Biotechnology research is traditionally focused on individual microbial strains that are perceived to have the necessary metabolic functions, or the capability to have these functions introduced, to achieve a particular task. For many important applications, the development of such omnipotent microbes is an extremely challenging if not impossible task. By contrast, nature employs a radically different strategy based on synergistic combinations of different microbial species that collectively achieve the desired task. These natural communities have evolved to exploit the native metabolic capabilities of each species and are highly adaptive to changes in their environments. However, microbial communities have proven difficult to study due to a lack of suitable experimental and computational tools.With the advent of genome sequencing, omics technologies, bioinformatics and genome-scale modeling, researchers now have unprecedented capabilities to analyze and engineer the metabolism of microbial communities. The goal of this review is to summarize recent applications of genome-scale metabolic modeling to microbial communities. A brief introduction to lumped community models is used to motivate the need for genome-level descriptions of individual species and their metabolic interactions. The review of genome-scale models begins with static modeling approaches, which are appropriate for communities where the extracellular environment can be assumed to be time invariant or slowly varying. Dynamic extensions of the static modeling approach are described, and then applications of genome-scale models for design of synthetic microbial communities are reviewed. The review concludes with a summary of metagenomic tools for analyzing community metabolism and an outlook for future research. Recent advances in omics and genetic engineering technologies have resulted in novel techniques to interrogate and manipulate biological processes at varying levels of detail. The availability of such tools to analyze system-wide changes at the biomolecular level of genes, proteins and metabolites has created significant opportunities to understand cellular functions and to ultimately design processes that achieve a desired objective . Such tools have been increasingly used to understand, manipulate and model microbial communities for several practical applications including biorefineries, bioelectricity generation and bioremediation , 34.Microbial communities are ubiquitous, and microorganisms rarely function in isolation in the environment, even though most microbiology research is focused around the study of pure culture representatives. Hence, it is valuable to investigate the interactions between microorganisms so that we can extend our understanding of the physiology of pure culture representatives to communities. Genome sequencing of whole communities in natural environments (metagenomics) is now possible  and a weExtending modeling approaches to microbial communities is challenging partly due to the range of metabolic interactions that are possible, including competition, cross-feeding, syntrophy and mutualism. Additionally, the isolation of individual members of the community can be challenging despite advances in physiological techniques . While gIn this review, we focus on studies that have used genome-scale modeling approaches for analyzing and manipulating metabolism in microbial communities. We start with a brief introduction to lumped models, following which static approaches for genome-scale modeling of community metabolism are presented. Then extensions that account for community dynamics are discussed and the use of static and dynamic models for the design of synthetic communities are described. Finally, recent studies on analyzing metabolism in metagenomes are summarized and prospects for future research are discussed. This reviews aims to provide the reader with an overview of how metabolic modeling approaches are being applied for analyzing and engineering microbial communities. The reader is referred to other recent reviews , 15 for Escherichia coli was developed , have been developed. The dFBA approach links the steady-state intracellular metabolic flux distribution with the dynamic changes in the environment and allows the prediction of microbial growth, substrate utilization and product formation dynamics. Initially, dFBA was used to model the diauxic growth of bstrates , 33, 50.bstrates  and in sineering  of S. ce2 by metal reducers, sulfate reducers and methanogens, respectively. Hence, models of community metabolism have to account for the environment as a separate compartment in addition to the intracellular metabolic networks of individual organisms in the community. Most often, these interacting partners are assumed to grow together and this metabolic interaction is assumed to be at steady state. There have been several such “static genome-scale models” of microbial communities that are summarized in the next section.Microbial communities often involve species interactions where a metabolic by-product of one species is the substrate for other species and vice versa. In the environment, carbohydrates from organic matter are fermented to hydrogen, acids and alcohols by fermentative microbes and these electron donating byproducts are oxidized with electron acceptors such as Fe(III), sulfate and CODesulfovibrio vulgaris, and the methanogen, Methanococcus maripaludis . The autFreilich and Ruppin (2011)  extendedDesulfovibrio vulgaris, Geobacter sulfurreducens and Clostridium cellulolyticum. The predictions of carbon and electron balance were found to be consistent with experimental observations. The authors also investigated a hypothetical scenario where new species were added to this microbial community and methane production was simulated. These results suggest the potential for a model-based framework in designing synthetic microbial communities for practical applications in bioenergy and bioremediation.In most studies, the community objective has been assumed to be growth rate maximization of the individual microbes. Recently, Zomorrodi et al. (2012)  formulatWhile the static models described in the previous section have been shown to be valuable for understanding and analyzing metabolic interactions in microbial communities, the static approach does not allow prediction of the biomass concentrations of the individual species or account for time-varying changes in the intracellular and extracellular environments. Consequently, static genome-based models cannot easily predict the microbial composition in dynamic environments. Recently, dynamic flux balance analysis (dFBA) has been extended to microbial communities to predict time-varying interactions between species and their effects on the microbial composition. The extension involves coupling genome-scale metabolic models of the individual species, dynamic extracellular mass balances on each species, substrate and metabolic byproduct, and uptake kinetics for each substrate/species pair. Below several representative studies are summarized to illustrate the potential of the dFBA approach.Rhodoferax and Geobacter, in natural and acetate amended environments. In agreement with data, the simulations suggested that Geobacter species were fast growing, energetically inefficient organisms relative to the Rhodoferax species and would dominate the community at high acetate availabilities. The Rhodoferax species were predicted to be most efficient at utilizing acetate and to be dominant during acetate limitation. Salimi et al.   have devi et al.  used thiE. coli mutant strains ALS1008 and ZSC113, which have been engineered to have glucose and xylose only uptake, respectively. The simulations suggested that improvements in batch substrate consumption observed in a previous experimental study  engineering the metabolism of individual microbes in defined microbial communities; 2) manipulating the community by adding new microbes ; and 3) engineering the environment by introducing substrates that allow a specific species of interest to dominate  . Below set al. and a mercuric ion reducer  was required to degrade PCP in the presence of mercury as S. chlorophenolicum was sensitive to mercuric ion. Following a different approach, Klitgord and Segre  injection rates to allow sustained uranium bioremediation to occur in the environment. For the ZSC113/S. cerevisiae co-culture system, Hanly and Henson (Currently there are few reported examples of utilizing dynamic genome-scale metabolic models for designing the community environment. Zhuang et al.  have used Henson  used genThe studies reviewed in this paper clearly demonstrate the value of genome-scale models for understanding and engineering metabolic interactions in microbial communities. The availability of such rigorous and experimentally validated models will provide the opportunity to systematically optimize microbial communities for practical applications through the engineering of the individual species, addition of new species to existing communities and manipulation of the community composition and environment . IndividWhile this review has focused on organisms for which an annotated genome sequence is available, metabolic network reconstructions of metagenomes are increasingly possible. Such reconstructions of overall communities have been applied to environmental metagenomes and human microbiomes. For example, a metabolic network reconstructed from the metagenome of the English Channel has been used to define a metric that correlated the presence of metabolic enzymes with the metabolic turnover of the associated pathways . AdditioIn the future, we anticipate that such genome-scale models can be generalized to more than three species and therefore can be extended to the main members of natural communities. In addition, integration of these microbial community models with biogeochemical, physical processes in ecosystem models will allow improved prediction of community responses to environmental perturbations. Game theoretic approaches have been proposed to represent both the fitness of the individual members and changes in the environment due to microbial dynamics , 40. The


