peak dose level for efficacy.", "This information of the peak dose level for efficacy can then be used in conjunction with the dose level picked as the highest dose level that is safe, to select an optimal dose for safety and efficacy.", "For example, if the peak dose level identified for efficacy is equal to or lower than the highest dose level that is safe, then the peak dose level identified for efficacy is chosen as the optimal dose for safety and efficacy, assuming that the peak is above the specified efficacy threshold – if not, no dose level is chosen as the optimal dose.", "If the peak dose level identified for efficacy is higher than the highest dose level that is safe, then the highest dose that is safe is chosen as the optimal dose, only if the response rate at that dose is greater than or equal to the efficacy threshold – if not, no dose is chosen as the optimal dose.", "Thus, we cannot select a dose that exceeds the threshold toxicity, but if the maximum/peak efficacy of the drug is reached at a lower dose, we can select that dose as optimal assuming the efficacy threshold is crossed at that dose (Figs. 2 and 3).", "When we use isotonic regression on the differences in observed response rates between adjacent dose levels when there is no peak in the true response rates (for e.g. monotonically increasing true response rates), we find that no dose level is selected as the peak very frequently.", "For a plateauing response curve, we find that no dose level is selected as the peak quite frequently and the dose level at which the response rate starts plateauing is not chosen frequently as the peak dose.", "Thus, the plateau/peak is not clearly revealed by this technique.", "Hence, in these cases (monotonically increasing and plateauing response rates), applying isotonic regression on the response rates themselves provides better performance than applying isotonic regression to the differences in observed response rates between adjacent dose levels.", "We compared the extended mTPI and TEQR designs to the Eff-Tox design, the OBD Isotonic design and the TEPI design for accuracy of optimal dose selection for some scenarios of true efficacy and toxicity rates.", "We found that the extended mTPI design selects the optimal dose more accurately than the other designs for most of the scenarios considered.", "The extended TEQR design performs as well as or better than the Eff-Tox design in terms of the accuracy of optimal dose selection for most of the scenarios considered.", "In summary, we have proposed two designs that incorporate toxicity and efficacy in dose selection, and found that the extended mTPI design selects the optimal dose more accurately than the other designs for most of the scenarios.", "We found that isotonic regression itself applied on the differences in observed response rates between adjacent dose levels could be used to identify the peak of a dose-response curve with a clear maximum, such as a convex umbrella-shaped dose-response curve.", "For other dose-response curves, such as monotonically increasing or plateau, applying isotonic regression to both the observed DLT and response rates independently can be used to determine the optimal dose for toxicity and efficacy.", "Finally, we note that our models and isotonic regression method to identify an optimal dose for safety and efficacy can be used for other binary efficacy endpoints, such as the progression-free survival or overall survival at a landmark time (e.g, 3 months)." ]
[]
Survey of the operation and system study on wireless charging electric vehicle systems
This survey investigates the state-of-the-art in operations and systems-related studies of wireless charging electric vehicles (EVs). The wireless charging EV is one of emerging transportation systems in which the EV's battery is charged via wireless power transfer (WPT) technology. The system makes use of charging infrastructure embedded under the surface of the road that transfers electric power to the vehicle while it is in transit. The survey focuses on studies related to both dynamic and quasi-dynamic types of wireless charging EV – charging while in motion and while temporarily stopped during a trip, respectively. The ability to charge EVs while in transit has raised numerous operations and systems issues that had not been observed in conventional EVs. This paper surveys the current research on such issues, including decisions on the allocation of charging infrastructure; cost and benefit analyses; billing and pricing; and other supporting operations and facilities. This survey consists of three parts. The first provides an orienting review of terminology specific to wireless charging EVs; it also reviews some past and ongoing developments and implementations of wireless charging EVs. The second part surveys the research on the operations and systems issues prompted by wireless charging EVs. The third part proposes future research directions. The goal of the survey is to provide researchers and practitioners with an overview of research trends and to provide a guide to promising future research directions.
[ "COMP", "ENGI", "SOCI" ]
[ "Electric vehicles", "Research survey", "Transportation planning", "Transportation systems", "Wireless charging" ]
[ "1706", "2203", "2205", "3313" ]
[ "As electric vehicles (EVs) have become increasingly popular as a mainstream transportation solution, opportunities to recharge the vehicle away from home have become a critical issue.", "Wireless charging EV is a type of EV in which charging is done using wireless power transfer (WPT) technology, which does not require any physical contact in the process of transferring electric energy.", "WPT has been successfully applied for charging various handheld devices, such as medical devices, electronic toothbrushes, and smartphones.", "It has also been widely used for automated material handling systems in semiconductor fabrication and flat-panel display production lines (Covic and Boys, 2013).", "Wireless charging technology was first commercialized for automobiles to eliminate the conventional charging of ‘plug-in’ EVs – charged by connecting a wired cable from a charger to the vehicle.", "The first wireless charging technology to be deployed was stationary, the system having been designed to charge EVs in garages or public parking spaces, when the vehicle is not operating for an extended period.", "Because a physical connection is not required, there has been major interest in the possibility of charging EVs while they are in transit.", "Charging an EV while in motion is called dynamic wireless charging.", "A typical dynamic wireless charging EV is a pure, battery-only EV that takes its electrical charge in motion, remotely, from a wireless charger installed underneath the road surface.", "Roads capable of supplying electric power to wireless charging EVs are called electrified roads or charging lanes.", "There is also a third wireless charging category, quasi-dynamic wireless charging, in which the charging takes place when the EV decelerates to or accelerates from a resting position.", "For both dynamic and quasi-dynamic wireless charging, charging can be done while the EV is in transit.", "These new charging mechanisms extend the operational range of EVs with both rapid boost charging during brief station stops and dynamic or “on the fly” charging opportunities.", "Stationary wireless charging makes the charging process safer and more convenient (Cirimele et al., 2016).", "However, in terms of charging time, frequency, the operation of the vehicle, and charging station allocation, stationary charging is not significantly different from conventional plug-in conductive charging.", "In contrast, dynamic and quasi-dynamic wireless charging enable the EVs battery to be charged while in operation.", "This capability has raised new operations and infrastructural design issues that had never been raised for conventional plug-in EVs.", "These issues are the focus of this paper.", "Note that in this paper, references to “wireless charging EV” indicate dynamic and quasi-dynamic wireless charging EVs, if not specified.", "For the readers interested in the static charging technology, refer to the following work: (Abe et al., 2000; Birrell et al., 2015; Wu et al., 2012; Foley et al., 2010).", "It should also be stated that although the term wireless charging EV suggests a single vehicle unit, it should be understood as a system comprised of EVs and the charging infrastructure.", "Further terminological and categorical distinctions are discussed in subsequent sections.", "By contrast, research on operations and systems issues is still nascent.", "Definitions of operations and systems challenges remain somewhat broad.", "In this paper, however, they refer specifically to modeling, analysis, and methods that can be leveraged to support and enhance effective and efficient operations of wireless charging-based transportation systems.", "The term system is used explicitly because the research challenges are not limited to just vehicular components but also include: supporting facilities such as charging infrastructure; information handling in intelligent transportation systems (ITS); integration with smart grids; economic analyses for the pricing of charging; and other related managerial strategies and tactics.", "Studies that deal with these issues tend to address the following questions:", "What are the long-term and short-term economic benefits of wireless charging EVs relative to other existing transportation systems?", "How much charging infrastructure should be allocated to maximize the economic benefit of this emerging transportation system?", "What are the critical design parameters for wireless charging EVs, given transportation requirements, and how do we determine them?", "How can policymakers design operational policies, rules, and regulations for new transportation systems?", "How can we design information systems to optimize the systems benefits?", "How would this new transportation system affect existing traffic and how should this be analyzed?", "What are the environmental benefits of wireless charging EV over conventional transportation systems in terms of stainability?", "Operations and systems research for wireless charging EVs is a multidisciplinary research domain, the success of which depends on integrated efforts.", "There is little doubt that academic research on operations and systems perspectives is critical, particularly in the current stage of the technology’s development.", "WPT technology, the key enabler of the wireless charging EV, has been improving rapidly.", "As mentioned previously, several commercialized WPT systems have already been made available.", "Given that the technology has reached this phase so rapidly, systematic investigations of its operations and systems implications for wireless charging EVs are needed.", "Several reasons can be given for why such an investigation is appropriately timed.", "First, there is high demand for surveys from government sectors, particularly policymakers and regulators, in addressing the operations and systems challenges posed by wireless charging EVs, especially for understanding the state-of-the-art.", "For example, in South Korea, one of the first countries where wireless charging EVs have been commercialized for public transportation, traffic and transportation administration agencies have funded research on analyzing the economic value of such systems.", "Similarly, the Transport Research Laboratory in the UK investigated the feasibility of dynamic WPT for EVs and published Feasibility study: powering electric vehicles on England’s major roads (Highway England, 2015) on behalf of Highways England, the government agency in charge of operating, maintaining, and improving England’s motorways and major roads.", "The European Union has also launched FABRIC (feasibility analysis and development of on-road charging solutions for future electric vehicles) a project to investigate the technological feasibility, economic viability, and socio-environmental sustainability of dynamic on-road charging of EVs (de Blas, 2017).", "In 2012, the US Department of Transportation (DOT) recognized the emergence of WPT charging of vehicles and the need to understand the implications of in-motion charging of vehicles on highways (Miller et al., 2015).", "This research considered what approaches might be adopted to best analyze the economic benefits of the technology and served as a guideline for electricity pricing.", "International scientific and technical communities, such as the SAE and IEEE, have also created working groups to draft recommendations for standardizing parts and components of the wireless charging system and to write guidelines for system operations (Brecher and Arthur, 2014; Miller et al., 2015).", "This study will provide a comprehensive overview of the current research to provide policymakers and regulators state-of-the-art information about wireless charging EVs, as developed among experts in the academic community.", "Second, a system-wide understanding of the technology is needed to ensure that it moves in the right direction.", "Many disciplines are currently pursuing research on the development of wireless charging EVs.", "Progress and innovation are driven by each disciplines state-of-the-art knowledge and eventually will result in individually optimized solutions.", "However, because different disciplines sometimes fail to communicate with one another, it cannot be guaranteed that the summation of these solutions will result in an optimized solution as a whole.", "Hardware development and operations design should be considered as a whole at the earliest stage in the emerging technologys development cycle.", "Third, this survey will also provide a guide for academic researchers, orienting them to new research directions outside their own discipline.", "Research on the operations and systems challenges posed by WPT and wireless charging EVs has considerable value.", "By presenting the state-of-art of the current research, researchers will better understand the various components and dimensions to the new transportation system and be better able to identify promising new research approaches.", "The goals of the survey are clear.", "It summarizes the state-of-the-art in ongoing research on wireless charging EVs, promoting a systems view of the technology to foster interdisciplinary dialogue.", "It also organizes and categorizes the extant literature so that regulators, policymakers, and transportation engineers can orient to specific research areas within the broader literature.", "It concludes by identifying promising new directions for future study.", "The electrical and mechanical engineer literatures already include a limited number of literature reviews on wireless charging EVs.", "Some of the previous survey work addresses the following issues:", "Chen et al. (2015) provides a historical overview of the technological developments required for the electrification of roads.", "This is organized according to various aspects of electrified road construction, such as structural design, pavement type, maintenance issues, and performance measures.", "Gil and Taiber (2014) presents a literature review of recent advancements in stationary and dynamic WPT used for EV charging.", "This article addresses power limitations, electromagnetic interference regulations, communication issues, and inter-operability to identify technological and infrastructural barriers to transitioning from stationary to dynamic wireless charging.", "Brecher and Arthur (2014) provides a status review of emerging and existing WPT technologies that can be applied to electric bus and rail transit.", "The WPT technology options discussed, especially inductive power transfer, enable rapid in-station or opportunity (boost) dynamic recharging of electric bus batteries for range extension.", "Similar work Cirimele et al. (2016) presents an overview of the state-of-the-art in current research and industrial projects.", "Particular attention is devoted to describing a prototypal system for dynamic inductive power transmission, the goal of which is to extend battery range by fast partial recharging as the vehicle is in transit.", "Another similar paper Foote and Onar (2017) reviews current high-power WPT systems and describes the passive elements, devices, subsystems, and techniques that have been developed to achieve high-power levels.", "Each of these surveys contains a brief discussion of WPT, the state-of-the-art in deployments and projects, and an overview of issues generally expected to affect the spreading of the technology, to provide general introductions to the technology.", "None specifically address the operations and systems challenges posed by wireless charging EVs, which suggests a major gap in the literature.", "The paper is organized as follows.", "Section 2 provides a review of the terms and definitions found in existing studies on wireless charging EVs.", "This section also describes current developments and progress in wireless charging EVs.", "Section 3 reviews the state-of-the-art of the research.", "It first describes how the perspectives upon which the section is organized and then proceeds to review the literature based on these perspectives.", "Section 4 proposes new directions for future research, and the last section concludes the paper.", "This section provides a summary of common terms and definitions for wireless charging EV, a brief introduction to wireless EV configurations, and an overview of the current development and deployment of wireless charging EV systems.", "Because this emerging transportation is still new to transportation researchers and engineers, and the technology is rapidly evolving, several terms and definitions have not been standardized, nor have related research issues been well defined.", "For these reasons, this section will provide clear definitions for terms that will be used in subsequent sections.", "The introduction to wireless EV configurations has two purposes.", "First, it will orient the reader to terminologies for the already-standard components of wireless charging EVs, and second, it discusses the boundaries and abstraction level of wireless charging EVs from the perspective of operations and systems issues.", "The review of major developments in, and uses of, wireless charging EVs is also worthwhile because a significant portion of the extant literature was produced by researchers involved in these projects.", "Although this discussion will not be exhaustive, it nonetheless introduces the key themes that recur across major projects and indicate the import of operations and systems issues for future work.", "Wireless charging EV or EV with wireless power transfer are the most widely used terms to describe the application of WPT technology to EVs.", "Variations on these arise occasionally, such as: in-motion charging or charge-on-the-move (Wu et al., 2011; Nicolaides and Miles, 2015); contactless charging or contactless power transfer (Chopra and Bauer, 2013); on-road charging (Stamati and Bauer, 2013); electrified road (Chen et al., 2015); en-route charging (Ahmad et al., 2018); and charging-while-driving (Chen et al., 2017).", "Among these variations, inductive power transfer (IPT) charging is also one of the most widely used terms.", "Inductive power transfer originated in the technology used for wireless charging in general.", "Different WPT technologies are based on two types of electromagnetic field propagation – near-field and far-field transition (Valtchev et al., 2012; Kalialakis and Georgiadis, 2014).", "Near-field transmissions typically involve application of a magnetic field and inductive techniques to transmit energy across relatively short distances, typically shorter than 1 m. By contrast, far-field electromagnetic transmission methods allow for long-range power transfers and typically involve beamed electromagnetic power, such as lasers, microwave and radio-wave transmissions (Shinohara, 2011).", "Among the different near-field technologies, inductive coupling or inductive power transfer is the most widely used solution for EVs (Valtchev et al., 2012; Musavi et al., 2012; Chen et al., 2015), and particularly the wireless charging bus, which requires high energy transfer (Valtchev et al., 2012).", "As a result, the term Inductive Power Transfer EV is also widely used to denote wireless charging EVs.", "There is no firm agreement on how to categorize wireless charging EVs from the perspective of operations.", "This survey categorizes wireless charging EV into stationary, quasi-dynamic, and dynamic wireless charging systems.", "These are defined as follows:", "Stationary: The charging is done while the EV is parked and not operational for an extended period at a stationary point, such as a garage or parking lot.", "Quasi-dynamic: The charging is carried out when the EV is moving at a low speed while the vehicle decelerates to or accelerates from a resting position.", "Dynamic: The charging is done even the EV is in full motion.", "These categories are present in some previous work, such as (Cirimele et al., 2016; Foote and Onar, 2017; Jang et al., 2016; Mohamed et al., 2017; Filho and Suh, 2013; Miller et al., 2015; Amditis et al., 2017).", "Although there are some variations in the terms, such as semi-dynamic or stop-and-go charging for quasi-dynamic, or static for stationary, the categorization basis and definitions remain the same.", "It should be noted that most of the literature organizes wireless charging into just two categories – stationary and dynamic – and include quasi-dynamic as a subset of dynamic wireless charging.", "The three-term categorization is used in this survey because quasi-dynamic and dynamic EV systems require different approaches at the systems-level; planning for charging infrastructure, operations scheduling, initial investment calculations, and ITS system design all require different considerations, depending on whether the system will be dynamic or quasi-dynamic.", "For instance, the frequency of traffic lights or passenger pick-up/drop-off time at bus stops will be critical variables in planning for a quasi-dynamic system, whereas the traffic flow and vehicular speeds will be primary factors for a dynamic wireless charging system.", "The three-part categorization schema makes the most sense from an operations and systems perspective.", "Covic and Boys (2013) defines the components of wireless charging EVs as:", "the power-supply, which connects the system to the electric grid to receive power;", "the charging infrastructure or facility, called a power track, which is an elongated track driven by the power supply whose current results in a magnetic field that follows the track;", "the pickup, which intercepts some of the magnetic field and converts it into controlled electricity; and", "the load, which is the entity being charged by the electrical power.", "Suh and Cho (2017c) dissects the wireless charging system for OLEV into two primary components: the pickup system and power supply system.", "The pickup system, which is attached to an EV, collects the power while the power supply, installed beneath the road, wirelessly transmits the power.", "Other studies define the elements similarly (Chen et al., 2015; Xie et al., 2016; Highway England, 2015; Jang et al., 2016; Limb et al., 2016; Choi et al., 2015).", "Based on existing studies, a standard conceptual configuration of WPT is proposed as illustrated in Fig. 1.", "Although WPT has several different technological applications, their conceptual configurations differ little from the standard configuration depicted in the figure, which can also be applied to both dynamic and quasi-dynamic wireless charging EVs.", "The definitions and functions of the standard configuration are explained as follows:", "Power transmitter system is the charging unit that transmits the power by electromagnetic field from the grid.", "It consists of a transmitter unit, the elongated track usually installed under the road that transmits power, and the power supply unit, which supplies the power from the grid to the power track.", "Pickup system is the component that receives the power from the power transmitter unit and consists of a pickup unit, attached at the bottom surface of the vehicle to receive power, and auxiliary units, such as rectifiers and regulators, which transmit the power from the pickup to the motor and battery.", "Notice that the detailed elements in the power supply unit – the power supply, the rectifier, the invertor and controller are aggregated and lumped together as a single unit.", "Details of these discrete elements are not usually included in operations and systems studies because some abstraction is typical in such research.", "This is a sharp contrast with most engineering research on wireless charging EVs, which is concerned with the discrete elements of the power supply and transmitter units or the pick system.", "Nonetheless, recent work has shown that understanding the conceptual configuration and composition of WPT at the sub-system level can be crucial.", "Depending on the granularly of the analysis or model, details at the subsystem-level may or may not be needed.", "For instance, the study by Chen et al. (2016) assumes that the cost of the charging lane is simply proportional to the length of the lane and as such does not require details about the power transmitter system; the model focuses on high-level traffic behavior and as a result details about the configuration of the power transmitter system may not be necessary.", "By contrast, the models used in Ko and Jang (2013) and other following work did have to account for configurations at the subsystem-level.", "If the goal is to develop a detailed infrastructural plan for a specific bus route, for example, a detailed cost analysis of the power transmitter system and its internal components, the transmitter and power supply units would indeed be necessary.", "In this paper, traffic lanes with a power transmitter system for dynamic charging is called the charging lane, and bus stations or temporary stopping points with a power transmitter for quasi-dynamic wireless charging are called charging stations.", "The charging lane and stations are collectively called charging infrastructures or charging facilities.", "Numerous papers, including (Chen et al., 2015; Palen et al., 2000; Shladover, 2007; Suh and Cho, 2017b), indicate that the Partners for Advanced Transit and Highways (PATH) project, led by researchers at the University of California, Berkeley, developed the first working prototype of the wireless charging EV.", "A systematic pilot test of dynamic wireless charging EV solutions was performed throughout the 1980s and 1990s.", "Its wireless transferred power capacity was 60 kW, and the operational frequencies of the inductive power transfer were 400 and 8500 Hz, achieving a maximal measured efficiency level of 60% with an air gap of 2–3 in.", "(Kim et al., 2013; Shladover, 2007; Lechner et al., 1993; Empey, 1994; Ahn, 2013).", "Although the project demonstrated the possibility of WPT applications for transportation, it did not lead to a commercialization, owing to its low power transfer efficiency and the economic infeasibility of the requisite infrastructural overhauls at the time.", "The first commercialized dynamic wireless charging bus is the On-Line Electric Vehicle (OLEV), developed by KAIST (Ahn, 2013; Suh and Cho, 2017b; Kim et al., 2013; Foote and Onar, 2017).", "The OLEV adopts a technology called Shaped Magnatic Field in Resonance (SMFIR), which effectively magnifies the electric waves, and has achieved charging efficiencies of up to 80%.", "The design concept of the OLEV was first presented in a plenary presentation at the 2010 CIRP Design Conference (Suh et al., 2011).", "Since the first demonstration in 2009, the OLEV system has thus far been deployed at four sites in South Korea.", "The OLEV trolley originally installed in Seoul Grand Park as a pilot project in late 2009-early 2010 has been running as a commercial service since 2011.", "It travels on a 2.2 km circular route around the park, powered by an underground power supply totaling 372.5 m in four segments.", "In 2012, two OLEV buses serviced the international exhibition Expo 2012 in Yeosu.", "Two OLEV buses have also been serving as shuttle buses on the KAIST campus in Daejeon since 2012.", "The route configurations, velocity profiles, and other operating conditions are well described in Jang et al. (2016, 2015).", "In August 2013, OLEV was installed in Gumi City, one of the largest industrial cities in South Korea.", "The 35 km route is serviced by six buses and