Clinicians search PubMed for answers to clinical questions although it is time consuming and not always successful.To determine if PubMed used with its Clinical Queries feature to filter results based on study quality would improve search success .We invited 528 primary care physicians to participate, 143 (27.1%) consented, and 111  completed the study. Participants answered 14 yes/no therapy questions and were given 4 of these  to search using either the PubMed main screen or PubMed Clinical Queries narrow therapy filter via a purpose-built system with identical search screens. Participants also picked 3 of the first 20 retrieved citations that best addressed each question. They were then asked to re-answer the original 14 questions.We found no statistically significant differences in the rates of correct or incorrect answers using the PubMed main screen or PubMed Clinical Queries. The rate of correct answers increased from 50.0% to 61.4% (95% CI 55.0%-67.8%) for the PubMed main screen searches and from 50.0% to 59.1% (95% CI 52.6%-65.6%) for Clinical Queries searches. These net absolute increases of 11.4% and 9.1%, respectively, included previously correct answers changing to incorrect at a rate of 9.5% (95% CI 5.6%-13.4%) for PubMed main screen searches and 9.1% (95% CI 5.3%-12.9%) for Clinical Queries searches, combined with increases in the rate of being correct of 20.5% (95% CI 15.2%-25.8%) for PubMed main screen searches and 17.7% (95% CI 12.7%-22.7%) for Clinical Queries searches.PubMed can assist clinicians answering clinical questions with an approximately 10% absolute rate of improvement in correct answers. This small increase includes more correct answers partially offset by a decrease in previously correct answers. Medline indexed 760,903 new articles in 2012, bringing their total to just under 20 million articles. The number of journals indexed by Medline has grown by 50% in the past 20 years [The 6S pyramid of evidence from health care research describes a range of tools and resources to assist physicians in accessing or retrieving relevant research evidence. The pyramid is structured so that original studies form the base and are topped by, in ascending order of clinical usefulness, synopses of studies, syntheses (systematic reviews), synopses of syntheses, summaries (evidence-driven online texts), and systems  . In addiResearch has shown that published original studies and reviews can provide clinicians with answers to their clinical questions -13 and lOther studies have reported negative effects of information searching on physician responses to clinical questions. McKibbon and Fridsma  found thSearch filters have been developed to help clinicians search the primary literature. These filters are rigorously developed and validated to increase the yield of clinically relevant articles based on research methods or clinical content. The Health Information Research Unit at McMaster University has developed filters for detecting primary studies for therapy ,20, diagThe objective of this pragmatic study was to determine if differences exist in the rate of correct answers to clinical questions when primary care physicians use the PubMed main screen or the Clinical Queries feature of PubMed for searches. Specifically, do searches done by primary care physicians through the PubMed main screen or through Clinical Queries give different rates of correctness of answers to clinical questions related to therapy?To assess if PubMed provided correct answers to clinical questions related to therapies, standard clinical questions with answers based on recent systematic reviews were developed. The reviews were selected from a database of clinical research from 125 journals preappraised for methodological rigor  and rateIn all, 24 standard questions were devised and iteratively tested on 3 physicians. The physicians were 2 experienced general practitioners and 1 experienced general internist. A fourth general internist also reviewed the questions. The physicians provided input on clinical applicability, perceived difficulty of the question, and relevance to practice for each question. Revised questions were then piloted on 2 general practitioners who provided further feedback. Questions were dropped if they were perceived by the clinicians as being too difficult or easy to answer, not relevant to general practice, or if the answer was perceived to be controversial. The remaining 14 questions are presented in Practicing physicians self-identified as general practitioners, family practitioners, or primary care general internal medicine practitioners who were registered with the McMaster Online Rating of Evidence (MORE)  system wParticipants were sent an Internet link to the survey that required them to sign into our information production system of high-quality clinical articles using their system passwords, which started the task . After pBecause our questions were treatment questions, we used the therapy category of the Research Methodology filter of Clinical Queries. We were interested in clinicians searching for answers to clinical questions; therefore, we used the narrow Clinical Queries. The narrow search filters are designed for clinical care because they retrieve a good proportion of potentially relevant citations while keeping the number of nonrelevant citations to a minimum (sensitivity of 93% and specificity of 97% ). The brFor each question, participants were asked to enter search terms into a textbox. If participants were unhappy with the retrieval, they could alter their search terms and submit a new search.After each of the 4 searches, the first 20 results were presented. The participant was blinded as to which PubMed interface the retrieval came from. They could view the abstract of the article in the same window by selecting the title of the article. Participants were asked to select the top 3 articles most important to forming/supporting their answer. After the 4 searches were performed and articles were selected, the participants were given the 14 questions again and asked to answer them with yes or no. The study was approved by the McMaster University Hamilton Health Sciences/Faculty of Health Sciences Research Ethics Board.The primary outcome of the study was the difference in the proportion of correct answers before and after searching. Secondary outcomes were the proportion of questions searched that went from incorrect to correct and correct to incorrect, the proportion of questions without searches that went from correct to incorrect and incorrect to correct, and the time taken to complete the project tasks.Based on previous studies, starting proportions of answers to clinical questions were 27% correct (n=557)  and 40% We anticipated an approximately 10% change in correct to incorrect answers; therefore, we set a 5% absolute difference between search modes as clinically interesting. This gave us a sample size of 522 searches for the correct group and 459 for the incorrect group to detect a 5% difference in search modes with 80% power.The Mantel-Haenszel test for matched pairs, stratified by participant and by question, was used to determine the odds ratio of changing a response by using Clinical Queries searches versus PubMed main screen searches. A posteriori we recognized that question 6 was a prognosis question rather than a treatment question. Given that the Clinical Queries searches used a therapy filter, we performed our analysis including this question and the sensitivity analysis without this question. In the entire dataset, only 1 of 29 participants (3%) presented with question 6 changed their answer .Articles selected by the participants as being relevant to answering their question were independently assessed in duplicate for methodological criteria outlined below. Disagreements were resolved through consensus.A therapy study is methodologically sound if it meets these 3 criteria:Random allocation of participants to comparison groups;Outcome assessment of at least 80% of those entering the investigation accounted for in one major analysis at any given follow-up assessment; andAnalysis consistent with study design.A systematic review of therapy studies is methodologically sound if it meets these 6 criteria:Explicit statement of clinical topic;Question refers to treatment;Methods are described in report body (not just the abstract);More than one major database searched or Cochrane CENTRAL searched;Explicit inclusion/exclusion criteria; andOne or more articles meet criteria set out for therapy studies (listed previously).During recruitment, 528 physicians were invited to take part in the study; 143 (27.1%) provided consent, 110 of whom (21.0% of those invited and 77.6% of those who consented) completed the study tasks (24 abandoned the task after the first search and 9 did not perform any searches). Two participants (1.8%) answered all 14 questions correctly; consequently, they were directed to search for only 2 questions. At baseline, participants answered 62.3% (95% CI 59.8%-64.7%) of the questions correctly.Time to complete the tasks was calculated based on the time the participant signed in to the website until the time they submitted the survey. If the participant logged off without clicking the submit button, the timer continued to count. As such, 16 observations were more than 100 minutes, ranging from 119 to 103,786 minutes (72 days). We selected a cutoff of 100 minutes as a likely point at which the tasks were not completed in 1 sitting. The remaining 95 participants completed the tasks within 6 to 76 minutes .During the study, 440 searches were executed, 222 (50.5%) answered correctly and 218 (49.5%) answered incorrectly initially. For questions selected for searching, baseline responses were 50.0% correct in both groups by design. After searching, responses were correct for 61.4% (95% CI 55.0%-67.8%) of questions for the PubMed main screen group, and 59.1% (95% CI 52.6%-65.6%) for the Clinical Queries group. We found no differences in the rate of answers going from incorrect to correct for the PubMed main screen searches  compared with the Clinical Queries searches  . Both seP>.05)  . SensitiP=.002).For questions answered before and after searching but without intervening searches, an average of 65.4% (95% CI 62.8%-68.0%) were correct at baseline, 64.6% (95% CI 62.0%-67.2%) were correct at the end of the study across the 14 questions, 4.0% (95% CI 2.3%-5.6%) went from correct to incorrect, and 3.1% (95% CI 2.2%-4.1%) went from incorrect to correct. There was variability in baseline performance across questions . WithoutClinical Queries were developed to retrieve clinically useful studies based on study design. Therapy filters retrieve citations based on the article being a randomized controlled trial. Therefore, we were interested in determining if the participants identified studies with strong methods  when presented with the first 20 retrievals. The participants were asked to identify the 3 most important articles that provided evidence to answer the clinical question they were addressing. Overall, the PubMed main page group tagged 334 articles as important and the Clinical Queries group tagged 321 articles. Although we sought to show that searches with PubMed Clinical Queries were associated with more correct answers to clinical questions than were PubMed main screen searches, we did not find any differences. This may be because we did not meet our sample size of approximately 1000 searches as originally calculated. Another explanation for these results may be the training and experience of the study participants. All were practicing clinicians and were registered with the MORE system, wherein they evaluate and rate clinician-ready health research studies. Also, this study was done on the Internet. The participants likely had strong computer and Internet skills and were probably skilled users of PubMed and the clinical research literature. Therefore, the study participants may be the least likely group of clinicians who could benefit from using Clinical Queries. Naïve users or new clinicians, such as interns and residents, or those clinicians less skilled at the assessment and application of research findings may derive more benefit from the Clinical Queries searches. Time is also a major factor in seeking answers to clinical questions. If the time to seek answers had been more tightly constrained in the study (we did not have time limits on the tasks) we may have found a larger difference between the correctness of the answers found with standalone PubMed searches and searches using Clinical Queries.P=.60).However, this study does show that PubMed, either on its own or using Clinical Queries, helps clinicians answer clinical questions with increased accuracy. For questions that clinicians answered twice without searching, the rate of correct answers stayed the same (65.4% correct at first answer and 64.6% correct on second answer). With searching, clinicians improved their rate of correct replies. Their answers went from 50.0% correct (set by the study) to 60.2% correct .The questions we used in the study were based on strong evidence from current systematic reviews, and they were pretested with various physician groups. However, despite these strengths, our questions were not questions that arose in the participants’ daily practices.Future research needs to be done to improve the quality of search tools and their ability to maximize the correct answers to questions while minimizing the answers that go from an initial correct answer to an incorrect answer. Focusing on specific groups of clinicians  or in certain situations  may also address the potential for automated assistance of PubMed searching. Other research has shown that an interface in PubMed leads to better question answering if the search entry screen required clinicians to enter concepts related to patients or populations, intervention, comparison, and outcome (PICO) aspects of the questions.  ComparisWe have shown that complex studies of searching can be done through the Internet. We also have reinforced that clinician searching in PubMed produces an absolute improvement of approximately 10% in clinician ability to correctly answer clinical questions. This 10% improvement is consistent with other similar studies ,17,18 an


In order to characterize the relationship between genes and inserted elements, we have examined the distribution of retrotransposons and their internal motifs within tissue-specific and housekeeping genes. We have identified that the previously established retrotransposon antisense bias decays at a linear rate across genes, resulting in an equal density of sense and antisense retrotransposons near the 3′-UTR. In addition, the decay of antisense bias across genes is less pronounced among tissue-specific genes. Our results provide support for the scenario in which this linear decay in antisense bias is established by natural selection shortly after retrotransposon integration, and that total antisense bias observed is above and beyond any bias introduced by the integration process itself. Finally, we provide an example of a retrotransposon acting as an eQTL on a coincident gene, highlighting one of several possible avenues through which insertions may modulate gene function.Retrotransposons comprise approximately half of the human genome and contribute to chromatin structure, regulatory motifs, and protein-coding sequences. Since retrotransposon insertions can disrupt functional genetic elements as well as introduce new sequence motifs to a region, they have the potential to affect the function of genes that harbour insertions as well as those nearby. Partly as a result of these effects, the distribution of retrotransposons across the genome is non-uniform and there are observed imbalances in the orientation of insertions with respect to the transcriptional direction of the containing gene. Although some of the factors underlying the observed distributions are understood, much of the variability remains unexplained. Detailed characterization of retrotransposon density in genes could help inform predictions of the functional consequence of  Retrotransposons (RT) make up approximately half of the human genome RTs contain DNA motifs that can act as binding sites for polymerases Previous studies have taken advantage of the high copy number of RTs to study their relationship with the host genome. These studies have begun to reveal the capacity of various genomic contexts to withstand retrotransposition. Within genes, certain regions are more susceptible to disruption by RTs than others The distribution of retrotransposons throughout the human genome shifted towards a non-uniform pattern over evolutionary time Given that polymorphic Alu and human-specific Alu already appear over-represented in the antisense orientation, we next wanted to determine how this antisense bias might be modified over time. To address this question, we examined the rate of change in both L1 and Alu frequencies within genes and the ratio of ASO/SO over time. All L1 and Alu annotated in the Hg19 reference genome were used for this analysis. As a proxy for time, RTs can be ranked by relative age of retrotransposition. To order subfamilies in approximate order of subfamily age, we made use of the average nucleotide distance of subfamily members to their consensus sequence . As detaWe first separately examined the frequency of L1 elements in each orientation across time. Most L1 subfamilies were depleted within genes , with thIn contrast to L1, the majority of Alu were over-represented in genes . After gCombined with the L1 data, these results indicated that L1 and Alu density within genes had increased over the course of evolutionary time in relation to intergenic frequencies, but the overall level of antisense bias remained largely unchanged.To determine how RT frequency was dependent upon an element's position in a gene, we calculated the frequency of all L1 and Alu within each gene decile both in the ASO and SO . We wereHere we further note that the slope of human-specific ASO Alu was significantly different from the slope calculated with all Alu , indicating that the decay in antisense bias across genes most likely arose over evolutionary time. Alternatively, and somewhat less parsimoniously, recently inserted elements may have significantly altered their insertion pattern compared to older elements due to shifts in RT targeting mechanisms.Since previous studies have shown that ASO RT density was inversely correlated with exon density, we next calculated the frequency of coding exons (CDS) within each decile. Indeed, CDS density increased at a similar linear rate across gene deciles . The firAlthough both are conserved, coding exons and UTRs have very different roles in the creation of a final gene product. It has been shown that exon density is highly predictive of RT frequency; however it is also possible that the 5′-UTR has a specific effect on RT abundance that is distinct from the effect imposed by coding exons. We therefore wanted to determine if the presence of a 5′-UTR was associated