powered by six charging pads under the road (Jeong et al., 2015).", "Gumi added two more OLEV buses to its system in May 2016.", "The latest site to install an OLEV system is Sejong, a recently developed city that houses many central government offices (Suh and Cho, 2017c; Ko and Jang, 2013; Jang et al., 2015).", "Developmental histories of OLEV can be found in Choi et al. (2015), Rim and Mi (n.d.), and Suh and Cho (2017d).", "The developmental history of SMFIR technology, along with its technical details, can be found in the recent book by Suh and Cho (2017b).", "Moreover, note that there are numerous patents on the technologies related to SMFIR and OLEV systems.", "They provide detailed technical information.", "PRIMOVE is the e-mobility unit of Bombardier Transportation, which is one of the largest suppliers in the rail industry.", "Its wireless charging solution is targeted to light rail, bus, and automotive fleet operations.", "Its solution concentrates on static charging, but it also has dynamic charging capability (Highway England, 2015).", "Through a partnership with German firms Viseon Bus and Gesellschaft mit beschrnkter Haftung(GmbH), PRIMOVE has developed a mechanical lifting and lowering mechanism to extend the onboard pickup coils to achieve the most efficient recharging (Brecher and Arthur, 2014).", "The PRIMOVE system for buses has been tested and deployed in many cities in Europe, including Mannheim and Berlin, Germany, and in Bruges, Belgium (Cirimele et al., 2016; Brecher and Arthur, 2014).", "Utah State University is actively performing research on dynamic wireless charging solutions.", "The university is also the home of SELECT (Sustainable Electrified Transportation Center), a research consortium of five partner universities and five affiliate institutions in the United States.", "SELECT, which was formed at the university in 2016, aims to validate and commercialize wireless charging and the supporting technologies through vehicle and roadway-scale demonstration and pilot projects.", "The demonstration track at Utah State University consists of a 400 m test track with 75 m precast trenches and power panels for embedding coils.", "The trenches are easily reconfigurable with different transmitters.", "The power level of each transmitter is up to 30 kW.", "The EV bus is equipped with a 60 kWh battery (Tavakoli et al., 2016; Möller, 2017).", "FABRIC (feasibility analysis and development of on-road charging solutions for future electric vehicles) is a European consortium of 24 partners including automakers Renault, Peugeot, Citroen, Fiat, Volvo, and Scania, coordinated by the Institute of Communication and Computer Systems (ICCS) in Greece.", "FABRIC aims to develop, test, and evaluate the efficiency of dynamic wireless charging prototypes to assess the feasibility of a wide deployment of dynamic wireless charging solutions (Amditis et al., 2017).", "During its four-year project period, starting from 2014, dynamic charging solutions on the prototype EVs under development have been tested in three test sites in France, Sweden, and Italy.", "The tested solutions include systems developed by Qualcomm-Halo, Polito, and Saet.", "The French test site primarily tests the Qualcomm-Halo solution on a 100-m-long charging lane.", "The Italian site, which consists of a 700-m-long track with a 200-m-long wireless charging infrastructure, tests the Polito and Saet systems.", "This demonstration track is also equipped with a simulation toll collection system and smart grid interfaces.", "The Swedish test site is primarily for a conductive solution for trucks (de Blas, 2017).", "The FABRIC project is on the verge of completion, and significant reports from the project are expected to be released in 2018.", "Oak Ridge National Laboratory (ORNL) is an active institution in the US, performing research on wireless charging EV technology with support from the US Department of Energys Vehicle Technologies Office (VTO) (Brecher and Arthur, 2014).", "ORNL has partnered with Hyundai America Technical Center and Toyota Research Institute of North America to develop stationary wireless charging solutions for various passenger vehicle models (Onar et al., 2016; Chehab, 2016).", "ORNL is also doing research and developing quasi- and dynamic wireless charging solutions (Brecher and Arthur, 2014; Miller et al., 2015).", "Onar et al. (2016) describes the concept and prototype of a WPT solution developed by ORNL and the mass transit vision of ORNL.", "UNPLUGGED was a European project-based consortium with a budget of 2.3-million euros, aiming to investigate how integrating wireless charging EVs with urban road systems could improve the convenience and sustainability of car-based mobility.", "The main objective of the UNPLUGGED project was to conduct a feasibility analysis of dynamic en-route charging technologies for long-term EVe range extension (Unplugged Fianl Report, 2015).", "UNPLUGGED examined the technical feasibility, practical issues, interoperability, user perception, and socio-economic impacts of wireless charging EVs from September 2012 to March 2015.", "The consortium was supported by the European Unions Seventh Framework Programme and was led by FKA (Forschungsgesellschaft Kraftfahrwesen.", "Aachen) and ENIDE Solutions S.L.", "It included 17 partners from the automotive and power supply industries, R&D centers, and transport operators.", "During the project period, two wireless charging systems were built in Zaragoza and Aachen.", "These innovative systems went beyond the current state-of-the-art in terms of high power transfer as they included a 50-kW charging station designed and manufactured by CIRCE.", "The system allowed for smart communication between the vehicle and the grid and was in line with the latest inductive charging standards, which considered interoperability.", "The results and outcomes of the project are documented in (Unplugged Fianl Report, 2015).", "VICTORIA (vehicle initiative consortium transportation operation and road inductive application), is one of the projects associated with FABRIC.", "The primary goal of the project was to investigate the feasibilities of WPT-based electric vehicle and road systems including stationary, quasi-dynamic, and dynamic wireless charging EVs (Bludszuweit, 2016).", "Thedemonstration system adopted a U-shaped track-type charging infrastructure similar to the one developed at KAIST OLEV.", "A maximum power transfer of 50 kW has been developed and operated on a 10-km bus route in Malaga, Spain, since December 2014.", "In the demonstration system, 10 segmented wireless charging infrastructure systems with a total length of 300 m were installed.", "In the EV bus, a self-guided control system automatically controls the position of the receiving panel, following the center of the road (Rim and Mi, 2017).", "Note that as mentioned earlier, the focus of the present paper is quasi-dynamic and dynamic wireless charging.", "While the architecture of a few dynamic wireless charging EV systems, such as, the KAIST OLEV, is solely targeted at dynamic wireless charging, most of the quasi- and dynamic wireless charging EV systems have been modified from static wireless charging systems or built upon the stationary technologies.", "As a result, it is not straightforward to clearly distinguish the type of wireless charging for some prototypes and demonstration models.", "This subsection introduces some of the solutions originally developed from the stationary type but widely mentioned in the wireless charging technology literature.", "IPT Technology, a spin-off of the company Conductix-Wampfler, has developed WPT solutions since 1997.", "The company has developed and deployed several generations of quasi-dynamic wireless charging buses, in which the buses charge when they are temporarily stopped at bus stops or parked for extended periods.", "The first version was an electric shuttle in the Rotorua Thermal Park in New Zealand, which started service in 1998.", "IPT Technology has since deployed their wireless charging solutions to public buses in Genoa and Turin, Italy in 2002 and 2003, respectively.", "In Turin, a fleet of 23 electric buses are in daily operation, using IPT Technology systems since 2003.", "The reported annual cost of electricity per bus was about $9 K, a substantial gain when compared against the $50 K cost for to fuel a diesel-powered bus (Brecher and Arthur, 2014).", "IPT Technology has also deployed wireless charging public buses in Utrecht, Netherlands and cities in the UK, including Milton Keynes, London, and Bristol, since 2014 (IPT Technology Official Website, 2017).", "A company spun off from a project at Utah State University, Wireless Advanced Vehicle Electrification (WAVE), has developed both stationary and quasi-dynamic wireless charging transit buses (Fisher et al., 2014).", "The first prototype, called Aggie Bus, was implemented as a campus shuttle, equipped with a receiver with the same dimensions as the transmitter embedded in the pavement of the bus stops (Cirimele et al., 2016; Bansal, 2015; Brecher and Arthur, 2014).", "This system allows the transfer of 25 kW at 20 kHz at each bus stop.", "The power transfer takes place over an air-gap of 15–25 cm, achieving 90% efficiency (Cirimele et al., 2016; WAVE Official Website, 2017).", "This is the first use of WPT in transportation by a North American entity (Bansal, 2015).", "WAVEs technology is also being adopted to charge electric buses in Long Beach, California, and to power the Monterrey-Salinas Transit trolleys, also in California (Brecher and Arthur, 2014).", "There are numerous projects currently underway for static charging personal vehicles, including Qualcomm-HaloIPT, Toyota Motors’ joint development with WiTricity, and Plugless Power Solutions, which is being tested by Nissan and Chevrolet.", "Because the static charging solution and applications are outside the scope of this survey, readers interested in these technologies should refer to the surveys in Cirimele et al. (2016) and Brecher and Arthur (2014).", "More on-going projects or list of past projects are found in the following literature: Brecher and Arthur (2014), Li (2016), Fisher et al. (2014), Foote and Onar (2017), Cirimele et al. (2016), Bi et al. (2016), de Blas (2017), Highway England (2015), Möller (2017), and Miller et al. (2015).", "The goal of this subsection is not to provide a complete list of demonstration projects, prototypes, initiatives, and consortia for wireless charging EV systems.", "Instead, it discusses the activities widely mentioned in the literature on the systems and operation of wireless charging EVs.", "Moreover, note that while the activities listed above are specifically for wireless charging EV systems, some of the activities deal with wireless charging as one of the many different subjects.", "For instance, eco-FEV was a European consortium investigating the economics of electric vehicles, system architecture, energy management, ITS, and other EV-related topics during the period from September 2012 until May 2015.", "The wireless charging EV system was one among many different topics.", "The wireless charging-related research literature tied to this project includes (Deflorio and Castello, 2017, 2015; Deflorio et al., 2015a,b).", "Furthermore, Highways Englands electrified road research with the Transport Research Laboratory has often been discussed in the literature, such as Chen et al. (2016), Liu and Song (2017), and Suh and Cho (2017d).", "The British government announced in 2015 that it would invest 500 million in research on and products related to dynamic wireless charging EV systems (Off road trials for electric highways technology, 2015).", "However, at the time of writing, this project has not yet been realized and is therefore not discussed in detail in this survey (Green Target, 2016).", "Table 1 summarizes recent activities related to wireless charging EV systems worldwide, which are widely mentioned in the academic literature.", "The status of the current research is reviewed from the following five different perspectives.", "Charging infrastructure allocation: methods for allocating wireless charging infrastructure.", "Driving range extension analyses: analysis methods and evaluation of driving range extension with the wireless charging technology.", "Cost and benefit analyses: investigations of the cost and benefit of the wireless charging EV compared with other type of transportation systems.", "Supporting systems: models and analyses for supporting systems in operations of wireless charging EVs, such as construction and installation methods, power supply to the charging facilities, electric billing infrastructure, and billing approaches.", "Miscellaneous: other perspectives that are not covered in the above including regulations/standardization, traffic simulation, and system architecture issues.", "Each of these research perspectives is based on the topics and approaches driving current research on operations and systems issues for wireless charging EVs.", "It should be noted that the Miscellaneous category includes research areas and perspectives that are promising but not developed enough to warrant their own category.", "Please note as well that the perspectives listed here are not mutually exclusive.", "As a result, a single research article may be covered across more than one perspective.", "The charging infrastructure allocation problem is one of the most actively investigated topics in systems and operations studies of conventional EVs.", "This is also true for the case of wireless charging EVs.", "The challenge presented by the allocation problem is to provide logistical insights to support decisions geared toward an optimal distribution of charging infrastructure.", "The allocation problem is categorized into two different types based on the scope of the modeling – the microscopic allocation model and the macroscopic allocation model.", "The microscopic allocation model, or simply micro-allocation, seeks to find an optimal location for charging infrastructure on a given route or path of a vehicle.", "The model considers cases in which the wireless charging EVs are traveling only on a given route or path.", "An example of the model is a public transit bus.", "Because the vehicle travels on a predetermined route, traffic conditions, velocity profile, and energy consumption are fairly predictable.", "More detailed modeling can then be constructed based on this micro-allocation.", "Specifically, micro-allocation approaches are useful when engineers or route planners need to decide the specific locations for wireless charging lanes or for charging points during the planning stage of wireless charging transit bus.", "The goal of the model is to provide a practical engineering tool to support system-level decisions for the wireless charging-based transit system.", "In contrast, the macroscopic allocation model, simply macro-allocation, considers a more high-level view.", "The primary goal of this type of modeling is to provide scientific insights for the wireless charging EV itself; to evaluate overall traffic behavior affected by the introduction of wireless charging EVs into broader transit systems.", "Typically, macro-allocation modeling considers the routing of passenger vehicles or commercial trucks that have more freedom in selecting their routes.", "Because the scope of the model is broader, high-level approximation is unavoidable.", "Most macro-allocation models adopt a network-based modeling approach, in which the traffic flow is modeled with nodes and arcs.", "This approach is derived from the well-known location problem, which seeks an optimal location for a facility with a given constraint (Church and Velle, 1974).", "Typically, in this modeling approach, travel flow from origin to destination is assumed to be known in advance.", "Jang and his colleagues were the first to formalize the micro-allocation problem (Chen et al., 2017), and most subsequent work follows the modeling assumptions and considerations provided in this and earlier work, including Ko and Jang (2013) and Jang et al. (2015).", "The modeling assumptions and considerations are as follows (Table 2):", "Each bus line in the system operates on a fixed route.", "Each bus line has a base station, where all buses start and end their service loops.", "Once a wireless charging bus completes a service loop, it will be fully charged at the base station before it starts another service loop.", "The speed profile and the number of boarding/disembarking passengers at bus station are predetermined.", "In an early application of the micro-allocation problem, Ko and Jang (2013) proposed an optimization-based approach, first, for the distribution of charging infrastructure, and second, for determining the right battery size for a public transportation system operating on a single route in a closed environment, where vehicles typically operate under regulated velocity and less traffic.", "The OLEV EV bus currently operating at KAIST is a good example of this type of closed-environment system.", "The model considers the route as a continuous spatial decision space.", "The optimization-based approach seeks the starting and end points of each wireless charging lane segment on a specified route in a continuous search space as shown in Fig. 2(a).", "The total number of segments is also a decision variable.", "In addition, the model considers the battery capacity as a part of its decision variables.", "The authors explain that these variables entail a trade-off, between battery capacity and number and length of charging lanes.", "If the EV bus is equipped with a large battery, fewer and shorter charging lanes need to be installed along the route and vice versa, indicating a clear trade-off between the cost of the battery and that of the charging lanes.", "The optimization-based approach thus seeks to determine a charging lane allocation in conjunction with an optimally sized battery.", "In terms of practical applications, this approach is most useful at the initial stage of planning for wireless charging EV-based transportation systems within closed-environments like that at KAIST.", "Jang et al. (2015) adopts assumptions and modeling conditions identical to those deployed in Ko and Jang (2013).", "The difference is that Jang et al. (2015) discretizes the route into multiple small segments, turning the optimization approach proposed by Ko and Jang (2013) into a discrete problem.", "The model is validated by comparing the results from Ko and Jang (2013).", "A similar modeling and optimization approach can be found in Ko et al. (2015), in which a nonlinear cost function and genetic algorithm are proposed to solve for the optimization.", "Jeong et al. (2015) is the first research to incorporate battery life as part of an economic analysis of wireless charging EV.", "The cost structure and modeling, which also considers a single-route transit bus case, are similar to those used in Jang et al. (2015), Ko and Jang (2013) and Jang et al. (2015).", "The idea to include battery life comes from the property of the lithium-based battery, the most widely used battery type for EVs.", "The performance of the battery (charging capacity and power amount) becomes worse if the depth of charging/discharging is greater and if charging is done infrequently, as compared to frequent and shallow charging/discharging.", "These conditions suggest that it might be more cost-efficient to install many short power tracks frequently rather than long tracks infrequently.", "In other words, the battery replacement cost also becomes a function of the power track allocations.", "Comparisons between modeling with and without consideration of battery life in the cost structure indicated the importance of battery life in long term economic analyses.", "The abovementioned studies only consider a single-route bus transit case.", "However, real-world transit systems are much more likely to contain multiple routes and bus stations, with some segments shared by more than one route, than they are to consist of a single-route.", "Particularly for downtown areas where major transit routes intersect, significant overlapping is to be expected.", "This adds a new set of considerations, as it becomes beneficial and efficient to allocate power tracks to segments where multiple routes overlap.", "Recent studies by Hwang et al. (2017) and Liu and Song (2017) incorporate this property.", "In particular, Hwang et al. (2017) offers an extension of Ko and Jang (2013), but relaxes the single-route assumption and provides a general modeling framework for multiple route considerations.", "Optimization is based primarily on the continuous variable framework in Fig. 2(a), but the model and solution algorithms are numerically validated with those from the segmentized discrete model.", "The OLEV Gumi case is used for empirical study such that the economic benefit of installation on shared routes can be analyzed.", "Liu and Song (2017) also considers the multiple route case.", "Compared to Hwang et al. (2017), the optimization is based on the segmentized discrete model.", "The study is significant in that it is the first to consider the issue of uncertainty in vehicles energy supply and consumption.", "Note that all of the work previously mentioned here assumes deterministic energy rates, where the amounts of energy supply and consumption are known in advance as deterministic parameters; these assumptions ignore the practical likelihood of uncertainty and may create a risk of not having energy supply to the battery.", "Liu and Song (2017) uses the set-based robust optimization technique to deal with uncertainty.", "This deals indirectly with the probabilistic parameters by introducing limits for each uncertain parameter.", "If the probability distribution of the uncertain parameter is not known then, it would be safe to assume that the parameter is within the worst bounds described in the set.", "Thus, the problem is formulated as a deterministic optimization but still considers uncertainty.", "This approach is more mathematically tractable than stochastic optimization approaches, which consider uncertain parameters as stochastic variables (Bertsimas et al., 2011).", "Liu and Song (2017) compares the solutions from the deterministic model, which do not consider uncertainty, to those from the robust optimization.", "As expected, the robust model requires higher cost in the initial investment to hedge uncertainty.", "But it is interesting to observe from the empirical study that robust optimization requires larger batteries to achieve robustness at the system-level, instead of requiring more power track installations.", "Applications of these robust optimization techniques as applied to electric vehicles can be also found in Sarker et al. (2015) and Mak et al. (2013).", "The macroscopic model seeks to address the high-level charging infrastructure by considering traffic flow, driver’s route choice, and other traffic-related issues.", "This modeling perspective is not limited to an individual vehicle traveling on a specified route.", "Instead, it considers the social benefit of the charging infrastructure to maximize the number of vehicles that use the infrastructure.", "The underlying assumptions of this approach are that the location of the charging infrastructure will affect routing choice and thus traffic flow.", "Riemann et al. (2015) investigates the optimal location of wireless charging facilities using a flow-capturing location model (FCLM), which seeks to maximize demand coverage by locating a set of facilities (Hodgson, 1990; Church and Velle, 1974).", "Riemann et al. (2015) seeks to address the optimal location problem of wireless charging facilities with a finite number of facilities available for deployment.", "This extended the arc-cover path-cover (AC-PC FRLM) model to explicitly capture the mutual interaction between the location of charging facilities and the resultant network traffic flow.", "In the optimization modeling, the links are candidate points for dynamic charging infrastructure.", "Specifically, it assumes that if a wireless charging facility is provided, it will be located in the middle of the links (the centroid node) in the network.", "The proposed model is also concerned with assigning traffic in the network onto the routes, with considerations for both travel time and the availability of charging facilities.", "The multinomial logit model stochastic user equilibrium principle is used to capture the routing choice behavior of drivers.", "It should be noted that the optimization modeling is based on some unrealistic assumptions, such as that the battery is fully charged if an EV is on a link with a power-tracked road, regardless of the battery charging level or that all vehicles traveling in the network model are wireless charging EVs.", "However, Riemann et al. (2015) is considered as a pioneering work, extending the conventional location model to wireless charging EV.", "Fuller (2016) suggests an optimization model to allocate charging facilities, using a liked-oriented set covering location modeling approach.", "The study evaluates the potential for dynamic wireless charging by considering travel to regional destinations in California.", "A 200-mile range EV is assumed to be used in coordination with stationary and dynamic wireless charging.", "An optimization model is constructed to evaluate the optimal allocations of dynamic wireless charging infrastructure at minimum total investment cost, while also meeting the travel requirements between cities (this is based on the location model developed in Wang and Lin, 2009).", "The proposed optimization problem essentially becomes a flow-based set covering problem.", "Traffic issues or driver behavior or choice of charging are not considered in the model.", "The primary focus is on cost analyses rather than charging facility allocation and optimization modeling; as a result, the study empirical findings are discussed below in the section on economic analyses.", "Chen et al. (2016) presents an optimization model and algorithm dealing with one of the most critical issues in wireless charging facility allocation.", "Their approach presented a novel user equilibrium model to describe EV drivers’ travel and charging choices when charging lanes are deployed.", "The model showed that slower speeds will allow more time to charge in the charging lane, but will yield longer travel times.", "As a consequence, there is a trade-off for EV drivers, pitting energy needs against travel time.", "With this trade-off, the relationship between traffic volume and actual travel time is no longer described by a single link performance function, as had been done in previous work.", "As a result, Chen et al. (2016) is considered the first to capture this trade-off behavior.", "Their proposed model explicitly considers the driver’s choice of operating speeds on wireless charging lanes by formulating a new user equilibrium model.", "Then, with the established user equilibrium conditions, the optimal deployment of charging infrastructure for a given budget is proposed.", "The algorithms are validated with empirical examples based on both a small-scale network from Nguyen and Dupuis (1984) and a large-scale network from He et al. (2014).", "Chen et al. (2017) presents a charging-facility-choice model investigating drivers choice of charging facilities, between plug-in charging stations and charging lanes with dynamic wireless charging, to explore the competitiveness of dynamic wireless charging.", "Both charging stations and wireless charging lanes are deployed so that EV drivers can choose either option.", "EV drivers who select charging stations must stop at the stations and thus encounter a charging delay, whereas those who use wireless charging lanes have to equip their vehicles with additional devices to enable wireless charging and may have to pay a higher charging price.", "However, these drivers will enjoy a less travel time because they will not need to stop for charging.", "The study first analyzes the charging-facility-choice equilibrium, and then discusses the optimal allocation of charging infrastructure, comparing public and private provisions.", "The public provision maximizes the social benefit, as if a government agency builds and operates the infrastructure.", "In contrast, the private provision is such that the infrastructure is built and operated by two competing private companies to maximize their own profits.", "This study differs from previously mentioned work, because its goal is to provide high-level insights about the competitiveness of dynamic charging against stationary charging; as a consequence