with altered RT frequency beyond what would be expected from exon enrichment alone.In our method of analysis the first and last deciles consistently contained a 5′- or 3′-UTR, whereas the internal deciles were variable as to the presence of a coding exon. The resulting frequency plots revealed that RTs were highly reduced within the first bin (containing the 5′-UTR) in comparison to the second bin. Since this could be a result of a decrease in the density of CDS exons as well as an altered effect by the 5′-UTR, we needed to distinguish the effects from one another. To accomplish this, we restricted analysis to genes that contained a second 5′-UTR in each decile. Examples of genes that contained a secondary 5′-UTR are depicted in L1 ASO, 0.04L1 SO, 0.49Alu ASO, 0.24Alu SO) , and so on for all ten deciles see . The fre4Alu SO) .Across all genes, we have shown that the second decile, that neighbours the 5′-UTR containing decile, contains the largest number of L1 and Alu. We therefore tested if L1 and Alu were similarly increased in the deciles surrounding the secondary 5′-UTRs. We found that the L1 and Alu were also reduced to a lower extent in the bins surrounding the 5′UTR . L1 nearWhen taken as a group, RT antisense bias decreased linearly across gene bounds. Different functional groups of genes, however, contain varied numbers of RTs. For example, housekeeping genes have a predictably higher level of Alu frequency than other gene categories To determine if differences in gene expression across tissue types was confounding this result, we tested the correlation between ASO frequency and exon density as a function of relative expression in housekeeping and tissue specific genes. There was no link between expression level and the correlation between ASO and exon density . FurtherAlthough the consensus sequences for Alu and L1 contain many functional DNA binding motifs, each individual RT in the genome is variable as to whether or not it contains these motifs. The loss or gain of a motif may occur by mutation at the nucleotide level or through retrotransposition of a parent element that has already lost or gained the motif sequence. To help determine how the functional motifs within Alu and L1 affect their frequency within genes, we analysed a subset of functional motifs found in the respective consensus sequences . The binSince we have shown that, at the level of entire elements, L1 and Alu frequencies are correlated with relative location within a gene, we examined the dependency of individual motifs on the relative location within the gene. To reduce the number of statistical tests (and hence required corrections), we restricted analysis to the bins that contained the extremes of Alu and L1 frequency. These were the first and last bins within the linear range of Alu/L1 frequency, which contained the most and least Alu and L1 respectively. Motif frequencies were normalized by the count of ASO or SO elements within the bin and then compared between bins . ContrarTo account for differences between gene groups we further classified subsets of genes into functional groups  . The relOverall, the frequencies of motifs found within L1 followed slightly different patterns. Motifs within L1 were significantly depleted within genes compared to motifs within Alu (t-test: p<1.3e-05) . They diThese results indicate that not only are whole RT elements non-randomly distributed within genes, but also functional motifs within Alu and L1 are further depleted. Furthermore, this depletion is sensitive to orientation and location of the retrotransposon within the gene as well as the tissue specificity of the gene itself.The human L1 consensus sequence is approximately 6 kb, although it varies by subfamily. During retrotransposition L1 are often truncated, resulting in an average size of 900 bp To test whether the linear decay of antisense oriented L1 was related to the size of the element, we calculated the proportions of L1<1 kb, 1–2 kb, 3–4 kb, and 5 kb+ versus expectation based on all L1. Fischer's exact test p-values were calculated between the count of observed size-specific L1 and the expectation within bins 1 and 2 since these bins had the largest effects of antisense bias. We found that full-length L1 (5 kb+) were greatly reduced in frequency near the 5′ region of the gene compared to expectation . This waPutku et al. have previously linked the presence of polymorphic RTs to differential gene expression of the WNK1 gene These results highlight the presence of predictive relationships between retrotransposons and the genes in which they reside. Primarily, we identified a linear decay of ASO RTs across the length of a gene that was correlated with exon density. The failure to observe a similar decay in polymorphic elements suggests this distribution is not present at the initial time of insertion and instead developed as a result of selective pressure relatively soon after insertion. The extent of antisense bias (ASO/SO), independent of gene decile, remained constant over time although the overall frequencies of RTs in genes steadily increased with increasing age. This is supported by previous cell line work which has shown that Alu may exhibit preferential insertion in the antisense orientation within a gene One factor strongly affecting the linear decay of ASO L1 retrotransposons is the length of the element. L1 that were truncated to <1 kb were substantially increased in frequency near the 5′-UTR when compared to full length L1. Although we postulate here that this was likely due to the size of the L1, it is also possible that the presence or absence of the bidirectional Pol-II promoter plays a role in maintenance of L1 near the 5′-UTR. Our analyses here cannot readily disentangle the effects of length, promoter presence/absence, and presence of motifs along the length of the element. Additional subfamily-specific analyses, adjusting for variability in promoter positions among L1 consensus sequences, will be required to tease apart these variables. Ideally, such analyses would be accompanied by experimental data examining the impact of various L1 insertion scenarios.Contrary to previous findings, RTs near 5′-UTRs were reduced in frequency, most significantly for L1 in the antisense orientation. Our analysis was capable of distinguishing this trend by removing the confounding effect of low exon density that is commonly present near 5′UTRs. Since the linearly decreasing antisense bias is highly correlated with CDS exons, this indicates that the commonly reported increase in antisense oriented RTs near the 5′-UTR is more likely due to a lack of CDS exons as opposed to a function of the UTR itself, while the overall reduction in elements in either orientation is attributable to the UTR itself.As previously reported, we observed a relative increase of RTs within genes over time. We consider it most likely that the increase in relative frequency of subfamilies within genes compared to intergenic is not a result of population fixation within genes, but, rather, a decrease of element numbers within intergenic regions. This could be explained by the relaxed purifying selection against deletion events within intergenic regions compared to genic One of the primary results of this study indicated that the bias towards antisense RTs near the 5′ UTR and the decay of bias toward the 3′ region of the gene occurred relatively soon after insertion, while the elements remained polymorphic within the human population. This was evidenced by the observation that the linear decay from 5′ to 3′ of the gene was not evident from more recently inserted, polymorphic elements. As noted above, we did not, however, detect a difference in the overall genic ASO/SO ratio between the polymorphic and non-polymorphic elements. This discrepancy could be attributed to the fact that the change in slope of decay was a more sensitive measurement than overall bias across the gene. If this explanation is correct, additional polymorphic element data from population screening should ultimately allow for the overall ASO/SO genic bias to be detectable. Based on our results the selective bias towards a linear decay of antisense orientation appears to be separate from the antisense bias introduced during the insertion process itself.One possible avenue, through which polymorphic elements may be influencing fitness, is through acting as eQTLs. It remains to be determined, however, if the elements in this case are directly impacting expression levels or simply tagging another functional variant in linkage disequilibrium with the element. The trends in RT distribution observed here may be helpful in predicting the effects of RTs insertions on a co-localized gene. Factors identified to be important in RT distribution were RT type, orientation with respect to the gene, proximity to a CDS, proximity to a 5′-UTR, and tissue specificity. While the biological mechanisms underlying these observations remain undetermined, it is plausible that the juxtaposition of ASO elements near intron-exon junctions interferes with splicing regulation Retrotransposon (RepeatMasker) coordinates were downloaded from the UCSC genome browser . Polymorphic Alu were taken from the supplemental data in the original publication Gene (RefSeq flat file track), and Retrotransposon (RepeatMasker) coordinates were downloaded from the UCSC genome browser . Each gene was subdivided into ten bins of equal size where bin1 began at the transcription start and decile 10 ended at the transcription stop. This method of binning resulted in a concentration of exons in the first and last bins. Further calculations took this bias into account. Repeats analyses were limited to recent inserts only where mentioned.L1 were ranked first by subfamily group by the