of this orientation, it used a highly simplified model, proposed by Nie and Ghamami (2013), in which identical EVs travel on a hypothetical traffic corridor.", "The model is not intended to specify an allocation of charging stations or power tracks.", "Instead, it provides a mathematically tractable means to characterize the deployment and operations of charging lanes and stations.", "The empirical study indicates that dynamic charging is competitive when compared against plug-in charging for drivers with higher value of time (VOT) in the public provision scenario.", "Although these findings depend on the input data, the finding is still significant in that the study is first to show, with a logical model, that dynamic wireless charging should be deployed initially for commercial fleet vehicles, such as OLEV, for which the VOT is much higher than that of passenger vehicles.", "Another consideration is whether the availability of wireless charging infrastructure will alter customers’ purchasing behaviors of EVs.", "More dynamic charging infrastructure can attract customers to purchase more dynamic charging EVs, which would result in an increase in the number of dynamic charging EVs on the road.", "This dynamic is captured in the study by Liu and Wang (2017), whose optimization model considers the tri-level relationship between government, vehicle choice, and driver.", "Investment decisions for plug-in charging and dynamic wireless charging facilities are determined at the government level; the purchasing of the EV type – conventional plug-in or dynamic wireless charging – is considered at the level of vehicle choice; and the route choice for a trip is selected at the driver level.", "Decisions on each level are interrelated.", "The tri-level optimization model is developed, and a solution algorithm is presented.", "The primary goal of the study is to assist government planners in the optimal placement of multiple types of EV charging facilities to satisfy the needs of different EV types within a given budget to minimize the social cost.", "Ushijima-Mwesigwa et al. (2017) uses a network-based modeling framework and algorithm to find an optimal allocation of wireless charging lanes.", "The primary goal of the model is to minimize the number of infeasible routes for a given budget.", "As a result, the model is able to determine a minimum budget to achieve zero infeasible routes – understood as EV drivers arriving at their destinations with a battery charge above a certain threshold.", "Numerical study is carried out with GIS data of Manhattan.", "Another category of studies considers the allocation of charging infrastructure as a way to investigate wireless charging systems extension of EV driving ranges.", "These studies use a standard driving-cycle, determined by a velocity profile of a specific type of vehicle provided by a government or organization that represents driving patterns.", "Studies in Pantic et al. (2009) and Lukic and Pantic (2013) are an examples of this category, in which a general standardized driving cycle is used to estimate the cost of charging infrastructure under the assumption that the charging infrastructure is optimally allocated.", "To the extent that this research deals with the general travel of a vehicle, rather than specific trips, it is similar to macro-allocation model.", "Pantic et al. (2009) uses a simple optimization technique to allocate charging lanes over a standard driving-cycle.", "This research supposes multiple scenarios based on a number of variables, including vehicle type (small/large/SUV); driving cycles, defined as low-demand urban driving cycles (UDDS), high-speed highway driving cycles (HWFET), and high-demanding mountain driving cycle (VAIL2NREL); and three different power rates for the power track (20, 40, and 60 kW).", "Optimization is constructed to minimize the total length of the charging lane and maximize battery life.", "The model shows that the required coverage for power tracks significantly varies depending on vehicle-driving combination.", "For example, the 8kWh battery-equipped compact car needs only 0.46% coverage under the urban UDDS driving cycle scenario, whereas the heavy SUV in a mountainous region requires as much as 64.3% coverage.", "Although the study does not analyze monetary costs, it does discuss the idea of power track allocation in terms of road coverage to achieve a target driving range.", "Stamati and Bauer (2013) conducted a simulation study using a standard highway driving cycle (Highway Fuel Economy Driving Schedule - HWFET).", "The simulation investigates the extension of EVs driving range with varying dynamic charging infrastructure coverage, between 10% and 100% of the highway, at a power rate between 10 and 60 kW.", "The paper concludes that a 500 km driving range could be achieved for an EV with a 24 kWh battery, 25 kW dynamic charging, and 40% roadway coverage.", "Chopra and Bauer (2013) also conducted a simulation-based study to investigate the driving range extension of EVs using wireless charging for urban and highway driving scenarios.", "The two case studies show that considerable driving range enhancement can be achieved by using wireless charging infrastructure for charge replenishment when an EV is idling at a traffic signal in the urban driving scenario, or when it is in motion in the highway driving scenario.", "The study shows that for an EV with a 24 kWh battery, 90% efficiency of the power supply from the charging infrastructure, and 20% road coverage, the expected driving range can be extended by an estimated 12% for 10 kWh batteries and 217% for 40 kWh batteries, respectively.", "Shekhar et al. (2016) provides a case study to analyze the initial investment cost of a fleet of wireless charging electric buses that must achieve a driving range of 400 km.", "The study proposes a high-level investment cost estimation model.", "For the cost estimation of the power track, simulations and regression are used.", "Standard driving cycles with different parameters are simulated first.", "Then, based on the simulation results, linear regression is used to estimate the relationship between the percentage of coverage for the charging facilities over the required road and the battery level at the end of the trip.", "Various cases are investigated with different parameter settings, such as the road coverage of charging facilities, the battery size, stopping times, and charging power level.", "The case study indicates that a 500 kWh battery is optimal.", "However, this result – the very large battery capacity – somewhat contradicts those of Bi et al. (2015), which indicated that wireless charging technology is promising due to the potential of downsizing the battery.", "Bi et al. (2015) claims that their empirical study shows that wireless charging solutions can reduce the size of the battery by one-third the weight of a benchmark plug-in battery.", "Of course, no direct comparison can be done across these two simulations, because they rely on different assumptions and parameter settings.", "Further investigation is still needed to propose an appropriate size for the battery for wireless charging EV.", "García-Vázquez et al. (2017) compares a dynamic wireless passenger EV equipped with a 24 kWh battery driving on three different traffic road scenarios (motorway, highway and urban stretch), each characterized by a different traffic flow (intensity and speed) and length.", "The results show that in the urban stretch, the energy transferred to the EV presents an average value of about 0.6 kWh/km, whereas on the highway it is 0.25 kWh/km.", "Cost and benefit analyses are concerned with estimating investment (or operational cost) and economic benefit of wireless charging-based transportation.", "There are some studies exclusively focusing on these cost and benefit evaluations, whereas others deal with these issues as part of validating optimization techniques, traffic models, or other economic assessment methods.", "In this section, both cases are discussed.", "Cost-benefit analyses can be divided into initial investment cost analyses and operational cost analyses.", "The initial investment analyses estimate the installation cost of the wireless charging EV, focusing mostly on transit fleets.", "Operations cost analyses are concerned with evaluating the costs of operating wireless charging EVs over a time.", "Compared to the previous two categories, environment assessment – lifetime assessment and environmental impact analyses – includes not only the cost of investment or vehicle operation but also the cost of the entire energy logistics – energy generation, conversion, and transmission.", "Table 3 lists papers based on these categories.", "Fuller (2016) conducted a case study to analyze the potential for a wireless charging infrastructure by considering travels to different regional destinations in California.", "Different scenarios in which static and dynamic wireless charging infrastructures are installed on major freeways in California are considered.", "Large cities and leisure destinations, along with likely travel routes between respective origin and destination pairs, are modeled as a travel network.", "The optimization model, based on work by Wang and Lin (2009), evaluates the allocation of infrastructure.", "Different combinations of wireless charging power (dynamic charging level from 20 to 120 kW) and various vehicle ranges (between 100 and 300 miles) are evaluated.", "The case study indicates that travel between popular destinations could be achieved with a 200-mile EV and a 40 kW dynamic wireless charging infrastructure at a cost of approximately $2.5 billion US dollars.", "This cost can be reduced to less than $1 billion US dollars if the charging power is increased to 100 kW or greater.", "Dynamic charging, coupled with static charging, proves to be more cost effective than gasoline over a 10-year period.", "The case study also shows that even at very low battery prices of $100 per kWh, dynamic charging can be more cost effective for extending range than increasing battery capacity.", "Ko and Jang (2013), Jeong et al. (2015), and Jang et al. (2016) all investigate the initial investment costs to implement a wireless charging transit bus system for a single route.", "The initial investment includes the cost of the infrastructure for the charging lanes and the total battery cost for the fleet of buses.", "Battery cost is included as a part of the initial investment because the battery size is directly related to the power track allocation requirements.", "Each bus needs to equip one pack of batteries; the total battery cost for the route is therefore determined by the cost of a pack of battery multiplied by the number of buses in the fleet.", "This assumes that all buses that operate on the route use the same battery size.", "The charging lane cost is broken into variable and fixed costs; the variable cost depends on the length of charging lanes, whereas the fixed cost is incurred regardless of the length of the charging lane.", "The variable cost captures the fact that longer charging lanes entail higher material and construction costs.", "The fixed cost includes the cost of connecting a facility in a charging lane to the power grid, the inverter cost, and other material costs that are incurred when installing a charging facility, regardless of the length.", "With this cost structure, it might be more cost-efficient to construct a smaller number of long charging lanes than a larger number of short lanes.", "Ko and Jang (2013) estimates the minimum cost of installing an OLEV-based trolley at Seoul Grand Park using an optimization technique.", "The cost is about $640,000, which includes cost of batteries and charging infrastructure, but excludes the bus units, construction, and labor.", "The cost sensitivity analysis shows that as the cost of battery goes up, the optimization mechanism recommends the installation of more charging infrastructure and vice versa.", "The primary focus of Jang et al. (2016) is to analyze the initial investment cost figures between stationary, quasi-dynamic, and dynamic wireless charging transit bus systems.", "Using the optimization methods and algorithms developed by Ko and Jang (2013) and Jang et al. (2015), this study extensively compares the three different types of dynamic wireless charging transit systems.", "The OLEV system at KAIST (short trip) and at Gumi City (long travel) are analyzed empirically.", "The interesting finding here is that dynamic wireless charging is less cost efficient for a short trip service.", "Dynamic wireless charging can be competitive with the stationary or quasi-dynamic options if the bus operates a minimum travel distance.", "The study also found that the OLEV-based transit system at Gumi City needs to have at least 18 buses in operation to be economically competitive with the stationary and quasi-dynamic solutions.", "Jeong et al. (2015) uses an initial cost estimation technique similar to that in Ko and Jang (2013), but it considers the cost of battery life over the lifespan of the service for OLEV at Gumi City.", "The proposed cost model includes battery performance over the battery lifespan because it is continuously charging and discharging.", "This paper points out that frequent-shallow charging with dynamic charging is better than infrequent deep-charging with plug-in or stationary charging when considering battery lifetime.", "It estimates a cost of approximately $11 million dollars for stationary charging transit buses, which could be reduced by around 20% to $9 million if replaced with dynamic charging.", "This cost reduction derives not only from the shared resource of the wireless charging infrastructure, but also from the lifetime improvement of battery life with dynamic charging.", "Park and Jeong (2017) reports on the economic benefits of the OLEV by comparing it with the operational costs for Plugin Hybrid EV (PHEV).", "The cost analysis shown in the report is significant because it provides one of the few figures based on actual operations costs for a wireless charging transit system.", "The data is collected from the OLEV system at Gumi City, where the No. 7 transit line is operated by OLEV buses and compressed natural gas (CNG) buses, which are widely used in South Koreas public transit systems as an alternative to the conventional diesel buses.", "Because these two systems operate on the same routes (approximately 259 km), an operational cost comparison can be done easily; such a comparison revealed that the yearly costs of operating OLEV and CNG buses are $14,136 and $52,932, respectively.", "These costs are calculated based on energy efficiency (km/kWh vs. km/l) and energy cost ($/kWh vs. $/l), suggesting that OLEV buses save 73% of energy costs when compared to a CNG bus.", "The study also projects that, under the same maintenance cost, the 10-year lifetime cost of the OLEV and CNG buses, including the bus costs, are $591,369 and $729,320, respectively, without government subsidies, suggesting that OLEV is more competitive than CNG buses in terms of both short-term operations and long-term investment.", "Of course, this figure does not include the charging infrastructure cost; nonetheless, the cost of an OLEV is expected to decline significantly, the study authors argue, if they were to be mass produced.", "The report goes on to estimate cost figures for OLEV-based passenger cars, noting that a medium-sized vehicle, equipped with OLEV technology at 5 kWh, would cost approximately $32,090.", "It also projects that the construction and installation of wireless charging infrastructure would cost $726 million, if an installation cost of $0.5 m/km on the 6% of the total traffic lanes in Seoul can be assumed.", "These suggestions imply that such an investment could serve 60% of the total travel by passenger cars within Seoul.", "Nicolaides and Miles (2015) investigates whether the adoption of wireless charging EV infrastructure at the national scale is technically feasible.", "With a qualitative analysis based on existing technical documents, surveys, reports, and academic work, the study concludes that the wide-scale adoption of the technology is feasible and can be a major driver towards a more sustainable, environmentally friendly and socially responsible transport sector.", "It should be noted that the data used across the various studies considered here are quite variable.", "For instance, the cost of a wireless charging lane in Fuller (2016) is $4 million per lane-mile, whereas in Chen et al. (2017) a lane-mile is estimated at 0.8 million which, in turn, is based on the figure used in Jang et al. (2016).", "As such, it is worth mentioning that the rapid development of wireless charging technologies can significantly affect the empirical data and that the findings and conclusions drawn from the results should therefore not be treated as conclusive.", "Bi et al. (2015) is considered one of the first studies to investigate the lifecycle energy and greenhouse gas emissions of wireless charging EVs.", "This study uses lifecycle assessment (LCA), the same method used to evaluate the potential environmental impact on the total lifecycle of a product or system.", "With this method, they compare lifecycle energy and greenhouse gas emissions across plug-in and wireless charging for an all-electric bus system.", "The bus system modeled for the assessment, consisting of 67 buses operating 21 routes, is based on an actual transit bus system serving the Ann Arbor and Ypsilanti areas in Michigan, called TheRide.", "In the model, all bus fleets operate with either wireless charging-based or plug-in based buses.", "For the analysis of plug-in buses, each bus is assumed to be equipped with a battery that has enough capacity to serve the length of the route, which is 458 kWh.", "For the wireless charging buses, it is assumed that a total of 428 stationary and quasi-dynamic wireless chargers are installed at major bus stops, transit centers, and parking lots.", "The results indicate no significant differences between the plug-in and wireless charging systems in terms of cumulative energy demand and global warming impact.", "The wireless charging system consumes 0.3% less energy and emits 0.5% less greenhouse gases than plug-in charging over the total lifecycle.", "However, the study also indicates that energy and greenhouse gas emissions of the wireless system can be further reduced by improving wireless charging efficiencies.", "Building upon Bi et al. (2015), Bi et al. (2017) integrated the LCA approach with a lifecycle cost model (LCA-LCC) to model a wireless charging bus system.", "LCC analysis provides a holistic evaluation of costs, from capital investment to use-phase operation and finally to the end of the lifecycle.", "Bi et al. (2017) studies the same bus system modeled in Bi et al. (2015).", "Using the integrated model, the cumulative costs and total costs per bus-kilometer are analyzed across wireless charging, plug-in, and conventional diesel-based bus fleets.", "The study concludes that a wireless charging system has a lower overall LCC compared to a plug-in charging system.", "Moreover, wireless charging electric buses are found to be more economically competitive than conventional diesel, diesel hybrid, and plug-in charging electric buses for the scope of an entire lifecycle.", "However, the article also emphasizes that there is considerable uncertainty for the results, which remain dependent upon favorable battery pricing, charging efficiency, and other related costs.", "Both Bi et al. (2015) and Bi et al. (2017) do not consider the optimal allocation of the wireless charging points.", "The bus routing model used in both analyses is assumed to have wireless charging infrastructure at almost every major stopping point.", "Moreover, they do not consider that utilization will affect the life and maintenance cost of the charging infrastructure.", "It is critical to understand the construction issues that wireless charging EV infrastructure entails before installation and maintenance costs can be adequately evaluated.", "Unfortunately, very little of the literature discusses construction issues, owing in large part to the limited number of implemented wireless charging systems, other than as testbeds.", "Jung (2017) provides a report on the installation of the charging infrastructure based on first-hand experience of installing an OLEV system, offering descriptions of the installation process, requirements, and construction steps.", "Pictures of the various stages of the installation and test procedures conducted during the installation process are also presented.", "Chen et al. (2015) also discusses expected challenges and issues for the road-installed power tracks and road maintenance.", "This paper also proposes potential methods to improve the structural integrity of the road with power tracks.", "Gill et al. (2014) suggests a business model to fund the wireless charging infrastructure.", "The model specifies the role of public and private agencies and provides a payment scheme for both EV and non-EV drivers.", "Although the model remains at the proposal level and has received no rigorous analysis based on either quantitative methods or case studies, it is nonetheless considered the first paper to discuss the infrastructure funding issue.", "Pricing the electricity for the wireless charging EV is a central issue in the commercialization of the technology.", "In particular, if the wireless charging EV is expanded to personal vehicles and charging infrastructure is shared with non-EVs, the pricing issue will be critical in the operation of the system.", "The literature on pricing suggests that it must include both a billing policy and the requisite infrastructure for billing.", "Sarker et al. (2017) considers the challenges of power-load balancing and congestion.", "The study investigates the power-load and congestion caused by wireless charging EVs using simulation and proposes a game theory model, based on a distributed power schedule framework, to find an optimal schedule between wireless charging EVs and the smart grid.", "This work assumes that the smart grid is capable of processing data and communicating with others and of course is also connected with the charging sections.", "While driving, wireless charging EVs receive energy from the smart grid via power tracks.", "He et al. (2013) proposes two mathematical models to determine optimal pricing schemes for electricity and the roads.", "The first is concerned with a first-best condition, in which a government agency controls the operations of both the regional transportation and power transmission networks and aims to maximize social welfare associated with both, defined as minimizing the sum of the total travel time cost and generation cost of electricity.", "The second scenario considers a more practical second-best case, in which the government agency only manages the transportation network while the power network is operated by an independent system operator (ISO) using locational marginal pricing.", "The ISO accepts supply and demand bids submitted by market participants, which are buyers and generators, and is responsible for determining the power supplies to meet the demands with the dual objectives of minimizing power generation costs and ensuring system security.", "In this case, the price that the ISO charges for electricity at each location will be the locational marginal price.", "Although the traffic authority has no control over the power market, the agency can participate in the wholesale market as a buyer; it will pay ISO, at locational marginal prices, for the dispatched power used to charge electric vehicles.", "By contrast, the agency can adjust the retail price of electricity at each link to affect drivers route choices.", "Under this second-best scenario, He et al. (2013) seeks an optimal pricing strategy to maximize social welfare associated with the transportation system.", "Manshadi et al. (2017) addresses the issue of the interdependency between traffic routing and the payment schemes for the wireless charging EVs.", "The study proposes a decentralized optimization framework to consider the effects of wireless charging on electricity and transportation networks.", "The framework enables one to simultaneously evaluate: (1) the effects of electricity demand imposed by the strategic wireless charging of the EVs on the price of electricity, and (2) the effects of electricitys pricing and availability on the EVs traffic flow within the transportation network.", "The underlying assumption is that the EVs behavior on the network will be captured by a deterministic user equilibrium, in which all paths that connect an origin and destination pair have the same total travel cost.", "The case studies are conducted under the assumption that the power is generated from a renewable source such as thermal generation or wind units.", "It should be noted that this study did not consider that fact that greater congestion on a traffic path can provide more charging opportunities for the EVs on the path, resulting in more power usage.", "As Hussain et al. (2015a) has addressed, a billing policy is challenging.", "Unlike road usage-based payments for a conventional toll collection on the highway, billing for wireless charging EVs should consider the amount of electricity used by each vehicle.", "A vehicle with a fully charged battery should not be charged the same amount for a vehicle with an almost empty battery, even if they are driving on the same charging lane.", "Seminal work by Hussain et al. (2015a) and Hussain et al. (2015b) propose a secure and privacy-aware fair framework for billing and authentication of wireless charging EVs.", "The proposed framework is based on the assumption that the power track consists of multiple segments and billing is done on segment-by-segment basis, where each segment delivers a constant amount of energy.", "Kalialakis and Georgiadis (2014) and Kalialakis et al. (2016) provide surveys of the regulatory frameworks pertinent to WPT systems.", "Readers can find the state-of-art on standardization and regulatory activity around wireless charging EVs, including the International Electrotechnical Commissions (IEC) Technical Committee on Electric vehicle (IEC, TC 69, 2017), the ISO Road Vehicles-Electrically Propelled Road Vehicles dealing with vehicle operation conditions, vehicle safety, and energy storage installation (ISO/AWI PAS 19363, 2017; Pereirinha et al., 2016), the Society of Automotive Engineers (SAE) standardization principles, the work of a taskforce called J2954 on wireless charging for vehicles (SAE J2954 Overview and Path Forward, 2017), and the work of the IEEE Industry Standards Technology Organization Electric Vehicle WPT, with a focus on motion wireless charging (IEEE ISTO, 2017; IEEE Standards, 2017).", "Cho (2017) and Jung (2017) report on safety regulation requirements and standards according to which testing and evaluations were performed during the installation and deployment of the OLEV system.", "These reports also provide how existing standards and regulations should be adapted for commercialization of the system.", "Building on Deflorio et al. (2015a) and Deflorio and Castello (2017) proposes a simulation framework for medium-sized freight vehicles operating on wireless charging lanes.", "A specific traffic model is developed based on a mesoscopic traffic simulation model that incorporates both the traffic behavior and energy state of each EV, based on the assumption that vehicles are operating in a fully cooperative manner, in which advanced driver assistance systems control both the speed of each vehicle and lane selection.", "When a wireless power transmission system is designed, it has to be done in a systems context.", "Specifically, the power transmission is the subsystem of the system, which is the EV and as a result, the subsystem should be carefully designed to meet the requirements defined at the systems level.", "That is, the design of the wireless charging system in isolation from the requirements of an electric transportation system may lead to a wrong conclusion.", "The OLEV was designed using a systematic design method, axiomatic design (Hong and Park, 2010; Suh, 2012; Suh et al., 2011).", "Suh and Cho (2017a) describes the detailed process of the system design issue and provides the process of defining the system-level functional requirements (FRs) and how the WPT system is designed to meet these system-level FRs using an axiomatic design method.", "This is one of the few articles to address the system design issue and describe the detailed design process of the WPT system in the context of system design.", "The system architecture is a high-level structural configuration that characterizes the system.", "In particular, the system architecture of EV is defined as the structure of the charging and energy transferring mechanics from the energy grid to the vehicles motor.", "A primary goal of architectural analysis is to provide a high-level perspective of the energy flow from a structural perspective (Emadi et al., 2005).", "For example, hybrid EVs are distinguished into two classes from the perspective of system architecture, serial- and parallel-hybrids, which reflect different ways that the internal-combustion (IC) engine receives energy flows.", "Jang et al. (2016) first defined the architectural characteristics of wireless charging EVs, identifying how they differ from conventional plug-in EVs.", "However, the term architecture here is based on the charging type rather than energy flow generated from the engine, meaning that system architecture equates to charging architecture.", "Jang et al. (2016) also presents use as the ratio of the vehicles drivable time to available time: the EV is drivable only if it has enough energy in the battery.", "For the conventional plug-in EVs, the vehicle is not drivable when it is being charged.", "Therefore, the charging frequency, determined by a driving range, together with charging time, directly impact the vehicles use.", "The study argues that, in recent decades, EV developers and engineers have been working to improve this use by improving the battery density (thus reducing the charging frequency) or by reducing its charging time.", "The hybrid-EV is also a result of this effort under this architectural paradigm.", "The study claims that wireless charging EVs are a new type of EV altogether because they decouple charging and driving.", "The improvement of the use of an EV can be achieved by architectural redesign – dynamically charging while the EV is in motion.", "The study provides the technological value of the wireless charging EV in terms of its system architecture.", "Although Pantic et al. (2009) does not exclusively investigate the system architecture issue, they use a system architecture perspective to compare wireless charging EVs with batteries with those with an ultra-capacitor.", "Note that ultra-capacitors are widely used for energy buffers.", "Its energy holding and access capacities differ greatly from those of a battery.", "This means that its energy flow mechanisms are different and, as a consequence, so too are the considerations for allocating wireless charging facilities.", "The OLEV at KAIST also investigated a new architecture for wireless charging EVs – combining the battery and ultra-capacitor (OLEV Annual Progress Report, 2010).", "The researchers at KAIST compare developmental costs and operational costs between battery-only and combined battery and ultra-capacitor wireless charging EVs, although this remains only a high-level feasibility study with no conclusive results.", "This section proposes promising new research directions.", "Note that the goal is not to provide a complete list of potential topics, but rather to offer possible directions based on the survey of research presented in the previous sections.", "These are organized into two topic groups, one that further investigates key operations and systems issues identified by the literature review (incorporating uncertainty, battery life considerations), and the other focused on extending the study domain (into passenger vehicles, ITS, and allocations).", "Although there is uncertainty in charging and discharging, very few studies incorporate this uncertainty in the modeling, with the exception of the work down by Liu and Song (2017).", "As mentioned in the previous section, Liu and Song (2017) uses a robust optimization approach to consider the uncertainty introduced by charging and discharging.", "This model deals indirectly with the parameters of uncertainty by introducing the concept of uncertainty limits for each variable, a technique that is appropriate if no information about the probability distribution of uncertain or stochastic variables is available but minimum and maximum bounds are known.", "This means that no study has dealt with uncertainty directly, despite the inevitable uncertainty raised by vehicle speed, road conditions, driving alignments, and other road or driving conditions.", "Because no quantitative investigation has been reported, further investigation – whether rigorous simulation or experimental study – is needed to determine what types of models would be appropriate to describe or model such uncertainty.", "The approach taken in Birrell et al. (2015) might be a good example in experimental analysis of charging uncertainty.", "The study focuses on parking misalignment for a stationary charging EV as a source of uncertainty.", "Although the work does not directly address dynamic wireless charging EVs, its approach and methods are informative.", "Uncertainty here was approached both from a human factor perspective, focusing on human driving behavior, and from a traffic pattern approach with standard driving cycle data.", "Regardless of which approach is used in further research, a logical foundation is needed to build statistical charging models as a basis for developing advanced charging infrastructure allocation simulations and for recommending and validating safety factors in the design of wireless charging EV systems.", "Although it is widely accepted that wireless charging EVs can use smaller batteries, their use may not guarantee long-term economic efficiency of the system.", "Studies have shown that batteries that are too small have a negative effect on overall battery life because they are prone to deep-discharging cycles that aggravate battery degradation (XU et al., 2013; Bi et al., 2015; Jeong et al., 2015); and if batteries are short-lived, they will need to be replaced frequently, leading to increased battery costs overall.", "As such, the use of larger batteries may be more economically sound from a long-term perspective.", "This quick example suggests the importance of considering battery life when designing wireless charging EV systems.", "Empirical evidence suggests that the capacity of a battery will degrade significantly throughout its lifetime.", "As such, studies no longer consider battery capacity as a static variable, as had been done previously for modeling wireless charging systems, for example, in Jang et al. (2015), Ko and Jang (2013), and Hwang et al. (2017).", "Rather, battery capacity instead must be treated as a dynamic variable that decreases over the battery’s lifespan.", "Jeong et al. (2015) first investigated the issue of battery degradation, but few follow-up studies have yet been reported, possibly because incorporation of battery life creates a strong nonlinear property in the model, which makes the analysis mathematically intractable and the development of cost-benefit analyses and optimal allocation models particularly challenging.", "Furthermore, to consider battery life in the analysis requires expert knowledge on different battery types and capacities.", "This suggests that more collaborative work is needed between battery experts and operations and systems researchers.", "Most studies of charging infrastructure allocations focus on public transit buses and routes, in which vehicles are traveling on fixed and predefined routes.", "This is because, as mentioned earlier, most commercialized or prototyped versions of the dynamic wireless charging EVs are found in public transit buses.", "At the time of writing, automakers are beginning to introduce passenger-type dynamic wireless charging EVs, and governments have begun to fund wireless charging EV infrastructural projects; for example, the British government is funding an electrified road project in which one lane of a highway is being converted to a wireless charging lane with any type of wireless charging EV – whether a public bus or a passenger vehicle – capable of charging when on it.", "Automakers, policy makers, regulators, and traffic and transportation planners are currently seeking answers to questions such as:", "What is the appropriate battery size for a wireless charging passenger EV?", "How much wireless charging infrastructure should be installed in a city?", "Is installation of a small number of long charging lanes better than installation of a large number of short charging lanes, or vice versa?", "How do installed charging lanes affect traffic?", "A pioneering work has investigated some of these issues (Chen et al., 2017).", "However, plenty of work is still needed to investigate the application of wireless charging systems in passenger EVs.", "In the era of Big Data and attendant advances in intelligent transportation systems (ITS), it is a natural step to incorporate wireless charging EVs with ITS.", "Real-time traffic conditions and the locations of the wireless charging lanes and stations can be shared with EV drivers.", "The development of real-time routing algorithms for wireless charging EV drivers is one example of a topic with great potential; evaluation of optimal velocity profiles for drivers for a given route, with real-time traffic conditions, and information about charging lanes and stations, is another.", "In such systems, the operations of wireless charging EV are coupled with internal information, such as the drivers habits and patterns and the vehicles battery level, and with external information, such as traffic conditions and charging infrastructure locations.", "Because the role of ITS is to provide drivers with these internal and external data points, and eventually to assist drivers in making good decisions, a number of interesting topics and ideas on wireless charging operations can be proposed in conjunction with ITS.", "Environmental and energy issues are a great concern these days.", "Although there are numerous government reports and considerable media coverage discussing the environmental and energy effects of dynamic wireless charging EVs, analyses performed with a rigorous model and scientific approaches are limited.", "As mentioned earlier, Bi et al. (2015) and Bi et al. (2017) are the pioneers investigating the energy and environmental issues related to wireless charging EV systems with scientific modeling approaches.", "Extending their work is definitely a good research area.", "For instance, the battery replacement issues in the environmental assessment might provide insight into wireless charging EVs.", "Furthermore, the environmental and energy impacts of the entire wall-to-wheel process is a promising research area.", "There are numerous papers analyzing the effect of EVs on the power grid (Green et al., 2011).", "Most of the studies were performed for conventional EVs with a mobility pattern, and charging was mostly done at home overnight and occasionally at a public or commercial charging station during the day.", "However, this assumption of the charging pattern is no longer valid if wireless charging becomes the norm for EVs.", "Therefore, systematic studies are required to investigate the effect of wireless charging EVs on the power grid, such as the power demand pattern across time of day or geographical regions, and the relationship between the installation of the wireless charging infrastructure and the power load in the grid.", "Moreover, the power distribution design is expected to change when the wireless charging infrastructure becomes widespread.", "A higher power demand is estimated along roads with wireless charging.", "The distribution network design must address this issue.", "Another interesting area is the integration of renewable energy sources such as solar panels and wind turbines with the wireless charging infrastructure.", "The penetration of more renewable energy sources into the energy market is shifting the power generation and distribution industry.", "The effectiveness of the integration of the wireless charging infrastructure with wireless charging in the form of distributed energy is a promising area of research.", "It is known that the research consortium led by the Utah State University, SELECT, installed a test track of wireless charging EVs with solar panels.", "Installing such a testbed and investigating the effectiveness of the integration or developing an energy distribution logic or algorithm are also promising areas of study.", "The application of WPT in transportation is not necessarily limited to transit or passenger vehicles.", "Yoon (2017) introduces the application of WPT to special purpose transportation systems, such as cargo transportation in airports, container transports at harbors, and cargo transporters in warehouses.", "Helber et al. (2018) considers the location planning for wireless charging systems for electric airport passenger buses.", "Ko and Jang (2018) proposes a design method of an operation profile for wireless charging electric tram systems.", "To a large extent, material handling systems in factories are also transportation systems for which WPT technologies have been adopted for more than 10 years, particularly in semiconductor and LCD factories (Hwang et al., 2016; Jang et al., 2005).", "There is plethora of interest in research for these applications.", "Another area that can be explored is systems design for wireless charging-based autonomous vehicles.", "With the rapid development of autonomous driving technology, it is also projected that electric charging will be introduced for autonomous vehicles in the near future.", "At such point, it will be a natural step that autonomous EVs to adopt automated charging mechanisms (it would be somewhat absurd to imagine the driving being done autonomously but charging still being performed manually).", "Wireless charging will be integral to such vehicles operations.", "This survey provides the first overview of the state-of-the-art in operations and systems research into wireless charging EVs.", "The technology is still in early developmental stages, and operations and systems-related research has yet to fully mature, so investigations into current research activities is critical.", "This survey work has broad applications for the research community, industry, and regulators and policymakers.", "The survey first discussed the current state of wireless charging EV systems deployment and commercialization projects, so that readers understand the actual technological development status.", "It is worth understanding these activities because most operations and systems-related researchers refer to these projects to convince readers of the value of their proposed study.", "The notations, terms, and definitions are also reviewed in this section, before describing in greater depth the components of the wireless charging EV as used in the existing publications.", "Note that these terms and definitions have yet to be standardized, and as such clear specification is critical for surveys of technology at this stage.", "It also identifies how different terms and definitions are used in other studies.", "The main part of the survey focused on the state-of-the-art in wireless charging EV, reviews the existing publications from five different perspectives: charging infrastructure allocation; driving range extension; cost and benefit analyses; supporting systems; and miscellaneous perspectives.", "With the perspective-based review, a single paper can be reviewed from more than one perspectives.", "The most active research area is charging allocation analyses, which propose methods for optimal allocation of charging infrastructures.", "Cost-benefit analyses for wireless charging EVs and range extension analyses are also widely studied areas.", "The paper then moved to propose future research directions: the incorporation of uncertainty into modeling; research on passenger vehicles; battery life considerations; ITS; and applications.", "These areas are proposed based on the level of completeness of the existing literature (incorporating uncertainty, battery life considerations) and on extensions of the study domain (passenger vehicle, ITS, and allocations).", "Note that these proposed directions are not a complete list but are areas that demonstrate promise for extending existing research.", "Almost all of the papers discussed in this survey consider dynamic wireless charging technology to be a promising solution for future EVs.", "However, there is also a threat to this technology.", "For instance, Chen et al. (2012) argues that dynamic wireless charging EVs are not competitive when compared to battery-swapping EV buses in terms of technological maturity.", "In South Korea, some municipal governments are adopting battery-swapping EV buses as an alternative to conventional diesel-based EVs (Kim et al., 2015).", "Therefore, some researchers have claimed that the battery-swapping solution might be a threat to wireless charging EVs.", "At this point, it might be too early to conclude which technology is more competitive.", "A cost and performance analysis comparing these two technologies might be good research topics for the future.", "Some scholars have also argued that ultra-fast charging technology and the advancement of battery technology might make dynamic wireless charging redundant (Wireless Charging Road for Electric Buses Will be Built in Israel, 2017).", "However, it is also true that fast charging might significantly reduce the cost of wireless charging infrastructure because the charging amount per length of wireless charging lane would be increased more power could be transmitted with a shorter charging lane.", "The effect of fast charging on the economics of wireless charging EVs is also an interesting topic.", "In summary, there are numerous threats as well as opportunities related to WPT applied to EVs.", "Investigating these issues with analytical rigor is a task for the future.", "Research into the operations and systems of wireless charging EVs is still evolving.", "Most methods have not yet been tested on actual systems.", "The cost figures also may not be correct because wireless charging EVs have not yet been mass produced.", "Therefore, the numerical results and conclusions drawn in most papers should be treated as preliminary.", "However, this too should be considered a research opportunity; the validation of methods and results is a primary responsibility for the academic community." ]
[ "Focusing on the operation and system-related studies of wireless charging EV systems.", "Discussing the technology development and deployment of wireless charging EV systems.", "Surveying the existing research related to the deployment and operation.", "Proposing future research directions to researchers and practitioners." ]
Scapula kinematics of pull-up techniques: Avoiding impingement risk with training changes
Objectives: Overhead athletic activities and scapula dyskinesia are linked with shoulder pathology; pull-ups are a common training method for some overhead sports. Different pull-up techniques exist: anecdotally some are easier to perform, and others linked to greater incidences of pathology. This study aims to quantify scapular kinematics and external forces for three pull-up techniques, thus discussing potential injury implications. Design: An observational study was performed with eleven participants (age = 26.8 ± 2.4 years) who regularly perform pull-ups. Methods: The upward motions of three pull-up techniques were analysed: palms facing anterior, palms facing posterior and wide-grip. A skin-fixed scapula tracking technique with attached retro-reflective markers was used. Results: High intra-participant repeatability was observed: mean coefficients of multiple correlations of 0.87-1.00 in humerothoracic rotations and 0.77-0.90 for scapulothoracic rotations. Standard deviations of hand force was low: <5% body weight. Significantly different patterns of humerothoracic, scapulothoracic and glenohumeral kinematics were observed between the pull-up techniques. The reverse technique has extreme glenohumeral internal-external rotation and large deviation from the scapula plane. The wide technique has a reduced range of pro/retraction in the same HT plane of elevation and 90° of arm abduction with 45° external rotation was observed. All these factors suggest increased sub-acromial impingement risk. Conclusions: The scapula tracking technique showed high repeatability. High arm elevation during pull-ups reduces sub-acromial space and increases pressure, increasing the risk of impingement injury. Wide and reverse pull-ups demonstrate kinematics patterns linked with increased impingement risk. Weight-assisted front pull-ups require further investigation and could be recommended for weaker participants.
[ "HEAL", "MEDI" ]
[ "Biomechanics", "Kipping", "Repeatability", "Shoulder", "Skin-fixed scapula tracking", "Supraspinatus" ]
[ "2732", "3612" ]
[ "Pull-ups are a common training activity for a range of sports.", "A link between scapula kinematics and injury, most commonly shoulder impingement, is widely theorized and occasionally tested,1,2 particularly in overhead activities.", "Shoulder impingement is the compression of the rotator cuff and subacromial bursa on the anterioinferior aspect of the acromion coracoacromial ligament.3", "This can occur with extreme internal glenohumeral (GH) rotation during unloaded abduction and forward flexion.4", "Anecdotal evidence indicates that reverse pull-ups are easiest to perform, while wide-grip pull-ups are implicated with higher incidences of shoulder pathology.", "Climbing and gymnastics, which utilize pull-up-like techniques, are strongly linked to shoulder pathology—particularly shoulder impingement.5,6", "However, there is no quantitative discussion of the scapula and upper limb kinematics, or comparisons of the many different techniques, for pull-ups.", "Difficulties in measuring 3-D scapula kinematics, due to skin artefacts, contributed to the lack of quantitative literature.", "Non-invasive skin-fixed devices with multiple attachment points and optimal calibration have reduced errors at high angles of humeral elevation and throughout the ROM in dynamic tasks.7,8", "Pull-ups are a closed-chain activity; good motion repeatability is therefore theorized across the experimental group (inter-participant), allowing comparison of group averages.", "Large muscle contractions in the shoulder have been hypothesized to reduce the consistency of observed joint kinematics.9", "Pull-ups will provide a challenging environment in which to observe the intra-participant repeatability.", "The aim is to present a kinematics dataset that compares the humerothoracic, scapulothoracic, and glenohumeral rotations across three pull-up techniques and discuss potential injury risks associated with these techniques.", "A convenience sample of eleven healthy male participants with no history of shoulder pathology participated (age = 26.8 ± 2.4 years, BMI = 22.2 ± 2.2 kg/m2, height = 1.80 ± 0.06 m).", "Participants were performing pull-ups as part of a regular training regime (>3 years training experience).", "The local ethics committee approved this study.", "Kinematic data collection utilized 9-camera optical motion tracking (Vicon, UK) at 200 Hz and a force plate (Kistler, Switzerland) at 1000 Hz (Fig. 1).", "A Scapula Tracker (ST7) measured scapula kinematics.", "The device consists of a base attached to the mid-portion of the scapula spine and an adjustable foot positioned on the meeting-point between the acromion process and the scapula spine.", "This position is optimal for the attachment of the ST.8 The ST technical coordinate frame was calibrated with the anatomical coordinate frame of the scapula using the International Society of Biomechanics (ISB) recommended anatomical landmarks10 and measured directly using a scapula Locator.8", "Calibration was performed at 90° of humerothoracic (HT) elevation at 45° to the coronal plane: the mid-point of the overall motion.7", "The calibration transformation was applied to each trial of that participant.", "Errors associated with static palpation of landmarks are small (∼2°11).", "Twenty-one retro-reflective markers were used to track the thorax, clavicle, humerus and forearm.8,10", "Elbow epicondyles were defined as a rigid offset from the humerus technical frame with the arm at 90° elevation, 45° from the coronal plane, 90° elbow flexion and a vertical forearm.", "Least squares sphere-fitting was used without bias compensation12 to calculate the glenohumeral head centre during a functional task with low arm elevation, using the Locator to track the scapula.", "Three pull-up techniques were performed: ‘front’ with anterior facing palms and hands approximately shoulder-width apart, ‘wide’ with anterior facing palms and hands on the lateral sloped portion of the bar and ‘reverse’ with posterior facing palms and hands approximately shoulder-width apart (Fig. 1).", "The hand positions were not prescribed between participants.", "Five sets of three pull-ups were performed: each set was a random distribution of the techniques, giving five repetitions of each technique.", "Thirty-seconds rest was enforced between each set.", "Participants were instructed to perform a maximal upward movement covering their full range of motion (ROM), keeping legs to the posterior at 90° to the thorax (Fig. 1).", "The upward motion and the mean of three complete trials (randomly selected) for each participant were analysed.", "Intra-participant repeatability is presented with coefficients of multiple correlations (CMC13) and standard deviations (SD).", "CMC is a measure of waveform similarity and has been used in gait analysis14 and shoulder kinematics.13", "Inter-participant repeatability is calculated with Pearson's product moment coefficient of correlation (Pearson's r), because variables are centred and scaled according to their own means and standard deviations, thus waveform similarity is not sensitive to offsets in joint rotations expected with different participant anatomies.", "The average rotations for each participant are presented with an average inter-participant Pearson's r value for the three techniques.", "A low-pass fourth-order Butterworth filter (cut-off: 4.7 Hz) was used to remove noise from the kinematics data.", "The ISB recommended coordinate frames were used for the thorax, humerus and scapula.10", "ISB recommended Euler rotations were used, except at the GH joint where gimbal lock was observed and thus a z–x′–y″ sequence (adduction–flexion–internal rotation being positive) was used instead.", "A z–y′–x″ sequence (posterior tilt positive around z-axis) was used between the laboratory and thorax frames to determine thorax posterior tilt.", "A low-pass fourth order Butterworth filter (cut-off: 10 Hz) was used on the force plate data, after a spectral analysis of the signal.", "To compensate for the lack of a second force plate, the vertical force when the participant is hanging from the bar minus the vertical force when the participant and frame are on the force plate is subtracted from that participant's trial's vertical force.", "Half the force values, normalized to the participant's body weight, give the force at each hand.", "Data were normalized to the time of force time-points: zero percent was taken as the first peak in upward vertical reaction force occurring as the movement is initiated, 100% of the motion was taken as the major trough in this force (Fig. 1).", "A cubic spline interpolation was used to find the value of each measure at every 10% of the motion.", "A two-way repeated measures ANOVA tested for significant differences between the three pull-up techniques (SPSS).", "Pull-up technique (front, wide, reverse) and percentage of motion (0–100%) were defined as the within-participant factors and joint rotations as the dependent variables.", "Where a significant interaction existed between technique and percentage of motion, a one-way repeated measures ANOVA tested for significant differences between the three pull-up motions at each 10% of the motion; percentage of motion was the within-subject factor.", "A Bonferroni post-hoc test then performed pair-wise comparisons between the techniques.", "Mauchly's test for sphericity was used.", "When a significant violation of sphericity was found the Greenhouse–Geisser correction was used.", "The Shapiro–Wilk test verified that the quantitative variables did not significantly depart from a normal distribution.", "Mean intra-participant repeatability is presented for each joint rotation and each pull-up technique (Table 1).", "CMC and Pearson's r values below 0.4 represent poor reliability, above 0.75 excellent reliability and between 0.4 and 0.75 fair to good