average divergence from their respective consensus sequences, as calculated in 5′-UTR coordinates were selectively downloaded from the UCSC genome browser RefSeq Gene track. These coordinates were intersected with gene deciles to determine which 5′-UTRs were within gene boundaries. Genes were then grouped depending on which decile the 5′-UTR was located within.Counts of elements within each decile were tabulated using an in-house script that intersected gene locations with RepeatMasker annotations to tabulate strand orientation counts per decile. Frequencies of elements within deciles were defined as the count of elements in a decile  per total elements . For SO-only or ASO-only plots, subfamily counts were normalized by the intergenic frequencies of the respective subfamilies in order to account for variable amplification rates. Trend lines were obtained via linear regression. For the analysis of L1 and Alu maintenance in genes as a function of rank by milliDiv; milliDiv was calculated as the average base mismatches in parts per thousand, as tabulated in RepeatMasker . A minimum sample size for comparison between subfamilies was calculated using G*Power3 To correct for differences in genomic coverage within genes and intergenically, retrotransposon frequencies were calculated as a factor of their total length as opposed to count. Coverage in bp was tabulated for each element and summed per subfamily. Alu subfamilies were ranked according to age as per presence in humans.The values within genes were then divided by the total bp covered by similar elements intergenically. All values were normalized by the total bp in the queried region.Gene categories were grouped based on the calculations in To reduce multiple testing we restricted motif analysis to sequences that have been previously identified in Alu or L1. Exact matches, unless otherwise stated, were searched in the necessary region  using an in-house program. Counts of each motif were tabulated in each region and, where necessary, were normalized by the relative element density. Truncated L1 were identified by comparing every known L1  versus known L1 consensus sequences using a local BLAST alignment. The start position of each L1 was denoted as the start position within its respective consensus sequence.Polymorphic retrotransposons were filtered from the dataset in Dataset S1List of Polymorphic Retrotransposons and Human-Specific Retrotransposons adapted from and .(XLSX)Click here for additional data file.Dataset S2Ranked list of L1 and Alu subfamilies used in this study.(XLSX)Click here for additional data file.


The aim of the research article is to develop a representative volume element using finite elements to study the buckling stability of graphene/polymer nanocomposites. Research work exploring the full potential of graphene as filler for nanocomposites is limited in part due to the complex processes associated with the mixing of graphene in polymer. To overcome some of these issues, a multiscale modeling technique has been proposed in this numerical work. Graphene was herein modeled in the atomistic scale, whereas the polymer deformation was analyzed as a continuum. Separate representative volume element models were developed for investigating buckling in neat polymer and graphene/polymer nanocomposites. Significant improvements in buckling strength were observed under applied compressive loading when compared with the buckling stability of neat polymer. In the recent past, graphene has emerged as a potential candidate for developing nanocomposites with improved properties ,2. The eBuckling in isolated graphene sheets was modeled by several researchers -11. HoweIt is well established that the reinforcement of polymer with graphene increases the elastic modulus of the material which further improves buckling stability. The aim of this study is to propose a numerical model which can estimate the increase in buckling stability with different volume fractions of graphene and can further be extended to complex shapes and structures.It has been reported that achieving a uniform dispersion of two-dimensional graphene sheets in polymer is more challenging compared to the mixing of one-dimensional CNT. Moreover, the application of nanocomposites is not limited to simple structures, and the comprehension of material behavior in complex structures is restricted when employing experimental and analytical methods. Consequently, research efforts are increasingly focused on numerical approaches. To overcome some of the limitations that exist in experimental and analytical work, a multiscale representative volume element (RVE) is proposed in this paper to investigate buckling phenomena in graphene/polymer nanocomposites under the assumption that graphene is uniformly distributed in the polymer. To the knowledge of the present authors, no numerical model has been reported yet to study the effect of graphene on the buckling strength of nanocomposites. In the proposed technique, graphene was modeled in the atomistic scale, whereas polymer deformation was analyzed as a continuum.In this paper a finite element technique was employed in conjunction with molecular and continuum mechanics to simulate buckling in graphene/polymer nanocomposites. In the proposed RVE, the polymer, epoxy in this case, was modeled as a continuum phase whereas the deformation in graphene was evaluated using an atomistic description. Nonbonded interactions were considered as the load transfer mechanism or interphase between polymer and graphene. FE modeling was performed in the ANSYS (Version 13) software environment .To model graphene in the proposed RVE, it was assumed that graphene behaves like a space frame structure in which the covalent bonding between C-C atoms constitutes the load-bearing element while atoms act like a joint. A molecular-mechanics-based approach was employed to estimate properties for graphene in the atomistic scale. In the molecular form, potential energy of graphene can be represented asVbonded represents the bonded interaction between atoms and is considered primarily responsible for maintaining the structural integrity. The potential energy term ‘Vnonbonded’ includes electrostatic forces, e.g., van der Waals forces, which are considered weak in nature and can generally be neglected in comparison to bonded interactions. Li and Chou  when the structure is subjected to the reference unit load PR. In Equation 7 the total stiffness matrix [K0] is the assembled global stiffness matrix for the multiscale structure composed of beam, truss, and solid elements. Conventional assembly techniques can be used to obtain the global stiffness matrix.The pre-buckling analysis was performed in the finite element environment to compute the reference stresses Equation 8 represents the general solution that is obtained in the finite element analysis after solving the assembled matrices defined in Equation 7.Q*} obtained from Equation 8 are further post-processed to compute strains and corresponding stress values S* within each element using Equation 9, in which matrix [D] represents the elasticity matrix  while matrix [B] is a function of approximating polynomial or shape functions.The nodal displacement vectors {λ and v are the load factor and the eigenvector of displacements. The geometric stiffness matrix KG used in Equation 10 is defined with the help of Equation 11, where Γ is composed of derivatives of shape functions and S is a function of the stress S* estimated in the pre-buckling analysis.The post-buckling analysis corresponds to a general eigenvalue problem as defined by Equation 10, in which λmin can be determined, which is then employed to estimate the buckling force given by Equation 12.By solving Equation 10, the lowest possible value for the load factor The proposed RVE was employed to understand the buckling behavior of graphene in a polymer matrix when graphene was assumed to be uniformly distributed. A second separate RVE structure with equal dimensions was developed using finite element modeling to study the buckling in neat (homogenous) polymer.E) were then used in defining the homogenous rectangular plate with dimensions of the proposed RVE model. The buckling loads were calculated for this homogenous rectangular plate shown in Figure To validate the results of the proposed RVE, an alternative method (indirect method) was also employed in this paper. The proposed RVE model with boundary conditions as shown in Figure Px is the applied unidirectional compressive load, w is the displacement in the outward normal direction, and D is flexural rigidity . Boundary conditions were kept identical in all RVE models. Only the value of flexure rigidity was varied, which afforded the proposed RVE model the capability of yielding E for different graphene volume fractions.The analytical formulation to estimate the buckling load for a rectangular plate is given in Equation 13, where The boundary conditions along with dimensions for the proposed RVE model are shown in Figure The results obtained from the developed RVE structures were plotted in Figure In this study a representative volume element method was successfully employed to investigate the buckling phenomenon in graphene/polymer nanocomposites, where graphene was assumed to be uniformly distributed. Graphene was modeled in the atomistic scale and polymer as a continuum. A significantly enhanced buckling strength of graphene reinforced polymers was observed as compared to neat polymer, i.e., buckling strength of graphene/polymer nanocomposite improved by 26% with only 6% filler volume fraction.The authors declare that they have no competing interests.Both authors made equally valuable contributions to this paper. Both authors read and approved the final manuscript.