reliability.15", "The presented intra-participant variations are excellent; with no average CMC below 0.77 (and most ≥0.87).", "The wide and reverse pull-ups show the worst CMC values in ScT rotations (in protraction) and the reverse pull-up for HT rotations (in axial rotation).", "The force produced at one hand for all participants and motions had a maximum intra-participant SD of less than 5% body weight.", "The excellent Pearson's r values describe a clear trend in the measured values for the three pull-up techniques (Table 1).", "However, the posterior tilt during the reverse technique and, more significantly, the protraction during the wide technique show relatively poor correlations (Table 1) indicating differing scapula rotations and control between participants.", "The average kinematics across the three pull-up techniques are presented (Fig. 2).", "The results of the one-way ANOVA testing, and the Bonferroni post-hoc tests, highlight where the differences between specific techniques are significant (Supplementary 1).", "The HT plane of elevation and axial rotation shows consistently significant differences between the three pull-up techniques (Fig. 2a).", "The most significant scapular differences are in scapula pro/retraction where front, wide and reverse pull-ups have ranges of 22°, 10° and 17°, respectively.", "Significant but small differences are seen in the other two rotations, with average ranges of 10° and 35° in medial/lateral rotation and ant/posterior tilt, respectively.", "There was a significantly different pattern of GH internal/external rotation and plane of elevation between the three pull-up techniques (Fig. 2c).", "A novel dataset has been presented, describing shoulder kinematics during three pull-up techniques.", "There is no data in the literature with which to compare these results.", "In general, high elevation of the arm reduces sub-acromial space and increases pressure; thus increasing the risk of impingement.4,26,29", "The HT rotations are within acceptable limits22 and, from qualitative examination, seem to describe the observed pull-up motions (Fig. 2).", "Significantly different planes of HT elevation and axial rotations result from different hand positions in the pull-up techniques (Fig. 1).", "More ScT retraction towards the top of the front pull-up, compared to the reverse pull-up, is expected because the humerus plane is more coronal during front pull-ups, which acts to retract the scapula.", "The more coronal plane of HT elevation at the bottom of the wide pull-ups (Fig. 2a) led to significantly more ScT retraction than the other two techniques.", "The greater ScT lateral rotation at the bottom of the front pull-up technique is expected with the increased humeral elevation.", "Similarly, there is a reduced ScT lateral rotation during the reverse pull-up, in-line with reduced HT elevation.", "ScT posterior tilt is similar between the three techniques.", "Given the small ROM it is unlikely that the ST is able to precisely differentiate between the techniques,7 although significant differences do exist (Fig. 2b).", "Overall, the pattern of ScT motion and the observed ROM is comparable to a bone-pin study of multi-planar humeral elevation.22", "Rotator cuff pathologies, especially impingement are related to glenohumeral (GH) joint kinematics.1", "Impingement is prevalent in climbers and gymnasts,5,6 both requiring similar tasks to pull-ups.", "Therefore, speculation on vulnerable positions during pull-up techniques is a justified activity.", "There is a significantly larger range of GH internal/external rotation in the reverse technique, starting in a position of quite extreme external rotation.22", "Extreme external rotation with an elevated arm has been linked to impingement in athletic patients,25 high sub-acromial pressures4 and reduced sub-acromial space.26", "Thus, the reverse pull up technique potentially increases sub-acromial impingement risk in the hanging and initiation phase, an important consideration, given that it is anecdotally easier and thus prescribed for weaker participants.", "Further work could analyse weight-assisted front pull-ups as a lower risk alternative.", "In a cadaver study4 internal rotation of the humerus during abduction and flexion gave the highest supraspinatus compression forces.", "The limits of internal rotation22 are not observed in pull-up kinematics (Fig. 2c).", "However, during wide pull-ups 90° of arm abduction with 45° external rotation was observed (Fig. 2c).", "This position has been shown to give significantly smaller sub-acromial spaces than other abduction positions, although the acromion is not as close to the vulnerable part of the supraspinatus as in 45° of internal rotation.26", "Greater protraction of the scapula relative to the humerus frame has also been shown to reduce sub-acromial space.27", "A significantly reduced range of ScT pro/retraction (Fig. 2b), with a similar range of HT plane of elevation (Fig. 2a), in wide pull-ups may also increase sub-acromial impingement risk.", "Increased ScT ant/posterior tilt compensates somewhat, although the magnitude of this rotation is small.", "The wide pull-up may therefore be associated with an increased injury risk, a concern given the popularity of “Kipping” pull-ups (swinging and then performing a dynamic wide pull-up).", "The dynamic nature is likely to decrease scapula control, particularly in the starting position.28", "Studies looking at sub-acromial space and pressure have been performed in unloaded, passive conditions or in cadavers.", "Thus, conclusions may be different to the highly loaded pull-ups presented here.", "However, given the position of the hand loading, the GH head is expected to be pulled more upwards onto the acromion.", "The plane of elevation of the GH joint follows an expected pattern given the hand positions.", "The significant deviation of the humerus from the plane of the scapula during reverse pull-ups (up to 42°) and, to a lesser extent, front pull-ups (up to 28°), may require greater stabilization by the rotator cuff muscles, since the prime movers tend to move the GH reaction force outside of the glenoid rim in these poses.", "Modelling work could investigate these ideas.", "Examination of intra-participant repeatability allows analysis of both movement and measurement method consistency.", "CMC values are sensitive to small differences in ROM, such as in ScT posterior tilt values.", "The associated SDs indicate whether there is large movement variability or statistical sensitivity.", "The SD values for intra-participant repeatability of HT rotations were in-line with a palpation study of simple planar motions.16,17", "The CMC values were also excellent (>0.85).", "Thus variations can be considered small and participants to be consistently performing the same motion.", "The ScT rotations showed similar SDs to literature,17,19,20 and excellent CMC values (>0.75; Table 1) also in agreement with literature values.19,20", "The reduced repeatability in ScT protraction and posterior tilt may be due to lower accuracy of the ST in these rotations relative to lateral rotation.7", "These results indicate that the ST has similar repeatability to accepted measurement techniques when applied to athletic loaded activities, although caution should be used in analysing posterior tilt.", "The repeatability of the external force (Table 1) is important,18 as is participants’ posture.", "CMC values are excellent for both (>0.88), particularly external force (>0.9).", "SDs are also low relative to the parameter's magnitude (<5% for force).", "These indicate that each participant's motion and body acceleration profile was consistent between trials.", "Higher repeatability of forces relative to kinematics (Table 1), may imply pull-ups are a force-driven task.", "Inter-participant repeatability must be good to allow kinematic data averaging across participants.", "High inter-participant variability (Table 1) relative to intra-participant values (Table 1) is expected.17", "Large variations (SD up to 20°), of a similar scale to those observed in pull-ups (Table 1), exist in the HT plane of elevation and axial rotation during simpler and more controllable motions in the literature.16,17", "The generally good values of Pearson's r in HT rotations (0.64–0.99) also indicate that the pattern of the motion is repeatable between participants.", "Inconsistent positioning of the hands may cause variation in HT rotations.", "Higher correlations observed in the wide technique, where the sloping pull-up frame handles determined the hand positions (Fig. 1), supports this.", "The position is consistent for each individual participant (Table 1), but enforcing a fixed hand position between participants would lead to unnatural, awkward or high-risk movements.", "The ScT variability, measured by SD, is consistent with values found using established scapula tracking methods in the literature (7–12°9,17).", "The values of Pearson's r are low in posterior tilt (0.55–0.66) and protraction during the wide pull-up (0.38), although not dissimilar to literature values.9", "Pearson's r can be very sensitive to small differences where the range of movement is small, as in these rotations (Fig. 2).", "The implied difficulty in scapula control during the wide pull-up technique, shown by relatively poor intra-participant repeatability, may also help to explain this variation.", "However, given the good agreement with literature values the ScT inter-participant repeatability is considered acceptable for presentation of general kinematic patterns.", "It is theorized that the scapula's starting position may be important in determining subsequent scapula rotations.21", "This seems reasonable in highly-loaded activities, because it may be difficult and physiologically costly to move the scapula according to a ‘natural’ rhythm.", "In the current study, this may be manifested in higher intra-participant repeatability for force compared to kinematics (Table 1).", "Future investigation of scapula starting position's effect on kinematics and ‘natural’ scapula motion patterns would be valuable in the rehabilitation setting, where modification of scapula motion is implemented.1,20", "For pull-up training this potential relationship would imply that starting shoulder posture is key for injury prevention and targeting specific muscle groups.", "A setting of the scapula at rest and under loading has been theorized.21,24", "This may occur in the hanging phase of pull-ups: potentially causing significant scapula movement at the beginning of pull-ups, transitioning from a set position to a dynamic movement.", "Given that the start of the motion (0%) is taken as the initial peak in the hand force this is unlikely to be captured in the data presented.", "This dynamic process may contribute to the ST wobble observed in some participants.", "The pattern of hand force and thorax tilt is similar across participants (Table 1).", "Un-loaded planar motions give a smaller range of ScT pro/retraction than literature values: ≈10°22 compared to ≈20° during pull-ups (Fig. 2).", "The force acting at the hand may pull the scapula around the thorax by means of muscular and ligamentous attachments, thus increasing ScT protraction.", "Since general agreement exists in the inter-participant waveform patterns, performance of data averaging was valid.", "A low cut-off frequency was used to reduce noise observed in the ST at the beginning of some trials.", "This noise was possibly caused by ST wobble during the pull-up's acceleration phase.", "This filtering had minimal effect on other joint rotations.", "The level of filtering is in-line with literature studies and recommendations.23", "The low frequency noise may result, in part, from the moment produced by ST's marker cluster.", "Improved design would position markers at the ST's base.", "Limitations of the study include the small sample size and a sample of convenience, which makes extrapolation to other populations somewhat limited.", "A novel kinematics dataset has been presented.", "Potential links between wide and reverse pull-ups, and increased injury risk have been described.", "In reverse pull-up this is due to extreme external rotation with an elevated arm and a large deviation of the humerus from the scapula plane.", "In wide pull-ups it is due to a reduced range of ScT pro/retraction with a similar range of HT plane of elevation, and the achievement of 90° of arm abduction with 45° external rotation, and indicators of reduced scapula control." ]
[]
Improvement in electrical characteristics of Silicon on Insulator (SOI) transistor using graphene material
This paper presents the electrical characteristics of a short channel Silicon on Insulator (SOI) transistor with a graphene layer. The graphene sheet is used at the bottom of the channel close to the source side and a proportionally heavily p-type retrograde doping implanted in nearly middle of the channel. To increase the gate electrostatic control over the channel we incorporated a high-K material i.e. HfO2 as the gate oxide insulator. Due to Graphene growth and Retrograde Doping in the Channel, we called this structure “GRDC-SOI” transistor. Because graphene sheet has low band gap and high mobility, we used it to increase the on-state current. Engineered p-type retrograde doping utilized for both decreasing off-state current and increasing on-state current. These dopants cause impurity scattering in the depth of the channel and deflect electron movements and decrease off-current. On the other hand, these dopants which are located almost in the middle of the channel can play the role of base in an NPN Bipolar Junction Transistor (BJT), and turn it on and exceed the on-state current. An immense comparison among our proposed device and a device similar to GRDC-SOI but without Graphene sheet (RDC-SOI) and a conventional structure shows that our proposed device has superior electrical characteristics in terms of ION/IOFF ratio, transconductance, subthreshold slope, leakage current, breakdown voltage and short channel effects like hot carriers injection and DIBL. Our analyses demonstrate that GRDC-SOI transistor can open a window for utilizing Graphene material in digital circuits and system on chip applications.
[ "PHYS" ]
[ "Graphene", "High-K material", "Retrograde doping", "SOI transistor" ]
[ "3100" ]
[ "Since last decades, for expansion of Moore’s law and satisfying electronic industry requirements, shrinking of Metal Oxide Field Effect Transistors (MOSFET) have been continued [1].", "Silicon on Insulator (SOI) Technology has a huge role to increase the quality of these transistors [2].", "However as transistors are scaled, leakage current increases and some unwanted effects which are called Short Channel Effects (SCE), appear [3–8].", "Therefore, there is a common opinion in the community that MOSFET scaling is not appropriate anymore and it is necessary to utilize new materials and device concepts to continue scaling and performance improvement [9,10].", "2-D Graphene, which is made of a single layer of graphitic carbon is an interesting material for future electronics industry [11].", "Although high carrier mobility and 2-D structure of graphene are desirable for device designers, but lack of bandgap in it prevents the device to be completely turned off and this is a challenge in logic devices [10,12].", "RF (Radio Frequency) FETs usually are biased in active region in circuits and are standby for amplifying, therefore GFET (FET with gapless large-area graphene channel) has got attention for RF applications.", "On the other hand, RF circuits are not as much complex as digital integrated circuits and RF chips have more flexibility in utilizing new materials.", "In the meantime, variety of transistors and materials used in RF circuits including bipolar transistors, n-channel MOSFETs and High Electron Mobility Transistors (HEMTs) [13,14].", "To open a bandgap in graphene and making it a semiconductor, several techniques are proposed including using bilayer graphene and applying a perpendicular field [15,16], narrowing graphene sheet in one dimension to form graphene nanoribbon [16,17], and applying strain to graphene.", "In this work we tried to benefit intrinsic behavior of graphene sheet i.e. high carrier mobility and low band gap, to increase on-state current.", "In order to control leakage current, we found the optimum place for P-type retrograde doping in the channel to decrease leakage current.", "This is due to the fact that electron movement is deflected by adding these dopants and the resultant impurity scattering occurs from where the gate control is low in the channel.", "This engineered P-type doping profile also takes part in exceeding on-state current, by forming base junction of NPN bipolar Junction Transistor (BJT), in parallel to intrinsic n-channel SOI MOSFET where source and drain regions are also equal to “Emitter” and “Collector”, respectively.", "Furthermore, for more electrostatic control over the channel and further increasing the on-current in the device, we used HfO2 material with dielectric constant of k = 16 [18–21], as gate insulator in the proposed device instead of SiO2 with k = 3.9 [22] in the C-SOI device.", "It should be noted that in this work the electrical characteristics of GRDC-SOI MOSFET is compared with RDC-SOI and C-SOI MOSFET counterparts where the active region including source, channel and drain regions are made from silicon material.", "The rest of this work consists of three sections.", "Section two presents a cross section view of the structures under study with related parameters.", "In section three, the simulated and extracted results are reported and discussed.", "Finally in section four, we explain comprehensive conclusion for this study.", "Fig. 1(A–C) shows the schematic diagram of three considered structures i.e. C-SOI, RDC_SOI and GRDC-SOI MOSFETs.", "All parameters related to geometry and dopings of these structures are presented in Table 1.", "Device simulation have been carried out with 2-D Silvaco ATLAS software.", "We enabled FLDMOB, CONMOB, SRH, Auger, HEI, HHI, BGN, IMAPC SELB models where FLDMOB and CONMOB mobility models take into account field and concentration dependent mobility; SRH and Auger models consider recombination process in the device; HEI and HHI calculate hot electron and hole current density; BGN model applies the band gap dependency to doping density and IMAPC SELB models impact ionization generation phenomenon which mostly happens in the drain side of the channel in our simulations [22].", "Although graphene material is not defined in Silvaco Software, in order to have acceptable behavior in simulations, we utilized 3C-SiC material like the work carried out in the literature [23].", "The parameters of this material were modified including carrier mobility, bandgap, velocity saturation and carrier effective mass by “user.", "Material” in ATLAS to achieve a behavior close to graphene.", "Table 2 shows the basic parameters to define graphene in TCAD and Fig. 2 shows IDS-VGS characteristic of Graphene-FET.", "This confirms there is a good agreement between experimental [12], other theoretical works [24] and our simulation results.", "This section consists of two subsections.", "Part A presents comparative study of the devices shown in Fig. 1(A–C) in terms of DC and AC electrical performance.", "Part B considers impact of graphene nanoribbon (GNR) width on the important device electrical parameters.", "It is obtained from Fig. 3 that ION/IOFF current ratio is about 106 in RDC-SOI and GRDC-SOI and OFF-current and sub-threshold slope of these devices are much less than the C-SOI which is excellent in such a short channel length.", "These achievements are indebted to engineered doping in the channel and incorporating HfO2 material (with permittivity of 16) as the gate insulator for high electrostatic control over the channel.", "In fact, retrograde doping profile in the channel blocks electron passage due to impurity scattering, and reduces leakage current when transistor is off.", "On the other hand, it causes creation of easier path for electrons from top of the channel when transistor is in on-state and the channel is created.", "The reduced amount of off-current in GRDC-SOI with respect to RDC-SOI can be described by tendency of graphene to attract electrons passing from it.", "In addition to ION/IOFF current ratio, GRDC-SOI also shows improvement in transconductance (gm), due to more drain current.", "Transconductance that shows how much the gate voltage controls the drain current [28], is depicted in Fig. 4.", "It is obvious that GRDC-SOI has more gm value.", "Therefore, it can exhibit more intrinsic gain with respect to RDC-SOI and C-SOI at VGS = 0.3–0.7 V i.e. the usual biasing range for amplification.", "Fig. 5 depicts output characteristics of three structures.", "Based on this figure, GRDC-SOI and RDC-SOI have much more current with respect to C-SOI at higher bias voltages.", "This current increase in two devices can be described by parallelism of two transistors i.e. one MOSFET in top of the channel and a BJT in the place of retrograde doping.", "At VGS = 0.9 V which voltage is high enough for creation of the channel in the top of silicon layer, by increasing the drain voltage, more carriers tend to pass from the channel and this cause more electron concentration and current density in it and BJT transistor drives to turn on.", "In such a case, N-type source, P-type retrograde doping and N-type Drain play the role of “Emitter”, “Base” and “Collector” regions of assumed NPN transistor parallel to MOSFET transistor.", "More current of GRDC-SOI with respect to RDC-SOI is due to the presence of the graphene material at the bottom of the channel, which attracts more electrons towards itself and creates an easier path for them to flow.", "In MOSFETs, when electrons pass from the channel, they collide to silicon atoms, especially close to the drain region where they have high kinetic energy.", "These collisions create hot electrons and holes which increase the device temperature.", "These hot carriers may have enough energy to pass from the gate oxide insulator barrier and then enter into gate electrode which leads to gate leakage current.", "Figs. 6 and 7 both indicate that gate hot hole and drain hot electron currents in RDC-SOI and GRDC-SOI is much lower in comparison to C-SOI counterpart.", "These emphasize both devices (particularly GRDC-SOI) reliability against this short channel effect with respect to C-SOI MOSFET.", "It should be noted that gate hot hole current has been stated in absolute value, because some of hot holes after creation are propelled to gate positive voltage, affected by drain higher potential.", "Breakdown voltage is a good measure to consider device persistence against higher unexpected voltages.", "This parameter has seriously been considered in the literature [30–32].", "It is mostly mentioned in high voltage and smart-power applications, where they also offer the advantage of compatibility with VLSI processes.", "Fig. 8 depicts breakdown voltage of three MOSFETs under study.", "It confirms GRDC-SOI has more than 10 times higher breakdown voltage and reliability with respect to its counterparts.", "This higher breakdown voltage in our proposed device origins somehow from lower collisions, less hot carriers and reduced device temperature.", "Until now we considered the parameters related to DC performance of a device.", "In the following we are going to point to some preferences of our device that is a figure of merit in RF performance.", "To estimate the switching speed of the devices, drain-gate (CDrain-Gate) and substrate-source (CSubstrate-Source) capacitances were simulated at bias conditions of VGS = VDS = 0.5 V as shown in Figs. 9 and 10, respectively.", "In all cases, parasitic capacitances for GRDC-SOI are lower.", "Actually, the presence of retrograde doping and graphene material at depth of channel have mitigated electrical coupling between drain-gate and substrate-source, which these parasitic capacitances reduced.", "As a result, delay time and dynamic power dissipation are improved in our proposed device.", "Graphene sheet is a gapless material and when it is confined in the width direction, it is called graphene nanoribbon (GNR) which exhibits a non-zero bandgap.", "In order to test GRDC-SOI MOSFET performance in different GNR widths, we swept the device width from 1 nm to 1000 nm to consider its impact on the device performance.", "Due to the fact that GNR width directly affects the electronic properties like bandgap and mobility, it is necessary to incorporate correct values for this case.", "Table 4 shows incorporated values for GNR simulation in different widths.", "According to the Fig. 11, GRDC-SOI has the highest ON-OFF current ratio with respect to other counterparts.", "It is clear in this figure that as device width increases, this ratio does not change too much.", "This is because the amount of gate electrostatic control over the channel will not be varied as a result of the graphene nanoribbon width.", "Fig. 12 shows as device width increases, transconductance also increases for all devices.", "But this increment is more intense for GRDC-SOI.", "This property in GRDC-SOI is indebted to graphene material which leads to more proportional drain current increase in this device with respect to RDC-SOI.", "Subthreshold slope (SS) parameter states device speed to turn on from off states and it is stated with the amount of gate voltage increment to increase the drain current one decade in subthreshold regime.", "It is observed from Fig. 13, that SS in RDC-SOI and GRDC-SOI MOSFETs are close to the ideal value of 60 mV/dec for all of the examined device widths.", "This improvement in these devices owes to retrograde doping in their channels.", "The proposed GRDC-SOI MOSFET demonstrated superior electrical characteristics with respect to RDC-SOI and C-SOI MOSFETs.", "Furthermore, this engineered device can turn on the NPN bipolar transistor by retrograde doping in the depth of its channel and increase its on-state current using the advantage of graphene material.", "GRDC-SOI also shows excellent persistence against short channel effects like DIBL and hot carriers injection, which these confirms this device reliability.", "According to abovementioned benefits, GRDC-SOI can open a window for using graphene material in digital and low power integrated circuits, while scaling transistors for system on chip applications." ]
[ "A novel architecture for Silicon on Insulator (SOI) transistor is proposed.", "Utilizing graphene material in the source side and retrograde doping in the channel improves the electrical characteristics.", "Simulations are performed to obtain transfer and output characteristics, transconductance, and hot electron current.", "All of important electronic figures of merit are compared between three structures including conventional SOI.", "Theoretical study confirms that the proposed structure is more persistent against Short Channel Effects (SCEs)." ]
NMR and structural data for Connexin 32 and Connexin 26 N-terminal peptides
In this article we present 1H and 13C chemical shift assignments, secondary structural propensity data and normalized temperature coefficient data for N-terminal peptides of Connexin 26 (Cx26), Cx26G12R and Cx32G12R mutants seen in syndromic deafness and Charcot Marie Tooth Disease respectively, published in “Structural Studies of N-Terminal Mutants of Connexin 26 and Connexin 32 Using 1H NMR Spectroscopy” (Y. Batir, T.A. Bargiello, T.L. Dowd, 2016) [1]. The mutation G12R affects the structure of both Cx26 and Cx32 peptides differently. We present data from secondary structure propensity chemical shift analysis which calculates a secondary structure propensity (SSP) score for both disordered or folded peptides and proteins using the difference between the 13C secondary chemical shifts of the Cα and Cβ protons. This data supplements the calculated NMR structures from NOESY data [1]. We present and compare the SSP data for the Cx26 vs Cx26G12R peptides and the Cx32 and Cx32G12R peptides. In addition, we present plots of temperature coefficients obtained for Cx26, Cx26G12R and Cx32G12R peptides collected previously [1] and normalized to their random coil temperature coefficients, “Random coil 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG” (G. Merutka, H.J. Dyson, P.E. Wright, 1995) [2]. Reductions in these normalized temperature coefficients are directly observable for residues in different segments of the peptide and this data informs on solvent accessibility of the NH protons and NH protons which may be more constrained due to the formation of H bonds.
[ "MULT" ]
[]
[ "1000" ]
[ "Tables 1–3 contain 1H and 13C chemical shift assignments for the Cx26, Cx26G12R and Cx32G12R peptides respectively.", "Fig. 1 is a plot of secondary structural propensity along the sequence of the peptide for Cx26 and Cx26G12R, and Cx32 and Cx32G12R peptides.", "A positive score indicates helical propensity, a negative score indicates β sheet propensity and values close to zero indicate random coil structure.", "Fig. 2 is a plot of temperature coefficients for NH protons normalized to their fully solvent exposed, random coil values for Cx26, Cx26G12R, Cx32G12R peptides.", "N-terminal peptides of Cx26 (MDWGTLQTILGGVNKHSTSIGK), Cx26 mutant G12R and Cx32 mutant G12R (MNWTGLYTLLSRVNRHSTAIGR) were synthesized by the New England Peptide Co. using Fmoc chemistry [3].", "Samples were prepared and data collected and analyzed as described previously with all peptides dissolved in 0.1 M KCl in 10% 2H2O/90% H2O at pH 7.0 with 100 mM 3-(trimethylsilyl)propionic acid (TSP) used as a chemical shift reference.", "Long (80 ms) and short (15 ms) mixing time TOCSY spectra and a NOESY spectrum (225 ms mixing time) were collected to assign all protons.", "A natural abundance 13C HSQC spectrum was collected to assign all Cα and Cβ carbons for each amino acid in all peptides.", "Data were processed using NMRPipe [4] and analyzed using NMRView [5].", "Resonances were assigned using standard procedures previously described [6].", "The resonance assignments for the Connexin 26, Cx26G12R and Cx32G12R peptides are presented in Tables 1–3.", "Secondary 13C chemical shifts (Δδ=δobserved−δrandom coil) are used as a measure of secondary structure.", "Secondary structural propensity reports the difference between secondary shifts (Δδ13Cα−Δδ13Cβ) and is also a measure of secondary structure with the advantage of canceling out chemical shift referencing errors and because both α helical and β sheet propensities can be well distinguished.", "The secondary structural propensity scores (SSP) for each peptide were calculated using the SSP program [7] and the 13Cα and 13Cβ chemical shifts.", "The secondary structural propensity plots (SSP) in Fig. 1 show the plots of secondary structural propensity scores for the Cx26, Cx26G12R, Cx32 and Cx32G12R N-terminal peptides along the sequence of the peptides.", "The positive values indicate helical structure and negative values indicate β sheets with values of approximately 2 representing fully formed structure while lower values indicate partial structure or propensity and more flexibility.", "Values close to zero indicate unstructured, more flexible regions.", "Fig. 1 shows the comparison between Cx26G12R and Cx26 and values of secondary structural propensity.", "Although the Cx26G12R peptide had a slightly higher structural propensity the plots were similar showing partial helical structure from residues 3–11 with very low ssp values from residues 11–16 for both N-termini supporting flexible turn regions or unstructured regions in this area as seen in the NMR structures [1].", "The remainder of the peptide from residues 16–22 has low ssp values also indicating an unstructured region which is in agreement with calculated NMR structures [1].", "The Cx26G12R peptide had a higher structural propensity but did not have as many NOEs and did not have well defined helical structure [1] suggesting it is a dynamic, flexible structure.", "The plot comparing the Cx32 and Cx32G12R secondary structural propensity values shows helical propensity for both peptides within residues 3-8 with a greater propensity for helicity for Cx32G12R from residue 9–13 as compared to Cx32.", "This shows less flexibility within residues 9–13 for Cx32G12R, in agreement with the NMR calculated structure which shows a more constricted helical turn in this region.", "The NMR structure of Cx32 has a more open turn in this region, in agreement with the low SSP values.", "Thus N-termini of Cx26, Cx26G12R and Cx32G12R show values very close to zero within the turn regions (residues 13–16) supporting a flexible turn, as seen in the NMR structures, while this region in Cx32G12R is a little higher and suggests more structure and less flexibility.", "This data agrees with the calculated structures from NMR data we have presented in the manuscript [1].", "Long mixing time (80 ms) 1H TOCSY spectra were collected at 278, 283, 288 and 293 K for the temperature coefficient data.", "The NH proton ppm values were recorded at each temperature for all amino acids in the peptides.", "Temperature coefficients were calculated from the slope of plots of NH chemical shift vs temperature (K) [1].", "The temperature coefficients give structural information as well and have been measured and reported for each amino acid in a solvent exposed, unstructured, flexible random coil conformation [2].", "A coefficient which is reduced from this value suggests the amino acid is in a region which is not solvent exposed or, if the temperature coefficient is lowered to a value of 6 ppb/K or less, the NH proton for that amino acid may be in an H bond and more constrained.", "Here we present temperature coefficients measured experimentally for each amino acid [1], which were then normalized to their random coil temperature coefficients [2] and plotted for Cx26, Cx26G12R and Cx32G12R peptides in Fig. 2.", "The fractional reduction of the temperature coefficient from its random coil value (which is amino acid dependent) can clearly be seen with the data presented this way.", "The normalized temperature coefficient plots show that for all 3 N-terminal peptides the NH temperature coefficient is reduced starting from residue 3 and continuing through residue 7 with values reaching less than 80% of the random coil temperature coefficient value.", "This indicates NH protons in a less solvent exposed region and maybe involved in H bonds.", "In the NMR structures we see this area is in a helical like turn for the 3 peptide structures and the NH protons are less solvent exposed due to interactions from neighboring hydrophobic side chains.", "Possible H bonds from the structural analysis are discussed in the NMR study [1].", "The NMR structures show larger unconstrained turns within residues 11–15 for Cx26 and Cx26G12R and the normalized temperature coefficients for almost all of the NH protons of these residues are at least 90% of their random coil values with many at the random coil value.", "This suggests a flexible, unconstrained turn in this region similar to what is shown in the NMR structure [1].", "In contrast, the NMR structure shows a tighter helical turn constricted by hydrophobic sidechain interactions within residues 9–13 in Cx32G12R [1].", "Examining this region, 9–13, in the normalized temperature coefficient plot for the Cx32G12R peptide the NH protons in the region are very reduced to values of 80% or less of their flexible random coil value.", "These protons are in a more constrained turn which is less solvent exposed and NMR structural analysis shows a number of H bonds in this area [1].", "This data shows that secondary structural propensity analysis using 13Cα and 13Cβ chemical shifts and normalized temperature coefficients can supplement and support NMR structural data in peptides.", "It also suggests that valid structural information can be obtained by these methods alone in the case of a disordered peptide or a disordered region of a protein where NMR or x-ray crystallographic information is difficult to obtain." ]
[]
Effect of convection coefficient and thickness on optimal cure cycles for the manufacturing of wind turbine components using VARTM
The paper deals with the influence of the convection coefficient and laminate thickness on multi-objective optimisation of the vacuum assisted resin transfer moulding cure stage for the manufacturing of wind turbine components. An epoxy resin system widely used in the wind turbine industry has been chemically characterised and the correspondent finite element implementation validated. The optimisation methodology developed links the finite element solution with a genetic algorithm and identifies a set of optimal cure cycles for a range of thicknesses (10–100 mm) able to minimise cure time (tcure) and the maximum degree of cure gradient developed through thickness (Δαmax) during the cure stage as a measure of quality of the product. The results highlight that, by adding convection coefficient as design parameter of the process, significant benefits could be obtained when insulation is applied at the vacuum bag side for all thicknesses.
[ "ENGI", "MATE" ]
[ "A. Thermosetting resin", "B. Cure behaviour", "C. Numerical analysis", "E. Vacuum infusion" ]
[ "2211", "2503" ]
[ "The manufacturing of structural parts for wind turbine blades poses serious challenges to the composite manufacturing industry due to the large thicknesses involved.", "The main girder, which is made by either carbon or glass fibre laminates, of a wind turbine is responsible to carry the bending loads and therefore its quality is crucial.", "Typical laminate thicknesses for these parts are about 50 mm whilst the root section that connects the blade with the hub goes up to 100 mm.", "The preferred manufacturing process in the wind industry is Vacuum Assisted Resin Transfer Moulding (VARTM).", "Given the dimension of the wind blades (up to 88 m in length to date), the single-mould nature of the VARTM process allows to contain the tooling cost.", "However, with the thicknesses at play, the curing stage of the Vacuum Assisted Resin Transfer Moulding (VARTM) manufacturing process of such parts becomes complex.", "The final mechanical performance of a composite laminate highly depends on a successful cure stage.", "Due to the low thermal conductivity in transverse direction of the composite laminates detrimental exothermic reaction and overshoot temperature are likely to happen as the thickness increases.", "The occurrence of violent temperature overshoots leads to significant differences in thermal history and therefore differences in degree of cure through thickness.", "This introduces different chemical shrinkage and thermal expansion which affects mechanical performance and life span of the part [1–3].", "This phenomenon becomes even more pronounced when dealing with thick and ultra-thick components [4].", "It is therefore crucial that the thermo-chemical properties of the part are accurately modelled to simulate the cure stage and that the cure profile selected is optimised to avoid or reduce the gradient in degree of cure through the thickness.", "Furthermore process time considerations need to be taken into account as they drive process costs.", "Identification of optimal cure cycles to address these objectives is needed as a trial and error approach or experience driven design choices may fail in producing reliable outcomes.", "Researchers have been studying the optimisation of the cure stage by seeking optimal cure profiles to minimise either cost related objectives (i.e. process time) or quality related objectives (i.e. temperature overshoot, degree of cure/temperature gradients [1–3]).", "Attempts have also been made in the direction of addressing multiple objectives simultaneously.", "Optimal cure cycles for the minimisation of cure time have been addressed in literature for composite laminates made with epoxy resin and either glass or carbon fibre.", "The mechanical performance of the parts was ensured by setting quality control constraints such as maximum overshoot temperature allowed or cure uniformity through thickness.", "The thicknesses were in the range of 10–50 mm [5–13].", "The optimisation problem of maximising performance has been investigated either by maximising final degree of cure of the part or by minimising residual stresses and distortion for part thicknesses in the range of 4–60 mm.", "Cure time constraints were applied to ensure reasonable duration of the process [14–22].", "In the attempt to address both cost related and quality related objectives, researchers have been designing single weighted fitness functions to be minimised by finding optimal cure profiles.", "Thicknesses were in the range of 5–25 mm [23–28].", "A multi-objective optimisation problem to minimise cure time and maximum overshoot temperature by finding optimal cure cycles was carried out for 24 mm and 60 mm components.", "In addition to this, it was shown that variation in convection coefficient could significantly affect the outcome of the cure process [4].", "A stochastic model of the cure process using surrogate model representation had been undertaken; the model had been linked with a GA for multi-objective problems.", "Optimal cure cycles were identified and reductions of about 40% were achieved in both temperature overshoot and cure time compared to standard cure profiles.", "Furthermore the reduction was accompanied by 20% variability reduction in both objectives [29].", "However, little attention has been devoted to the influence of convection coefficient and to thicknesses reaching up to 100 mm on the VARTM process and on its optimal cure cycle solutions.", "The aim of the paper is to look into the effect of convection conditions on optimal cure cycles to minimise cure time (tcure) and maximum degree of cure gradient through thickness (Δαmax) experienced by the part during the curing process at each time step.", "To highlight the effect of the convection coefficient on the optimisation problem, three different thicknesses are considered.", "Solutions obtained when a constant natural convection boundary condition at the vacuum bag side (13.6 W/m2 °C) is applied and when an optimisable convection coefficient between insulation (1 W/m2 °C) and improved heat exchange with environment (20 W/m2 °C) is addressed, are compared.", "The effect of thickness on the outcomes of the optimisations will be discussed for both the natural convection coefficient and the optimisable convection coefficient scenario.", "Also the effect of different thicknesses (10 mm, 50 mm and 100 mm) on the optimisation problem and on the achievable trade-offs between the objectives will be addressed in detail in the paper.", "The study is limited to the manufacturing of glass fibre/epoxy composite laminate via VARTM process which is a single-mould process occurring on a hot tool closed with a flexible vacuum bag at the top end, mimicking the industrial application.", "An epoxy resin system widely used in the wind industry has been chemically characterised.", "The optimisation methodology linking multi-objective Genetic Algorithm (GA) with Finite Element (FE) model is adapted and implemented [4].", "A solution of the cure stage of a VARTM process is carried out.", "Optimal sets of cure profiles and convection coefficient capable to minimise tcure and Δαmax are identified and studied.", "The Pareto fronts obtained are also compared with the outcomes achieved when Manufacturer Recommended Cure Cycle (MRCC) is applied to the three different thicknesses.", "The materials considered in this study are a non-crimp biaxial E-glass fibres fabric and the two component Airstone™ 780E epoxy resin and 785H Hardener system [30] used in wind turbine blade manufacturing.", "The cure kinetics characterisation campaign of the resin system has been carried out.", "A Perkin Elmer® Differential Scanning Calorimetry (DSC) has been used for the characterisation campaign.", "Four isothermal tests at 50, 70, 90 and 110 °C and one dynamic test at 1 °C/min have been carried out.", "The development of the glass transition temperature of the system has been also characterised.", "The samples were heated in the DSC at 1 °C/min up to increasing final temperature and quickly cooled down to stop the cure.", "This was performed in order to produce different levels of partially cured samples.", "Subsequently the samples were heated at 10 °C/min to identify the glass transition temperature in the heat flow signal.", "Three repeated tests per final temperature were run.", "The degree of cure reached by each sample was extrapolated from the 1 °C/min dynamic experiment.", "To validate the degree of cure prediction of the cure kinetics some additional validation tests were required.", "Pure resin samples with different degrees of cure were manufactured using mould of rectangular shape made of two aluminium plates with a slit in between.", "Rectangular shape samples with dimensions of 170 × 110 × 2.5 mm were manufactured.", "The freshly mixed resin was poured in the slit between the two aluminium plates, a thermocouple was located in the epoxy resin and after that the mould was placed in the oven to cure.", "Four different cure profiles have been used to manufacture samples with different degrees of cure namely 45 min at 70 °C, 95 min at 70 °C, 255 min at 70 °C and 135 min at 110 °C.", "After that, small pieces for DSC analysis were scraped from the original samples.", "A DSC run at 10 °C/min was subsequently performed to identify the glass transition temperature of each manufactured plate.", "The effective thermal history that the resin underwent was measured by the thermocouple and fed to the cure kinetics model which provides the degree of cure of the sample.", "The thermo-chemical problem occurring during the cure of the composite part is modelled and solved using the Finite Element solver Marc.Mentat® [35].", "The elements used for the simulation are three-dimensional isoparametric 8-nodes brick composite for heat transfer analysis (Marc® element type 175) [36].", "Material properties and boundary conditions are implemented using user subroutines [37].", "With regard to the FEA models used for the optimisation procedure, since VARTM process is used by the wind turbine industry natural air convection boundary condition at the vacuum bag side is applied using UFILM user subroutine.", "The ambient temperature considered for the natural convection is 25 °C whilst the convection coefficient is equal to 13.6 W/m2 °C in the case where the convection coefficient is kept constant [38].", "Since curing during the manufacturing of wind turbine blades occur on a hot tool, time dependent fixed temperature boundary condition is applied at the nodes in contact with the mould following the thermal profile set by the optimisation with FORCDT user subroutine.", "Initial temperature condition equal to ambient temperature is applied to all the nodes in the model.", "The cure kinetics were implemented using the UCURE user subroutine whilst thermal properties such as specific heat and thermal conductivity were implemented using USPCHT and ANKOND user subroutines.", "Fig. 1 depicts a schematic of the model boundary condition application.", "Although the models are implemented in a 3D analysis the temperature evolution is as a matter of fact one dimensional.", "This is achieved by using a single element for both in plane directions which alongside the implied zero heat flux boundary condition on the corresponding boundaries results in infinite length and width.", "In the through-thickness direction the model comprises 16 elements and 68 nodes.", "A mesh convergence analysis has been performed for the 100 mm thick component.", "The number of elements was kept the same also for the thinner parts as the computational time for one run was in the order of minutes.", "The multi-objective optimisation problem has been set in order to find optimal cure cycles and convection condition that minimise tcure (cost related objective) and Δαmax (quality related objective).", "The MRCC for the resin system under study imposes a one dwell profile at 70 °C.", "The optimal cure profiles have been sought among two dwells cycles [3].", "Therefore four parameters have been identified for parameterisation, namely: temperature of first and second dwell (T1, T2), duration of the first dwell (Δt1) and ramp rate (r).", "Furthermore the convection coefficient (h) has been added as parameter of the optimisation.", "Fig. 2 reports the general shape of the MRCC and the shape of the proposed parameterisation of the two dwell profile.", "A GA capable to deal with multi-objective optimisation problems has been adapted and tuned for the problem under study.", "The GA adopted has been tested against standard benchmarks problem and its reliability and results reproducibility assessed [4].", "Table 2 reports the range investigated in the optimisation runs for each parameter.", "Regarding T1, a temperature of 30 °C has been chosen as lower limit since it would be not feasible curing at temperature lower than ambient.", "The upper limit is set at 70 °C as this is the MRCC temperature.", "The upper limit of T2 is set at 105 °C due to technological limitation of the moulds.", "The first dwell duration range has been selected in order to allow the GA to select one dwell scenarios in the case of tcure shorter than Δt1.", "Ramp rate range has been decided according to technological limitation of the mould used in industry.", "As for the convection coefficient, according to [36], the standard natural convection coefficient is equal to 13.6 W/m2 °C therefore the range has been chosen in order to allow scenarios where insulation is applied (h < 13.6 W/m2 °C) and scenarios where improved heat exchange with the environment is applied (h > 13.6 W/m2 °C).", "The tcure is defined as the time at which the minimum degree of cure reached in the model is 93% therefore the duration of the second dwell is not a parameter of the optimisation.", "The 93% degree of cure threshold is chosen as this is the degree of cure reached by the epoxy resin with an isothermal DSC run at the cure temperature of 70 °C.", "A total of three test cases have been taken into account.", "The components are flat panels with different thicknesses.", "The thicknesses addressed are 10 mm for the thin case, 50 mm for the thick case resembling the girder of the wind blade and 100 mm which is typical thickness of root insert sections.", "Initial and boundary conditions applied to the models are as described in Section 3.", "The optimisation methodology uses an interface built in C++ to link the GA optimiser and the FE models built in Marc.Mentat.", "Fig. 3 illustrates the function of the interface.", "The GA generates a new set of four or five parameters at each iteration depending on whether the convection coefficient is considered.", "The interface creates a copy of the Marc.Mentat input file by copying line by line the old input file and updating at the correct location the new file inserting the new parameters generated.", "After that a command to execute the FE simulation with the modified input file is run.", "During the simulation run the objective of interest are made available by user subroutines [37].", "The UPSTNO user subroutine read the temperature and degree of cure at each increment of the simulation and the Δαmax for the corresponding increment is stored into a variable.", "Once the minimum degree of cure threshold is reached the UEDINC user subroutine withdraw the corresponding tcure and store the value into a variable.", "At the end of the run the interface writes the values of tcure and Δαmax in two text files.", "The interface opens the text files, reads the values and sends them to the GA.", "At this point a new iteration starts.", "Table 3 reports the GA optimisation parameters used for the three cases.", "The number of individuals per population, of individuals used per reproduction and elite individuals has been doubled for the thick and ultra-thick cases with respect to the thin case.", "The non-linearity of the cure process increases with thickness making the landscape of the problem riddled with more local minima where the GA can get trapped [4].", "Increasing the size of the initial population helps the GA to not get trapped in local minima.", "In order to validate the cure kinetics and the through-thickness heat transfer model developed, two laminates have been manufactured.", "The laminates were manufactured using non-crimp biaxial E-glass fibre with 812 g/m2 and the NEG 2001/2002 sizing and the Airstone™ epoxy system characterised in this study.", "The lay-up was [45/−45]4s and volume fibre fraction 54%.", "The thickness of the manufactured laminates was 31.5 mm.", "The two laminates were manufactured following two different cure cycles.", "For symmetry reasons, an aluminium plate was placed at the top of the laminate.", "The first cure cycle prescribes a ramp at 0.83 °C/min up to 50 °C, 50 min dwell at 50 °C, ramp at 0.83 °C/min up to 100 °C, 400 min dwell at 100 °C, the second cycle was the MRCC which dictates a ramp at 0.33 °C/min up to 70 °C and an isothermal dwell at 70 °C for 240 min [30].", "The aim of the tests is to monitor the temperature evolution through the thickness and compare the data from the experiment with the cure simulation prediction.", "One thermocouple was placed at 15 mm and one at 1 mm through thickness.", "The laminate infusion occurs at room temperature.", "The infusion time for the two laminates was in the range of 101–104 min.", "To validate the heat transfer model and the cure kinetics through the thickness of the laminate a convection boundary condition has been applied at the top and bottom of the two aluminium plates with a sink temperature following the cure profile and a convection coefficient of 13.6 W/m2 °C [38] since the curing occurred inside the oven.", "The cure model with the aforementioned boundary conditions has been run and the thermal history predicted by the model for the nodes at 15 mm and at 1 mm through thickness have been compared with the ones detected by the thermocouples placed at the same locations during the experiment.", "Fig. 5 shows the comparison between the predicted and measured temperature at 1 mm and 15 mm thickness of the laminate.", "The agreement between model prediction and measurements is highly satisfactory.", "The accuracy with which the FE model predicts the temperature evolution through thickness guarantees that the thermal properties adopted in this study are adequate for the resin system studied and that the exothermic reaction described by the cure kinetics is accurate.", "Figs. 6–8 report the results of the multi objective optimisation for the thin (10 mm), thick (50 mm) and ultra-thick (100 mm) case respectively.", "In all cases, the optimisation converges to a Pareto front highlighting the existence of optimal trade-offs between the objectives selected.", "The optimisation methodology is able to identify and quantify the efficiency solutions existing within the landscape of the problem.", "The L-shape of the Pareto fronts points out the competitive nature of the cost and quality related objectives, enforcing the necessity of a multi-objective analysis.", "It also suggests that a division of the objective space in two regions exist.", "One in which cure time is prioritised over the Δαmax and significant improvements can be obtained in the Δαmax with small changes in cure time (vertical part) and one where high importance is given to the Δαmax and significant improvements in cure time can be achieved with little change in the Δαmax (horizontal part).", "The maximum temperature reached by the solutions in the final Pareto fronts never exceeds 200 °C.", "The degradation temperature for the current system is 330 °C, therefore each solution belonging to the final Pareto fronts can be accepted since the maximum temperature stays well below the degradation temperature of the resin.", "In the following sections the results of the 10, 50, and 100 mm optimisations will be discussed in more detail.", "Afterwards, the effect of adding the convection coefficient to the optimisation and the effect of thickness of the laminates will be described.", "Finally, a comparison of the optimisation results with the results when the manufacturing recommended cure cycle is applied will be described.", "The optimisation results for the 10 mm flat panel are reported in Fig. 6.", "For this case convergence of the Pareto is achieved after 15 generations.", "A comparison between the Pareto obtained using only the cure profile parameters and with the addition of convection coefficient as optimisation parameter (WCCOP in Fig. 6) is shown.", "The introduction of the convection coefficient as design parameter brings significant benefits, shifting the Pareto towards shorter curing time and lower degree of cure gradients, achieving approximately 50% reduction in Δαmax and 75% reduction in cure time.", "All the individuals in the final Pareto present convection coefficients close to 1 W/m2 °C meaning that insulation at the vacuum bag side brings benefits in both objectives.", "From Fig. 6 it is possible to identify three main zones in both graphs around which individuals tend to cluster.", "These zones translate into specific design choices.", "A quality conservative zone that allows longer process time to ensure small degree of cure differences through thickness, a quality non-conservative zone that allows higher degree of cure gradients during the process to favour process time and a transitional zone in which mixed behaviour can be identified.", "Table 5 reports the design parameters of candidate individuals (corresponding to A, B, C, A′, B′, C′ in Fig. 6) picked out of each zone for the 10 mm flat panel case.", "All the individuals show a high second dwell between 102 and 105 °C.", "Specifically when only the four cure cycle parameters are optimised (i. e. without optimising the convection coefficient), conservative zone individuals (i.e. A) are characterised by a low first dwell temperature (i.e. 43 °C), long first dwell duration (i.e. 208 min) and very mild ramp rate (i.e. 0.13).", "The design parameters for this zone point toward mild development of the reaction switching to the higher second dwell once the chemical potential of the resin has been exploited and an increase in temperature will not trigger violent exothermic reaction.", "As a result the final part has the lowest Δαmax (i.e. 0.07) but a long tcure (i.e. 803 min).", "Moving toward the other two zones (i.e. B, C) the trend shown is to seek faster tcure.", "This is achieved by increasing the temperature of first dwell (i.e. 57.8 °C for the transitional and 68.2 °C for the non-conservative zone), decreasing the duration of first dwell (i.e. 138 min for the transitional and 2 min for the non-conservative zone) and by increasing the ramp rate (i.e. 0.87 °C/min for the transitional and 3.4 °C/min for the non-conservative).", "This results in shorter tcure but higher Δαmax during the process (i.e. 0.12 for the transitional and 0.22 for the non-conservative zone) and therefore progressively lower quality of the part.", "When the convection coefficient is added as optimisation parameter, optimal individuals (i.e. A′, B′, C′) show a value close to 1 W/m2 °C meaning that insulation on the vacuum bag side of the process is beneficial in terms of both objectives.", "The introduction of insulation at the top allows more aggressive design parameter choices (i.e. higher ramp rate and first dwell temperature and shorter first dwell duration) compared to the case without it and resulting in significant improvements in both objectives.", "Fig. 7 reports the optimisation results for the flat panel 50 mm cases.", "Convergence is reached after nine generations.", "Observing the design parameters that led to the Pareto front it is possible to identify three categories of individuals in the Pareto behaving similarly.", "Candidate individuals (i.e. A, B, C, A′, B′, C′) are picked out the Pareto front and highlighted in Fig. 7.", "Table 5 reports the details of these individuals.", "All the individuals present a high second dwell in the range of 101–105 °C.", "When convection coefficient is not used as optimisation parameter the conservative individuals (i.e. A) show a first dwell temperature of about 52 °C, with duration of about 50 min and 2.3 °C/min ramp rate.", "The cure ends after 180 min with the lowest Δαmax.", "As the thickness increases it becomes more challenging to keep the cure time long and therefore develop mild reaction.", "An increase in first dwell temperature together with a decrease in first dwell duration marks the passage from conservative zone towards transitional and non-conservative zones (i. e. B, C respectively).", "These individuals present first dwell temperature higher than 60 °C, shorter first dwell duration (46 min for individual B and 4 min for individual C) and a quick ramp up to the second dwell (3.5 °C/min).", "This results in shorter tcure (90 and 50 min for individual B and C respectively) but higher Δαmax, 0.38 and 0.73 for individual B and C respectively.", "In this case, the introduction of convection as parameter brings benefits (up to 50% reduction in Δαmax) for the individuals in the conservative zone whereas the benefits are negligible in the non-conservative zone.", "Optimal individuals (i.e. A′, B′, C′) showed to have a convection coefficient of about 1 W/m2 °C meaning that the application of insulation is beneficial also for the 50 mm thick panel.", "However it is only in the case of conservative individuals (i.e. A′) that differences in cure profiles can be found whilst in the case of transitional and non-conservative individuals (i. e. B′, C′ respectively) the cure profile look similar to the case without convection coefficient as parameter.", "It is interesting to notice that in this case due to the larger thickness at play, individual A′ presents a first dwell temperature of about 42 °C which is 10 °C less than individual A.", "This is opposite behaviour compared to the 10 mm scenario suggesting that for thicker parts the effect of applying lower convection coefficients needs to be counteracted by a decrease in first dwell temperature and longer first dwell duration (i. e. 77 min for individual A′).", "To understand why there is a difference in the cure behaviour for A and C, the cure and temperature profiles of the 50 mm case will be investigated in more detail in Section 5.3.4.", "Fig. 8 illustrates the optimisation results for the flat panel 100 mm cases.", "The convergence is reached after 11 generations and proved to be the most demanding optimisation problem with a computational time of 2820 min.", "The Pareto front seems to show a change in convexity, however this has to be attributed to the minimum temperature allowed in the optimisation (i.e 30 °C).", "Lower curing temperatures would result in the standard L-shaped Pareto, however these temperature are not possible in practice therefore are excluded in the first dwell temperature range.", "Analysis of the design parameters of the Pareto individuals allow to identify common trends within same clusters of individuals.", "Candidate individuals (i.e. A, B, C, A′, B′, C′) are selected and highlighted in Fig. 8 whilst the corresponding design parameters are reported in Table 5.", "All the individuals present second dwell in the range of 100–104 °C.", "When the convection coefficient is not used as parameter, the first dwell temperature is at its lowest compared to the 10 and 50 mm case.", "Individual A in the conservative zone has a first dwell temperature of about 30 °C, Individual B 40 °C and individual C 52 °C.", "The strategy for these points is dictated by the high thickness at play and makes the degree of cure develop at a low temperature to lower the chemical potential of the reaction when the ramp to the second dwell occurs.", "The first dwell duration for individual A and B are in the range of 80–90 min whilst the individual C has a short first dwell which brings the profile to the high second dwell temperature resulting in the shortest tcure.", "The introduction of convection coefficient as optimisation parameter introduces benefits for the individual in the conservative and transitional zone (i.e. A′, B′).", "The optimal convection coefficient is in the range of 1–3.4 W/m2 °C which means application of insulation at the bag side.", "In specific individuals A′ and B′ show shorter first dwell duration and higher second dwell temperature compared to individual A and B. Furthermore, non-conservative zone individuals in both 50 and 100 mm case (i. e. C, C′) are obtained essentially applying one dwell cure profile leading to a fast process but larger amount of process induced defects.", "From the observation of Figs. 6–8 and Table 5, it is possible to notice that the convection coefficient always tends towards insulation of the vacuum bag side which is in contrast to results shown in [4].", "In that study, the effect of different convection coefficients on Pareto fronts has been investigated in the case of curing inside a fan oven.", "The results in this case showed Pareto front worsened when a lower convection coefficient is applied.", "In that case, unlike the problem currently under study, the sink temperature of the convection boundary condition followed the cure profile since the cure process occurred in the oven.", "Therefore application of insulation in this case would trigger uncontrolled exothermic reaction leading to worse thermal gradients through thickness.", "In the current study, the effect of convection coefficient is investigated when the curing occurs by means of hot tool plate for a single mould process.", "In this scenario the sink temperature of the convection boundary condition is equal to ambient temperature.", "Therefore, applying a lower convection coefficient at the vacuum bad side influenced the process positively shifting the Pareto towards shorter tcure and lower Δαmax.", "However the magnitude of the benefits brought differs.", "For the 10 mm case the benefits are similar throughout the different zones whilst for the 50 mm and 100 mm case only the conservative zone individuals see a significant benefit.", "In order to understand this behaviour it is necessary to look at the temperature and degree of cure evolution through thickness of individuals belonging to different zones.", "Fig. 9 illustrates the temperature and degree of cure evolution of the non-conservative points for the 50 mm flat panel in the case when convection coefficient is not used as optimisation parameter (a and b respectively) corresponding to point C in Fig. 7 and in the case when it is an optimisation parameter (c and d respectively) corresponding to point C′ in Fig. 7.", "The application of a convection coefficient equal to 1 W/m2 °C (i.e. insulation) in this case does not generate different curing behaviours.", "The two solutions are equivalent and only the magnitude of the temperature overshoot generated at the vacuum side changes, being 10 °C higher for individual C′.", "In both cases, due to the exotherm generated, the last region to reach the target degree of cure is the one in contact with the tool.", "Fig. 10 presents the temperature and degree of cure evolution of the conservative points for the 50 mm flat panel in the case when convection coefficient is not used as optimisation parameter (a and b respectively) corresponding to point A in Fig. 7 and in the case when it is an optimisation parameter (c and d respectively) corresponding to point A′ in Fig. 7.", "In this case the benefits brought by the application of a convection coefficient equal to 1 W/m2 °C (i.e. insulation) are significant, about 30% reduction in tcure and about 65% in Δαmax.", "It is possible to notice in the temperature evolution plots that when insulation is applied, the region at the vacuum bag side experiences a significant temperature overshoot.", "This reflects in a different cure evolution compared to the non-insulated case.", "From the degree of cure evolution it is possible to highlight that when the vacuum bag side of the flat panel experiences temperature overshoot, the last region to cure is the one touching the tool whilst when no temperature overshoot occurs it is the vacuum bag side that completes the cure last.", "This highlights that exothermic effect when controlled and understood can be very beneficial.", "Fig. 11 reports the temperature and degree of cure evolution for the individual A of the 50 mm flat panel case.", "In one simulation, Fig. 11(a, b) a convection coefficient of 18 W/m2 °C was applied whereas in the second simulation the convection coefficient was equal to 9 W/m2 °C.", "The convection coefficients were chosen to be equally spaced from the standard convection coefficient (i.e. 13.6 W/m2 °C).", "The simulations are intended to isolate the effect of convection coefficient on degree of cure evolution.", "The evolution of the degree of cure can be compared with Fig. 10(a, b) in which convection coefficient is 13.6 W/m2 °C and the process ended with a Δαmax equal to 0.33.", "It can be noted that increasing the convection coefficient (i.e. 18 W/m2 °C) leads to a larger temperature gradient through thickness and consequently in degree of cure (i.e. 0.36) whilst a decrease in convection coefficient (i.e. 9 W/m2 °C) leads to smaller temperature and degree of cure gradients (i.e. 0.26).", "Furthermore, it points out that the midpoint temperature is slightly affected by the change in convection coefficient and that is the vacuum bag side region to experience the biggest change in temperature due to the change in convection coefficient.", "This leads in the case of Fig. 11(a, b), to fail to reach the 93% minimum degree of cure target for the point at the vacuum bag side strengthening the significant role played by the convection coefficient in the process.", "Fig. 12 depicts the comparison of the Pareto front for the three thicknesses when natural convection is applied at the bag side.", "All the individuals belonging to the non-conservative zone end up with a short first dwell duration (about 2 mins) making the cure profile a quasi-one dwell.", "The results show a clear influence of thickness on the Pareto front.", "A previous work showed that higher thicknesses leads to Pareto fronts shifted towards higher tcure and maximum Δαmax for thicknesses in the range of 3–24 mm [41].", "This holds for the 50 and 100 mm Pareto fronts.", "The 100 mm Pareto fronts is about 50 mins shifted towards longer tcure and 0.03 towards higher Δαmax.", "The Pareto front for 10 mm case does not fall within this framework.", "This can be explained considering that the cure behaviour of the 50 mm and 100 mm case are alike both involving temperature overshoots whilst the 10 mm case involves a cure evolution with no temperature overshoot occurrence hence a comparison with the other Pareto fronts is not meaningful in this case.", "Furthermore, the Pareto fronts for the 50 mm and 100 mm show the majority of their solutions in the non-conservative zone whereas the 10 mm Pareto front has the majority of the solutions in the conservative zone.", "This is related to the selection of parameters ranges and the nature of the resin of the study.", "The first dwell lowest temperature selected is 30 °C.", "The selection of first dwell temperature lower than 30 °C would probably fill the conservative zone of the Pareto fronts for the 50 mm and 100 mm case.", "However curing temperatures lower than 30 °C are not feasible in practice and therefore have not been considered in this study.", "On the other hand the selection of temperature ranges proves to be very conservative in the case of 10 mm.", "This highlights the importance of appropriate selection of design parameters and the necessity of optimisation methodology at the design stage.", "Table 6 reports the results obtained when MRCC has been applied to the different thicknesses cases.", "The MRCC fails to reach the 93% degree of cure requirement for both the 10 mm and 50 mm case stopping at 88% and 87% respectively.", "It is not meaningful therefore to quantify the benefits introduced by optimal solutions in terms of tcure and Δαmax reduction since the process as a matter of fact failed to meet the quality requirements when MRCC was applied.", "With regards to the 10 mm and 50 mm cases the exothermic reaction generated by the MRCC is not strong enough to overcome the cool down due to the convection on the vacuum bag side; as a consequence the top part experience a colder thermal history that is not sufficient to reach the 93% requirement.", "For the 100 mm case the MRCC generated a solution meeting the set degree of cure requirements ending in 370 min and with a Δαmax equal to 0.56.", "When compared to the solution obtained with the MRCC, the selection of Pareto points belonging to the conservative zone could lead up to 60% tcure reduction and up to 30% Δαmax reduction.", "Any point in the Pareto brings tcure reduction greater than 60% therefore an engineering choice could be to maximise the reduction in Δαmax hence selecting individuals in the conservative zone.", "In the present paper the chemical characterisation involving cure kinetics and Di Benedetto equation of the two components system Airstone 780E epoxy resin and 785H Hardener has been successfully undertaken.", "The relevant material models have been built.", "A heat transfer model considering heat generation from the cure process has been developed, implemented and validated through test.", "The cure simulation implemented in the FE solver Marc.Mentat constitutes the first published work describing the cure evolution of the system under study.", "The multi-objective optimisation methodology developed is able to identify efficient design points for the manufacturing of thin, thick and ultra-thick components for the wind turbine industry, represented by 10, 50 and 100 mm in this study.", "However the results can be extended also to other fields.", "The multi-objective set-up optimises the two objectives (cure time: tcure and quality: Δαmax) independently without the need to assume a priori the weights/benefits associated to each objective.", "The final Pareto fronts contain solutions with different prioritisation of the objectives each one would be the solution of a specific weighted fitness function.", "The Pareto fronts show the competitive nature of two objectives selected.", "The multiple optimal design points in the Pareto can be ranked and selected according to the quality/cost ratio required by the end user.", "The results highlight that MRCC generates parts with lower final degree of cure and results in unnecessary long cure times.", "In the case of the 100 mm thick flat panel tcure reduction up to 60% and Δαmax reduction up to 30% can be achieved compared to the solution obtained with MRCC.", "Moreover, an investigation of the influence of thicknesses on Pareto fronts demonstrated that higher thicknesses shift the efficient solutions towards higher Δαmax level and longer tcure unveiling the hidden relationship between thickness and minimum Pareto front achievable.", "Furthermore, the study suggests the idea of using convection coefficient as a new design parameter to consider alongside thermal profile.", "Introduction of the convection coefficient as additional parameter in the optimisation showed that the convection coefficient tends towards insulation values for all points studied (<13.6 W/m2 °C).", "The application of insulation at the vacuum bag side can lead to significant improvement in both tcure and Δαmax reduction especially for points belonging to the conservative zone of the Pareto fronts.", "This could be achieved in first instance by placing insulating blankets at the vacuum bag side to decrease the heat exchange with the environment.", "Different thicknesses for the insulating material would generate different levels of insulation.", "Characterisation of the aforementioned blankets would be required before its use in industrial application.", "The measurement technique adopted in [38] can be implemented for this purpose.", "The same study shows that the measurements of convection coefficient shows local variability therefore challenges are foreseen with regard to this type of characterisation.", "Detailed analysis of the temperature and degree of cure evolution of individuals belonging to two different zones in the Pareto front of the 50 mm thick flat panel proved that the insulation is particularly beneficial when it introduces changes in the cure mechanisms by triggering exothermic phenomena through the thickness intense enough to allow the vacuum bag side to cure faster than tool side.", "A further benefit of about 30% reduction in tcure and 65% reduction in Δαmax compared to optimal solutions obtained when natural convection coefficient was applied at the bag side can be achieved.", "It has to be noted that the strategy selected contributes to a uniformity of the cure through thickness which reflects into higher mechanical performances.", "The findings of the paper pointed out that exothermic effect can be used to improve the quality and efficiency of the manufacturing process when predicted and controlled and that appropriate insulation can help achieving it.", "More than this, the methodology implemented is an important step forward towards a first time right design of the process which can reduce scrapped parts and waste material, improve the quality of the final part, reducing cost and facilitating scheduling.", "Further investigations require definition of stability areas and robustness of the solutions in the Pareto fronts.", "Considerations regarding deviations from nominal values for boundary and initial conditions are necessary.", "Discarding optimal solutions that presents high level of variability will lead to the generation of more robust Pareto fronts hence robust design choices." ]
[]
Dataset of working fluid parameters and performance characteristics for the oxy-fuel, supercritical CO2 cycle
This article provides the dataset of working fluid parameters and performance characteristics for the oxy-fuel, supercritical CO2 cycle by the thermodynamic optimization procedure employed in the research article “Thermodynamic optimization and equipment development for a high efficient fossil fuel power plant with zero emissions” (Rogalev et al., 2019) [1]. The performance characteristics of the different air separation units, which were used for the investigation of an influence of the produced oxygen purity on power consumption, are presented. Moreover, the methodology for the evaluation of gross and net efficiency is described. The working fluid parameters and performance characteristics are subdivided into several tables differing from each other by the values of initial pressure.
[ "MULT" ]
[ "Air separation unit", "Oxy-fuel combustion cycle", "Supercritical carbon dioxide", "Thermodynamic calculation" ]
[ "1000" ]
[ "This article provides the dataset of working fluid parameters and performance characteristics for the oxy-fuel, supercritical CO2 cycle, given in Ref. [1].", "The concept of the oxy-fuel, supercritical CO2 cycle shown in Fig. 1 was proposed in Ref. [2].", "The mathematical model of the cooled gas turbine described in Refs. [3–5].", "The data for the air separation units (ASU) providing oxygen pressure, temperature, mass flow and purity values, impurities composition and specific power consumption is presented in Tables 1 and 2.", "The data for the oxy-fuel, supercritical CO2 cycle providing for each combination of the turbine inlet pressure and temperature the flow parameters, powers and efficiency levels is presented in Tables 3–7.", "In the following the configuration of the power plant is firstly described, then the parameters for ASU are given, the method to identify efficiency is described and finally the working fluid parameters and performance characteristics for the oxy-fuel, supercritical CO2 cycle are presented.", "The flow chart of the oxy-fuel, supercritical CO2 cycle shown in Fig. 1 is similar to the chart [6].", "The gas fuel compressor 1 supplies fuel to the combustor 2 where is also supplied the high purity oxygen produced in the air separation unit 9 and compressed up to the proper pressure by the oxygen compressor 10.", "The oxy-fuel mixture combustion increases the flow temperature.", "The high temperature flow enters turbine 3, expands and drives the turbine and the electric generator 4.", "The exhaust gas flow enters the multi-flow high temperature regenerator 11, where it transfers its heat to the following three flows:", "oxygen and carbon dioxide mixture traveling to the combustor 2;", "carbon dioxide recirculation flow upstream the combustor 2 to control the maximal temperature;", "carbon dioxide flow to the turbine 3 cooling.", "The regenerator 11 also transfers the low potential heat of the compressed hot air from the air separation unit 9.", "After the regenerator 11, the cooled exhaust gas enters the condenser 12 where the two-component mixture is cooled, water is condensed and removed from the cycle flow.", "After the condenser 12, the mixture enriched with carbon dioxide enters the multi-stage intercooled compressor 5.", "Upstream the compressor 5 some fluid is taken to the carbon dioxide compressor 13 for its further storage.", "Downstream compressor 5 the flow enters cooler 6 and then the first stage pump 7.", "Apart of the fluid flow is mixed with oxygen on its way to the oxygen compressor 10 and the other part travels to the second stage pump 8.", "After the final compression, the flow is split into two parts, one part goes to the combustor and another part is supplied to the turbine cooling.", "Thus, the cycle is closed.", "The article [1] shows the dependence of the ASU specific power on oxygen purity obtained by statistical analysis of the characteristics of the different models of air separation units [7–9].", "The data on characteristics review of high-performance ASU are presented in Table 1.", "According to the data, an oxygen purity ranges from 85 to 99.5%, an oxygen pressure differs in a range of 0.1–4 MPa, an oxygen temperature varies in a range of 15–30 °C and the ASU specific power ranges from 735 to 1512 kWe/(kg O2/s).", "The main impurities are argon and nitrogen, the ratio of which varies depending on the ASU scheme and parameters.", "The modeling of the oxy-fuel, supercritical CO2 cycle presented in Fig. 1 involves evaluation of the amount of low temperature heat released during air compression on the first stage of the oxygen production.", "To evaluate this value, the performance characteristics of the various industrial ASU were collected in Table 2.", "The ratio of the air and oxygen mass flows for the ASU presented in Table 2 is equal to 5.46.", "This value was used to estimate the amount of low-potential heat supplied in the regenerator.", "Unlike traditional gas turbine and combined cycle power plants the oxy-fuel, supercritical CO2 cycle is characterized by additional energy penalties on oxygen production and carbon dioxide compression before storage.", "The value of these penalties in the total losses structure is significant, so the net efficiency is the most suitable parameter to characterize cycle performance.", "However, gross efficiency was also calculated for the comparison reason.", "The equations for the energy efficiency coefficients used for calculation of the oxy-fuel, supercritical CO2 cycle are presented below.", "B – the natural gas mass flow, kg/s;", "QLHW – the lower heating value of fuel, MJ/kg.", "NASU – the air separation unit power consumption, MWe.", "NCO2 – the electric power for compression of carbon dioxide before storage, MWe.", "NP – electric power for all pumps, MWe.", "∑NFuel.C – electric power for the fuel compressor, MWe.", "∑NOxy.C – electric power for the oxygen compressors, MWe.", "The net efficiency data for the oxy-fuel, supercritical CO2 cycle presented in Ref. [1] comes from the thermodynamic calculations using AspenONE software.", "Tables 3–7 showed the data on thermal scheme calculation for the different values of the turbine initial parameters.", "The data on thermodynamic investigations of the turbine inlet parameters influence upon the oxy-fuel, supercritical CO2 cycle net efficiency shows that the maximum value of cycle net efficiency of 56.5% (including air separation unit penalty and carbon capture and storage at 100 bar) is achieved for the turbine inlet temperature of 1083 °C and pressure of 300 bar.", "The turbine outlet pressure value was fixed at 30 bar and turbine coolant temperature – at 200 °C during the optimization of turbine inlet parameters.", "The turbine inlet temperature optimum at a fixed pressure may be explained by specific features of the high temperature regenerator thermodynamic process.", "Usually, the turbine inlet temperature increase is followed by growth of the cycle mean integral heat intake temperature that increases the equivalent Carnot cycle efficiency, and the cycle thermodynamic efficiency also grows.", "On the other side, the regenerator analysis shows that the excessive increase of the turbine inlet temperature increases its exhaust temperature that changes the pinch point.", "Production and compression of the oxygen supplied to the combustor reduce the cycle net efficiency in average for 7.2%, and the compression of carbon dioxide before storage – for 0.4%.", "The low energy consumption of CO2 compressor is due to the high cycle minimal pressure, which is equal to 30 bar.", "According to the data presented in Tables 3–7 the maximal net efficiency of the oxy-fuel, supercritical CO2 cycle is achieved for the next combinations of turbine inlet parameters:", "at the 996 °C temperature and 200 bar pressure, the facility net efficiency is 54.6% and the coolant flow coefficient is 3.9%;", "at the 1054 °C temperature and 250 bar pressure, the facility net efficiency is 55.7% and the coolant flow coefficient is 6.6%;", "at the 1083 °C temperature and 300 bar pressure, the facility net efficiency is 56.5% and the coolant flow coefficient is 11.4%;", "at the 1164 °C temperature and 350 bar pressure, the facility net efficiency is 55.8% and the coolant flow coefficient is 11.4%;", "at the 1058 °C temperature and 400 bar pressure, the facility net efficiency is 55.8% and the coolant flow coefficient is 10.5%." ]
[]
Crop yield sensitivity of global major agricultural countries to droughts and the projected changes in the future
Understanding the potential drought impacts on agricultural production is critical for ensuring global food security. Instead of providing a deterministic estimate, this study investigates the likelihood of yield loss of wheat, maize, rice and soybeans in response to droughts of various intensities in the 10 largest producing countries. We use crop-country specific standardized precipitation index (SPI) and census yield data for 1961–2016 to build a probabilistic modeling framework for estimating yield loss risk under a moderate (−1.2 < SPI < −0.8), severe (−1.5 < SPI < −1.3), extreme (−1.9 < SPI < −1.6) and exceptional (SPI < −2.0) drought. Results show that there is >80% probability that wheat production will fall below its long-term average when experiencing an exceptional drought, especially in USA and Canada. As for maize, India shows the highest risk of yield reduction under droughts, while rice is the crop that is most vulnerable to droughts in Vietnam and Thailand. Risk of drought-driven soybean yield loss is the highest in USA, Russian and India. Yield loss risk tends to grow faster when experiencing a shift in drought severity from moderate to severe than that from extreme to the exceptional category, demonstrating the non-linear response of yield to the increase in drought severity. Sensitivity analysis shows that temperature plays an important role in determining drought impacts, through reducing or amplifying drought-driven yield loss risk. Compared to present conditions, an ensemble of 11 crop models simulated an increase in yield loss risk by 9%–12%, 5.6%–6.3%, 18.1%–19.4% and 15.1%–16.1 for wheat, maize, rice and soybeans by the end of 21st century, respectively, without considering the benefits of CO 2 fertilization and adaptations. This study highlights the non-linear response of yield loss risk to the increase in drought severity. This implies that adaptations should be more targeted, considering not only the crop type and region but also the specific drought severity of interest.