RH) and temperature error magnitudes of at most 2% RH and 0.3 °C respectively, and similar measurement time response, would be an adequate and less expensive substitute for a dewpoint hygrometer. Furthermore, a combination capacitive humidity instrument requires no servicing compared to a dewpoint hygrometer which requires a bias adjustment and mirror cleaning each week. These findings make unattended BREB measurements of sensible heat flux and evaporation cheaper and more reliable with the system easier to assemble and service and with reduced instrument power.The possibility of reliable, reasonably accurate and relatively inexpensive estimates of sensible heat and latent energy fluxes was investigated using a commercial combination thin-film polymer capacitive relative humidity and adjacent temperature sensor instrument. Long-term and unattended water vapour pressure profile difference measurements using low-power combination instruments were compared with those from a cooled dewpoint mirror hygrometer, the latter often used with Bowen ratio energy balance (BREB) systems. An error analysis, based on instrument relative humidity and temperature errors, was applied for various capacitive humidity instrument models. The main disadvantage of a combination capacitive humidity instrument is that two measurements, relative humidity and temperature, are required for estimation of water vapour pressure as opposed to one for a dewpoint hygrometer. In a laboratory experiment using an automated procedure, water vapour pressure differences generated using a reference dewpoint generator were measured using a commercial model (Dew-10) dewpoint hygrometer and a combination capacitive humidity instrument. The laboratory measurement comparisons showed that, potentially, an inexpensive model combination capacitive humidity instrument (CS500 or HMP50), or for improved results a slightly more expensive model (HMP35C or HMP45C), could substitute for the more expensive dewpoint hygrometer. In a field study, in a mesic grassland, the water vapour pressure measurement noise for the combination capacitive humidity instruments was greater than that for the dewpoint hygrometer. The average water vapour pressure profile difference measured using a HMP45C was highly correlated with that from a dewpoint hygrometer with a slope less than unity. Water vapour pressure measurements using the capacitive humidity instruments were not as accurate, compared to those obtained using a dewpoint hygrometer, but the resolution magnitudes for the profile difference measurements were less than the minimum of 0.01 kPa required for BREB measurements when averaged over 20 min. Furthermore, the longer-term capacitive humidity measurements are more reliable and not dependent on a sensor bias adjustment as is the case for the dewpoint hygrometer. A field comparison of CS500 and HMP45C profile water vapour pressure differences yielded a slope of close to unity. However, the CS500 exhibited more variable water vapour pressure measurements mainly due to its increased variation in temperature measurements compared to the HMP45C. Comparisons between 20-min BREB sensible heat fluxes obtained using a HMP45C and a dewpoint hygrometer yielded a slope of almost unity. BREB sensible heat fluxes measured using a HMP45C were reasonably well correlated with those obtained using a surface-layer scintillometer and eddy covariance (slope of 0.9629 and 0.9198 respectively). This reasonable agreement showed that a combination capacitive humidity instrument, with similar relative humidity ( Theoretical aspects of the BREB method in relation to data exclusion have been examined –3. The mCellier and Olioso  concludede facto standard for flux measurements, instrument cost and a greater power requirement may prohibit simultaneous measurements within the same or in different catchments over different canopy types or for simultaneous flux measurements above areas subjected to different management. Furthermore, EC measurements require many corrections [While the EC method is the rections  and therrections  with difrections . The amoBREB automatic height exchange  and fixeBased on previous experiences and personal communications with other researchers, the dewpoint hygrometer of the BREB system used for long-term field measurements may after some time yield unreliable data due to the sensor's inability to maintain a stable bias. Given other practical problems such as servicing and power requirements for dewpoint hygrometer systems employed in distant locations, the objective of this work was to investigate the use of a BREB system, by substituting the dewpoint hygrometer with a combination thin-film polymer capacitive relative humidity and air temperature instrument, for long-term and unattended estimation of sensible heat and latent energy fluxes. An error analysis is presented for two different combination capacitive humidity instrument models, which are compared to a dewpoint hygrometer, in the laboratory and in a mesic grassland, for their ability to measure water vapour pressure profile differences for an extended period. Furthermore, to test the adequacy of the combination capacitive humidity instruments used in the field for flux measurements, comparisons of BREB-measured sensible heat flux against that obtained using the path-weighting surface-layer scintillometer (SLS) method, dependent on Monin-Obukhov Similarity Theory (MOST), and the standard EC method are presented. Such comparisons have not been presented previously in the literature.2.2.1.A brief description of the BREB method for measuring sensible and latent energy flux is presented. The BREB method relies on the K-theory hypothesis for which sensible heat flux, for example, is proportional to an exchange coefficient for sensible heat flux and the profile air temperature gradient.LE (W m−2) is given by:ρ is the density of air (kg m−3), pc the specific heat capacity of air at constant pressure (J kg−1 K−1), γ the psychrometric constant (approximately 0.066 kPa K−1 at sea level), wK the exchange coefficient (m2 s−1) for latent energy flux, and z2 and z1 above the soil surface respectively where Bowen , buildineen used .H is given by:hK is the exchange coefficient for sensible heat flux (m2 s−1) and z2 and z1 above the soil surface respectively where H and LE fluxes are indicative of energy losses at the surface and are negative.Similarly, the sensible heat flux β is defined as:β can be determined by measuring air temperature and water vapour pressure at two levels in the atmosphere. Ignoring photosynthesis, advection and physically and biochemically stored fluxes, the surface energy balance is given by:nR is the net irradiance and S the soil heat flux. Combining wK = hK. The equality of these two exchange coefficients, referred to as the Similarity Principle, allows the BREB method to be used without the need for atmospheric stability adjustments. A limitation of the BREB method occurs when the denominator of et al. [Following Bowen , the rativen by:Rn=−LE−H−S)/(1+β)H=−β where the % unit is in RH units and not a percentage of the measured RH (for RH between 0 and 90%) and temperature accuracy is within 0.2 °C to 20 °C , for the HMP45C is given by:T ≥ 20 °Cfor T ≤ 20 °Cand for T≥ 0 °C is given by:and that for a CS500 for The HMP45C  combinatto 20 °C . For theδT| for the CS500 varies linearly from 0.3 to 0.62 °C respectively compared to 0.3 °C for the HMP45C for the same temperature range but with a minimum for |δT| of 0.2 °C at 20 °C. Therefore, the HMP45C has a more accurate temperature sensor than that used in the CS500.These relationships are depicted in e (kPa), is calculated from the measured capacitive sensor RH and the saturation water vapour pressure se (kPa) calculated from the adjacent temperature sensor using:T (°C) is the sensor-measured airstream temperature. Hence the error magnitude in water vapour pressure, δe (kPa), is estimated from the error in RH, δRH, and error in se, sδe (kPa), using:∂e/∂RH = se/100 and ∂e/s∂e = RH/100 and where s∂e = (s∂e/∂T)·δT = Δ·δT where Δ (kPa °C−1) is the slope of the saturation water vapour pressure vs temperature relationship at temperature T:The water vapour pressure of an airstream, δe for any relative humidity and temperature combination sensor is given by:Finally then, from se and δRH, is usually much greater than the second term except when temperature is low and RH is high. Thus the error δe, mainly governed by δRH, increases with temperature. The error δe is temperature