[ "ENVI" ]
[ "Agricultural production", "Climate change", "Drought", "Risk" ]
[ "2304", "2305", "2310", "2311" ]
[ "Global food demand is expected to roughly double by 2050s (Godfray et al., 2010; Tilman et al., 2011).", "To meet growing food demand in the context of global warming requires enhanced understandings of the climatic factors influencing food production.", "Important in this regard is to examine how crop yield responds to climate variability and extremes.", "Typically, farmers would be more capable of adapting to the gradual changes in local mean climate conditions than extreme events, calling for the need of improved understanding of the impacts of climate extremes on agricultural production (Lesk et al., 2016; Zampieri et al., 2017).", "Drought, as an extreme weather phenomenon, is one of the major climatic constraints to crop yield (Lesk et al., 2016; Lobell et al., 2014; Matiu et al., 2017; Mishra and Cherkauer, 2010; Zipper et al., 2016).", "Under drought conditions, crops close their stomata to limit evaporative water loss, thus reducing carbon uptake by photosynthesis and decreasing yields.", "Globally, it is estimated that a cereal (maize, rice and wheat) loss of 1820 million Mg has been caused by droughts during the past four decades (Lesk et al., 2016).", "Assessment of drought impact on agricultural production is challenging, especially given that drought itself is driven by complex interactions among precipitation, temperature, vapor pressure, and solar radiation.", "In addition, compound changes in other physical and agronomical factors influencing crop growth would complicate the assessment of yield response to droughts.", "For example, Leng (2017a,b) showed that agricultural management such as irrigation can mitigate the negative impacts of water stress on maize yield, though the study was limited to US central High Plains.", "Recent process-based modeling studies also revealed large uncertainties in crop yield simulations arising from our incomplete knowledge of the physical and agronomical processes underlying crop growth and yield (Asseng et al., 2011; Elliott et al., 2015; Folberth et al., 2016; Rosenzweig et al., 2014).", "Thus, the incomplete understanding of the physical processes make it hard to derive certain estimates of drought impact on crop yield.", "Previous studies have discussed the potential impact of drought on crop production in the United States (Lobell et al., 2014; Troy et al., 2015; Zipper et al., 2016), China (Shi and Tao, 2014; Yu et al., 2014), Australia (Madadgar et al., 2017), South Africa (Araujo et al., 2016), Republic of Moldova (Potopová et al., 2016), Czech Republic (Hlavinka et al., 2009), and the whole globe (Lesk et al., 2016; Matiu et al., 2017).", "Although these studies have provided valuable insights into the possible impacts of droughts, they are mainly based on deterministic approaches, reporting the overall yield fluctuation associated with drought.", "Recently, probabilistic methods have been adopted with the purpose of accounting for uncertainty from climate data (Tao et al., 2009; Tebaldi and Lobell, 2008).", "To date, probabilistic estimation of yield changes under a given drought of specific severity has not been conducted across global agricultural regions.", "This study fills the gap by assessing the risk of crop yield loss under droughts in global major producing countries in a probabilistic manner.", "Specifically, we address the following scientific questions: 1) what are the possible changes in crop yield in response to an individual drought of specific severity?", "Understanding the distribution of possible outcomes of crop yield under a given drought can help decision-makers and insurers in selecting appropriate strategies based on the likelihood of different outcomes.", "2) Does yield loss risk grow linearly with increase in drought severity?", "Examining yield sensitivity to droughts of various severity can better inform targeted adaptation and mitigations.", "3) How will the likelihood of yield reduction change in the future?", "Country level census data on yields of the four largest commodity crops (i.e. wheat, maize, rice and soybeans) are collected from the Food and Agriculture Organization (http://faostat.fao.org) for the period 1961–2016.", "These four crops provide ~75% of all calories consumed by humans (Lobell et al., 2011).", "To investigate future changes in yield loss risk under droughts, we obtained crop yields simulated by 11 gridded crop models (Table 1) driven by five global climate models (Table 2) under RCP8.5 emission scenario from the Agricultural Modeling Inter-comparison and Improvement Project (AgMIP) (Rosenzweig et al., 2013) and the Inter-Sectoral Impact Model Inter-comparison Project (ISI-MIP) (Warszawski et al., 2014).", "Gridded monthly climate data at 0.5° × 0.5° for the period 1961–2016 were obtained from the Climate Research Unit (Mitchell and Jones, 2005).", "Spatially weighted averages of gridded precipitation and temperature were computed for each crop and growing season based on weights defined by the crop area (Portmann et al., 2010), resulting in annual growing-season precipitation and temperature for each crop-country and crop-globe combination following (Lobell and Field, 2007).", "Growing seasons are determined from the crop calendar dataset developed by (Sacks et al., 2010).", "In this study, we focus on the top 10 producing countries that are highly relevant for global food market and security (Lobell, 2007; Matiu et al., 2017).", "The combined production of the top 10 productive countries accounts for 96%, 87%, 78% and 68% of global total production of soybeans, rice, maize and wheat, respectively.", "Yield variations in these major producing regions would therefore lead to significant anomalies at the global level, affecting global food market and security.", "Here, the FAO country-level yield data and observed climate are used to build the probabilistic model, based on which the historical sensitivity of crop yield to droughts is examined.", "To be consistent with observation-based analysis, the gridded crop model simulations are aggregated to country level based on which the projected changes in yield loss risk by the end of 21st century are investigated.", "Drought is multi-dimensional and can be grouped into meteorological, agricultural, hydrological and socio-economic droughts (Heim Jr, 2002; Huang et al., 2016; Leng et al., 2015; Mishra and Singh, 2010).", "In this study, we use the Standardized Precipitation Index (SPI) (McKee et al., 1993) to estimate the impacts of moisture supply originated from precipitation.", "A drought event is identified when the SPI value is below −0.8, following the U.S. Drought Monitor (http://droughtmonitor.unl.edu/).", "Yield changes under four drought categories (i.e. moderate, severe, extreme and exceptional droughts) are examined to quantify the sensitivity of yield response to the increase in drought severity (Table 4).", "Besides SPI, we use a drought index that includes the effects of temperature, given the critical role of temperature in crop growth and yield (Lobell et al., 2013; Lobell et al., 2014).", "We note that there are several indices that take into account the effects of temperature directly or indirectly, e.g., the standardized precipitation evapotranspiration index (SPEI) (Vicente-Serrano et al., 2010) and the Palmer Drought Severity Index (PDSI) (Palmer, 1965).", "Here, we select the SPEI for analysis, as it is an extension of SPI in the calculation procedures.", "The potential evapotranspiration (PET) is estimated based on the Thortnthwaite equation (Thornthwaite, 1948), instead of using other complex equations requiring more inputs.", "Comparing the results based on SPI and SPEI allows for examination on the compounding effects of temperature in modulating drought impacts.", "We first remove the linear temporal trend of crop yield using the least squares method to minimize the effects of slowly changing factors (e.g. agricultural management) (Hlavinka et al., 2009; Lobell et al., 2011).", "The five bi-variable copulas are then fitted to the de-trended crop yield and growing season SPI for each crop-country combination.", "We select the copula that has the highest statistically significant maximum likelihood as the best one (Sadegh et al., 2017), based on which the probability of yield loss (i.e. yield dropping below its long-term mean) under a given drought of specific severity is calculated.", "The statistical significance is estimated according to the two-tailed Student's t-test.", "We obtained similar results when using Bayesian Information Criterion (BIC) (Schwarz, 1978) and Akaike Information Criteria (AIC) (Akaike, 1981) as the evaluation metric.", "The above analysis is repeated based on simulated crop yields by crop models driven by global climate models for the historical period (1961–2016) and future period (2071–2100) to explore future changes in yield loss risk by the end of 21st century.", "We note that several reference periods have been used in climate change impact assessment, such as 1980–2010 (Schewe et al., 2014), 1971–2000 (Haddeland et al., 2014) and 1986–2005 (Lissner and Fischer, 2016).", "To examine the uncertainty from the reference period is out of the scope of this study.", "Rather, we tend to investigate the temporal change in the sensitivity of yield loss risk to drought severity under global warming.", "The RCP8.5 emission scenario is used to represent the upper bound of climate change impacts in the business-as-usual world.", "Global agricultural productivity has exhibited substantial variations during the past decades, and crop yield reductions are often observed when dry conditions occurred (Fig. 1).", "Overall, crop yield variability can be explained by the drought index (i.e. SPI) for the study period.", "Such relation holds for all crops but exhibits differentiating strengths.", "Specifically, year-to-year variation of soybean shows the highest correlation with the drought index, followed by rice, wheat and maize.", "This is consistent with previous studies reporting larger impacts of precipitation on rice and soybeans than wheat and maize (Lobell and Field, 2007).", "A substantial decrease up to 25% in crop yield is observed in dry conditions (SPI < −0.8) as compared to wet conditions (SPI > 0.8).", "The negative impact of drought on crop yields is in line with previous studies at regional and global scales (Antwi-Agyei et al., 2012; Li et al., 2009; Lobell et al., 2014; Richter and Semenov, 2005; Troy et al., 2015; Zipper et al., 2016), although the magnitudes of estimations differ to certain extent due to difference in the time period and dataset used.", "Notably, a large portion of yield variation cannot be explained by the drought index, since several other factors influencing yield are not considered.", "Nevertheless, these results imply that occurrence of a drought event may not necessarily lead to a reduction of yield, confirming the importance of re-assessing the drought impacts in a probabilistic manner, as pursued in this study.", "We then fit the joint distribution function between crop yields and SPI for the globe as a whole (Fig. 2).", "The Frank, Gumbel, Frank, and t models are selected for global wheat, maize, rice and soybeans, respectively, while the probabilistic model used for other country-crop combination is summarized in Table 5.", "The diverse model selections among crops and regions indicate that the relationship between yield anomaly and droughts depends on the region and crop of interest.", "Comparing the estimated probability distribution of yields with observations (red dots) shows that the majority of observed yields fall well within the high-density regions.", "Indeed, based on evaluation metrics including the maximum likelihood, Bayesian Information Criterion and Akaike Information Criteria (see Methods), the probabilistic model developed in this study is reliable for investigating crop yield responses to droughts of various severity.", "Based on the selected copulas, the conditional probability distributions of crop yields under dry and wet conditions are estimated (Fig. 3).", "A consistent increase of yield loss likelihood is observed for all crops from wet to dry conditions, as indicated by the shift of probability density curves to the left of long-term average.", "Under wet conditions, the probability of wheat yield loss (i.e. yield dropping below its long-term average as indicated by the vertical dashed line) for the globe as a whole is 41%, while the risk would increase by 17% to 58% when experiencing a moderate drought.", "Similar changes in yield distribution curves are found for maize, rice and soybean yields, exhibiting an increase in yield loss probability by 22%, 9% and 22%, respectively.", "How will yield loss risk change with increase in drought intensity?.", "Here, the top 10 producing countries are selected for detailed analysis (Fig. 4), since they are highly relevant for global food market and security.", "Results for other small countries can be found in the Supplementary Tables.", "Globally, wheat is more vulnerable to droughts than maize, rice and soybeans, as indicated by the higher magnitude of yield loss probability under the four categories of droughts (i.e. moderate, severe, extreme and exceptional droughts).", "Regionally, the probability of wheat reduction is the highest in USA and Canada than other regions, and there is >80% probability that wheat reduction may fall below its long-term average when experiencing a drought at the exceptional category.", "As for maize, India shows the highest risk of yield reduction under droughts, while rice yield in Vietnam and Thailand are most vulnerable to droughts.", "Risk of drought-driven soybean yield reduction is the highest in USA, Russian and India, while relatively low risk is observed in other countries.", "The physical mechanisms behind the distinct spatial patterns are an open question since many factors could modulate drought impacts on farmers' fields.", "One obstacle has been lack of data on the physical and agronomical conditions that are relevant to crop growth and yield at fine scales.", "For example, agricultural management such as irrigation in US Great Plains can substantially modulate the response of maize yield to precipitation anomaly (Leng, 2017a).", "Nevertheless, the drought risk for crop yield in global major agricultural countries as revealed in this study provides valuable information for targeted adaptation and mitigation.", "Comparing yield loss risk among moderate, extreme, severe and exceptional droughts can also give us valuable insights into the sensitivity of yield loss risk to the increase in drought severity.", "It is observed that with increase in drought severity, yield loss risk tends to grow progressively independent of crop types and regions.", "When drought severity shifts from moderate to exceptional, the USA and Canada would experience an increase of wheat loss risk by 20%, while Romania, USA and India show an increase in yield loss risk by 15%.", "India and Vietnam show the largest increase of rice reduction risk by 20% when experiencing a growth in drought severity from moderate to exceptional, while yield loss risk remains relatively stable in Indonesia in response to increase in drought severity.", "The likelihood of soybean reduction in Brazil would increase from 52% under a moderate drought to 71% under an exception drought.", "Importantly, the rate of risk growth tends to become lower with increase in drought severity.", "That is, the risk difference between severe and exceptional droughts is smaller than that between moderate and extreme droughts.", "This trend suggests that yield response to droughts is non-linear with drought severity, implying that yield loss risk under a drought category cannot be extrapolated for estimation under droughts of other categories.", "The non-linear response of yield loss risk to droughts implies that targeted mitigation and adaptation strategies should consider not only the crop type and region, but also the drought severity of interest.", "How will the likelihood of drought-induced yield loss change in the future?", "Fig. 5 shows the projected yield loss risks under moderate, extreme, severe and exceptional droughts simulated by 11 state-of-the-art crop models driven by 5 climate models under RCP8.5 emission scenario.", "The multi-model ensemble mean gives estimates of yield loss risk that is comparable to those based on observations (Fig. 4) not only in the magnitude but also in the sensitivity of risk to drought severity, although considerable bias remains (Table 6).", "However, large discrepancy exists among crop models in simulating yield loss risk under droughts not only in the magnitude but also in terms of its sensitivity to increasing drought severity.", "This could be due to the differences in model structure, representation of environmental stress, agricultural management, and CO2 fertilization effect (Asseng et al., 2011; Elliott et al., 2015; Folberth et al., 2016; Rosenzweig et al., 2014).", "Such a validation suggests that it is reasonable to use the ensemble of process-based crop model simulations for assessing future changes in yield loss risk.", "Projections based on the ensemble of crop models show that yield loss risk will increase in the future, with the magnitude depending on crop and severity of drought.", "The largest increase of yield loss risk is found for rice, followed by soybeans, wheat and maize.", "Notably, an uneven growth rate of yield loss risk is observed among the four drought categories.", "For example, larger risk of wheat yield loss is projected with increase in drought severity, while rice, maize and soybeans tend to show less sensitivity to increasing drought severity in the future (Fig. 5).", "Overall, drought-driven yield loss risk is projected to increase by 9%–12%, 5.6%–6.3%, 18.1%–19.4% and 15.1%–16.1 for wheat, maize, rice and soybeans, respectively, without considering adaptations or CO2 fertilization effect.", "The substantial increase of yield loss risk points to the need for effective adaptive measures for ensuring resilience in agricultural production in a warming climate with greater likelihood of more frequent and severe droughts (Dai, 2013; Huang et al., 2017; Sheffield and Wood, 2008).", "In this study, we develop a probabilistic model for estimating yield loss risk given an individual drought of specific severity.", "Serval uncertainty sources have to be acknowledged when interpreting the results of this study.", "For example, five popular copula models were selected to describe the dependency structure between yield and a drought index.", "Inherent uncertainty from the copula model itself would propagate and affect the estimation of yield loss risk under droughts.", "Here, we use the Markov chain Monte Carlo simulation technique within a Bayesian framework to infer the posterior distribution of copula parameters, based on which the uncertainty range arising from the copulas is estimated (see Methods).", "Fig. 6 shows the range between the lower and upper bounds of estimated yield loss risk under droughts for the four crops.", "Overall, the uncertainty is small compared to the estimated yield loss risk, with the magnitude depending on the crop-country combination of interest.", "Notably, the uncertainty magnitude tends to increase with drought severity, suggesting that estimation of yield loss risk under a moderate drought would be more reliable than those under more severe drought categories.", "It is well known that drought is driven by several other factors including temperature, which is also critical for crop growth and yield (Lobell et al., 2013; Lobell et al., 2014).", "Here, additional sensitivity analysis is conducted using a new drought index (i.e. SPEI) that accounts for the effects of temperature.", "As shown in Fig. 7, difference is observed between SPI- and SPEI-based yield loss risks.", "This difference suggests that drought-driven yield loss risk does depend on the definition of drought.", "Specifically, inclusion of temperature effects leads to considerable difference in the estimated yield loss risk of wheat and maize.", "However, the results remain largely similar for rice and soybean in response to SPI- and SPEI-based droughts.", "This could be attributed to the fact that rice and soybean are more sensitive to precipitation than temperature (Lobell and Field, 2007).", "Thus, consideration of temperature leads to minor change in the estimated yield loss risk under droughts.", "Besides climate factors, crop yields would be influenced by agricultural management such as irrigation (Leng et al., 2014; Müller et al., 2018) and fertilization (Leng et al., 2016; Müller et al., 2018; Stewart et al., 2005), conservation tillage (Karlen et al., 2013), multiple cropping (Seifert and Lobell, 2015), tile drainage (Schilling and Libra, 2003) and soil mulching (Qin et al., 2015).", "In addition, the CO2 fertilization effects tend to be beneficial for crop growth, but remain a large source of uncertainty (Schleussner et al., 2018).", "To which extent these compounding factors would reduce or amplify drought impacts on crop yield is not within the scope of this study, especially given the lack of detailed information and incomplete knowledge of the complex mechanisms behind yield variations.", "We also acknowledge that the 11 process-based crop models and 5 climate models used in this study may not represent the full uncertainty range in the projection of future yield loss risk.", "In addition, future projections are conditional on the assumption of no adaptations, and thus may not be able to indicate the true future course of yield impacts.", "Therefore, future yield loss risks should not be interpreted as the correct magnitude of climate change impacts.", "Rather, the results are intended to provide a measure of the temporal evolution of yield loss risk under a warming climate, which has received little attention.", "Understanding the potential changes in the sensitivity of yield loss response to drought is a critical step towards planning and prioritizing effective adaptation options.", "This study provides a complementary method for assessing drought impact on global crop yields.", "Through developing a probabilistic model, we estimate yield loss risks (i.e. the probability of yield dropping below its long-term mean) under moderate, extreme, severe and exceptional droughts.", "Results show a significant association between droughts and yield reductions during the past decades.", "When experiencing an exceptional drought, the probability of yield loss could exceed 70% for soybean and maize, while the risk for wheat and rice is up to 68% and 64%, respectively.", "This prediction represents an increase of yield loss risk by 24%, 21%, 18% and 20% for soybean, maize, rice and wheat, respectively, when drought severity grows from moderate to the exceptional category.", "Notably, the rate of risk growth tends to become slower with increase in drought severity, suggesting the non-linear response of yields to droughts.", "Regionally, the risk of drought-driven wheat reduction is the highest in USA and Canada, where there is >80% probability that wheat reduction may fall below its long-term average given an exceptional drought.", "As for maize, India shows the highest risk of yield reduction, while rice yield in Vietnam and Thailand are most vulnerable to droughts.", "Risk of soybean yield reduction is the highest in USA, Russian and India, while relatively low risk is observed in other regions.", "Further analysis based on 11 process-based model simulations shows that yield loss risk will increase in the future, with the largest growth found for rice followed by soybeans, wheat and maize.", "Enhancing the resilience of agricultural system to droughts would greatly benefit from improved understanding of the full range of possible outcomes of crop yields under a drought of specific severity.", "The insights abstained in this study enable more targeted efforts to manage drought risk, through adaptations of agricultural and water management practices, spatial diversification of crop production and the design of insurance instruments.", "The non-linear yield response to the increase in drought severity implies that future adaptations should be more targeted, considering not only the crop type and region but also the drought severity of interest." ]
[ "The probability of yield loss under droughts is estimated for four major crops.", "Yield loss risk grows non-linearly with increase in drought severity.", "Yield loss risk is projected to increase in the future by an ensemble of models." ]