dependent since both se and Δ are temperature dependent and the information provided by the manufacturer shows that δT is also temperature dependent. For the HMP45C, combining T ≥ 20 °C or for T < 20 °CT ≥ 0 °C.In δRH = ±2% (for 0% ≤ RH ≤ 90%) and for the CS500 δRH = ±3% (for 10% ≥ RH ≥ 90%), |δe| was determined as a function of temperature between 5 and 50 °C and RH between 10 and 90 %. The calculations show : δRH = ±2 % and δT = ±0.4 °C . The RH and T errors for the CS215 are similar to those for the Campbell HMP35C combination instrument  except that the CS215 contains a single-chip element that includes both relative humidity and temperature sensors. The Campbell 155A combination instrument is based on a Vaisala model 180 Humicap capacitive humidity sensor and a platinum resistance thermometer. Measurement errors are specified in δe| values for the CS215 are marginally greater than those for the HMP45C  with an altitude of 671.3 m. The long-term annual rainfall for Pietermaritzburg is 928 mm (58 years of data) and the mean maximum and mean minimum air temperatures are 24.8 and 12.3 °C respectively (South African Weather Service). During winter, there was frequent dewfall and occasional frost. Management practices at the research site included mowing  and burning (in August when firebreaks are established and in October).3.3.BREB field measurements were conducted from January to July 2004. Water vapour pressure profile difference comparisons were made using a Dew-10 dewpoint hygrometer and two combination capacitive humidity instruments, the HMP45C and CS500. The minimum fetch distance for the site for the prevailing wind direction was 135 m for the BREB systems. The mixed grassland area to the south of the site was more exposed with a slight slope increase. The adjacent area had occasional trees. To the north of the study site, there was a residential area and occasional tall trees.−1. Air continuously sampled from each height was drawn to the respective mixing bottle housed in the instrument enclosure and then to the chamber containing either the Dew-10 or combination humidity instrument.BREB systems, modified from Campbell 023A systems  and connEach capacitive humidity instrument was sealed in a metal chamber. Unlike the dewpoint hygrometer chamber, the capacitive humidity chambers were heavily insulated using layers of mirror tape to dampen sudden changes in temperature. Tubing in the vicinity of the chamber was covered with adhesive aluminium tape in an attempt to further reduce temperature differences between the humidity and temperature sensors and that of the airstream. The Bev-a-line tubing  used for the water vapour pressure measurements was as short as possible to minimise the transit time of the air from the intake point to the instrument. Furthermore, the filter cover of the combination capacitive humidity instruments was removed to improve the time response to relative humidity. The inlet Teflon filter (1-μm pore size) at each of the two intake positions was however retained for both BREB capacitive humidity systems to prevent entry of liquid water and dirt. The voltages corresponding to the HMP45C relative humidity and air temperature were differential. The corresponding CS500 voltages were single-ended, as recommended by the manufacturer. Every 1 s, the airstream atmospheric water vapour pressure was calculated from the measured relative humidity and temperature. Averages and standard deviations were calculated every 20 min.In the first field experiment, the HMP45C and CS500 instruments were placed in individual humidity chambers. The outlet of the one chamber was connected to the inlet of the other. The inlet of the first chamber was connected to the inlet airstream from the mixing bottles and the outlet of the second bottle was connected to the inlet of the pump. The outlet of the pump was vented to the atmosphere. In this fashion, the two instruments were exposed to the same water vapour pressure.In a second experiment, measurements were made using two chambers connected in-line with the one chamber containing a HMP45C and the second chamber containing a Dew-10 dewpoint hygrometer. Most of the measurements were with the inlet air passing through the HMP45C chamber first and then entering the dewpoint hygrometer chamber and then out to the pump. This arrangement would reduce the influence of the dewpoint hygrometer measurement method on the HMP45C measurements. The dewpoint hygrometer required a weekly bias adjustment for convergence on the correct dewpoint. The capacitive humidity instruments required no attention.The air temperature differences between two levels were measured using a pair of naturally-ventilated and unshielded 75-μm type-E thermocouples. At each level, a parallel combination of 75-μm thermocouples was used. Extra insulation was used to cover the thermocouple connectors at the thermocouple join. Extra precautions were taken by covering and thermally insulating the point at which the thermocouple wires were connected to the datalogger. The thermocouples were inspected for damage, cleanliness, insects and cobwebs during each site visit and replaced or cleaned.H and LE  have been presented [H every 20 min by application of the semi-empirical MOST. A beam distance of 101 m was used. The SLS was positioned at a height of 1.68 metres above the soil surface. The SLS signal processing unit was connected to a computer via the computer serial port. Vegetation height, weighted over the beam path length [The methodology and details for the dual-beam SLS measurements  for calculating sensible heat flux . The sonic anemometer measurements were processed on-line. No coordinate rotations [w and sonicT, for determining H, was calculated using A three-dimensional sonic anemometer  was used as an EC system to measure otations  were pos4.Before the results of the flux measurements for the various field methods could be compared, a study was undertaken to investigate the possible substitution of the dewpoint hygrometer by a combination capacitive humidity instrument.4.1.de) were simulated using a LI610 dewpoint generator. The de values generated were compared against measurements in three separate experiments using a Dew-10 dewpoint hygrometer and HMP35C and CS500 combination capacitive humidity instruments. Judging by the root mean square error (RMSE) for the three measurement comparisons . Presumably, for aerodynamically rougher surfaces such as tree canopies, the magnitude of de and dT would decrease and therefore the separation distance between the upper and lower arms may need to be increased. This increase may require a greater fetch, limiting use of the BREB method if this is not possible.The measured  1.0 kPa . Measurede for the HMP45C and CS500 instruments were also good  for high evaporative demand conditions (vs 0.0009 kPa respectively) for the low evaporative demand conditions  the saturated water vapour corresponding to the sensor temperature sensorT. Therefore, assuming that sensorRH is unchanged, an increase in sensorT results in a non-linear increase in se(sensorT) causing an increase in airstreame. Similarly, a decrease in sensorT results in a decrease in se(sensorT) causing a decrease in airstreame.The variation in HMP45C-measured temperature in the chamber is shown for a 20- and 2-min period  for the The step-like variation in temperature , 7a is dThe water vapour pressure measurements of the CS500 are more variable than those obtained using the Dew-10 dewpoint hygrometer or the HMP45C (data not shown). The reason for this could be that the CS500 temperature sensor is less accurate than that used for the HMP45C . For exaWith the procedures specified and based on the data collected, it would appear that the HMP45C may be used as a substitute for the dewpoint hygrometer. Field measurements of water vapour pressure profile differences, with a resolution magnitude of at least 0.01 kPa, were possible using a capacitive humidity instrument .An advantage of the HMP45C is that it does have a switch control option to turn the instrument on, using datalogger control, just before measurements and then immediately off. The CS500 does not have a built-in switch control option and this results in an increased current drain if the instrument is continuously powered. This disadvantage for the CS500 is obviated when using a CR10X, CR23X, CR800 or CR1000 that can provide for switched 12 V control. However, the HMP50  is cheaper than the HMP45C and the Dew-10 .4.3.H obtained using capacitive humidity instruments and a Dew-10 dewpoint hygrometer is shown (A relatively long-term measurement comparison between is shown . As expeis shown . The comis shown . The HMPH measured using the BREB method using the HMP45C and the SLS (−2 and a slope value of less than 1. Of note is that the BREB estimate of H requires netR and S measurements (H. Besides measurement errors, measurement differences for the different methods are due to the differing theoretical bases for the methods, the spatial separation of the measurement systems, differing measurement heights, differing footprints and that the SLS measurements represent a path-weighted estimate [The comparison between  the SLS  and EC m the SLS  are reasurements  whereas estimate ,34 as weH for the EC and BREB methods, for days ranging from wet and overcast to cloudless conditions, also show reasonable correspondence. For some days (101 and 102 and 104 to 106, H compared to EC whereas on other days (32 and 34, H. During the night-time, usually when dew occurs, fluctuations occur in the data particularly for the BREB method (day of year 31 to 35, Temporal plots of H and LE, it is recommended that for water vapour pressure measurements, either the HMP45C be used, or a combination polymer capacitive instrument with similar or better accuracy for both RH and T. Alternatively, the CS500 or HMP50 could be used but with a more accurate temperature sensor.For BREB calculations of 5.RH and T respectively, yielded less accurate water vapour pressure differences with more measurement noise and an increased RMSE of 34.89 W m−2 in the sensible heat flux comparisons obtained using a dewpoint hygrometer compared to a RMSE of 8.06 W m−2 using the HMP45C. Modifications to the combination capacitive humidity instrument included removal of the instrument membrane filter and providing a more thermally stable environment for the combination instrument chamber and adjacent connecting hoses. This substitution makes the BREB system much cheaper with little servicing required by the capacitive humidity instrument compared to the dewpoint hygrometer used. The combination capacitive humidity instruments show more water vapour pressure noise than the dewpoint hygrometer but the 20-min average of the profile water vapour pressure differences compare well with the corresponding dewpoint hygrometer measurements. Further modifications could include increasing the equilibration time from 40 s to 60 s and using a thermocouple to measure the temperature adjacent the capacitive humidity sensor or using a single-chip relative humidity and temperature capacitive instrument placed in a chamber. Sensible heat flux obtained using a HMP45C instrument compared to either surface-layer scintillometer or eddy covariance estimates yielded a RMSE of about 38 W m−2 and a slope less than 1.Based on the laboratory and field experimental measurements, a combination polymer capacitive humidity instrument such as the HMP45C with a relative humidity magnitude accuracy of 2% or better and a temperature accuracy of at most 0.3 °C, perhaps 0.4 °C, is a reasonable substitute for the cooled mirror dewpoint hygrometer of a BREB system for measuring water vapour pressure profile differences. A less accurate combination capacitive humidity instrument, the CS500 or HMP50, with an accuracy of 3% and 0.4 °C for


Diagnostic and treatment approaches for sarcoidosis have changed dramatically over the past decade. Yet, the most recent reports of trends in hospitalizations of sarcoidosis patients are over ten years old. The objectives of this study were to determine the incidence of sarcoidosis among hospitalized patients and to analyze recent trends and seasonality of hospitalizations in sarcoidosis patients.We performed a retrospective cohort study of the Nationwide Inpatient Sample from 1998 through 2008. We identified all hospitalizations with a primary or secondary diagnosis of sarcoidosis (ICD-9-CM code 135). Incidence was modeled as a seasonal time series about a linear trend.Time series analysis of the monthly number of hospitalizations revealed a distinct positive linear trend. Over the study period, the number of hospitalized patients with sarcoidosis increased from 37,516 to 70,947 cases. Trends were most pronounced in patients older than 55 years (p < 0.0001), African Americans (p < 0.0001), females (p = 0.0289), and non-Medicaid populations (p < 0.0001). Hospitalizations are seasonal with highest incidence in January through March.Hospitalizations among sarcoidosis patients have almost doubled during the past decade, with disproportionate rate increases in African Americans, women, and older patients. The rate also increases among patients with insurance other than Medicaid. This study indicates the need for heightened surveillance of sarcoidosis patients given the unknown consequences of evolving treatment approaches. Our results point to a need for research investigating risk factors for hospitalization, including medications, co-morbidities, demographics, and socioeconomic status. Sarcoidosis is a systemic granulomatous inflammatory disease of unknown etiology which can cause significant morbidity ,2. It isPrior studies focusing on hospitalization trends among patients with sarcoidosis report conflicting results. A study of U.S. Navy personnel showed a steep decline in hospitalizations for sarcoidosis over the time period of 1975 to 2001, with the authors concluding that this was due to improvements in protection from occupational exposures that may have been causing sarcoidosis, or reflecting improvement in a ‘sarcoid-like’ disease from these exposures .Since these studies were published, diagnostic and treatment approaches have changed dramatically. More sensitive imaging technologies to detect and monitor organ involvement are now available, and increased use of immunosuppressive alternatives to prednisone  has altered clinical management over recent years -13. DespThe goal of this study was to determine the incidence of sarcoidosis among hospitalized patients and to analyze the recent trend in hospitalizations among patients with sarcoidosis. We also examined demographics, hospital mortality, length of stay, and seasonal disease patterns.th Revision, Clinical Modification (ICD-9-CM) code 135. We then aggregated all cases of sarcoidosis by month to produce a national sample of cases of sarcoidosis over time. Cases of sarcoidosis were assigned to a calendar month on the basis of the date that the patient was admitted to the hospital. The numbers of sarcoidosis cases were further converted to incidence rates to account for the variation in total overall number of all hospitalized discharges in each month.All data were extracted from the Nationwide Inpatient Sample (NIS), the largest all-payer database of national hospital discharges in the United States. The purpose of the NIS is to specifically identify, track, and analyze national trends in healthcare utilization, access, charges, quality, and outcomes. It is maintained as part of the Healthcare Cost and Utilization Project by the Agency for Healthcare Research and Quality (AHRQ) and contains data from a 20 % stratified sample of nonfederal acute hospitals . This saThe yearly series for hospital mortality rate and average length of stay were also extracted to monitor the characteristics of sarcoidosis patients over time. For each year, the hospital mortality rate is defined as the number of deaths during hospitalization over the total number of sarcoidosis cases in that year.We performed a time series analysis of the sarcoidosis rate over the study period. Specifically, we built a seasonal autoregressive integrated moving average (SARIMA) model, which accounted for the serial correlation and allowed us to test for a potential trend and for seasonality in sarcoidosis incidence. After obtaining a suitable model for the national data, we then applied the same model structure to each sub-series stratified by the dichotomous variables age (< 55 vs. > = 55), race (black vs. non-black), gender , and payer (Medicaid vs. non-Medicaid). We chose 55 years old as an age cut-off in order to capture the second bimodal peak of presentation of sarcoidosis patients in their 60’s that has been previously noted in epidemiological studies . In a siTo determine if there was a trend in hospital mortality rate, we fit a logistic regression model with the year as the independent variable. Generalized estimating equations were used for parameter estimation and an autoregressive correlation structure was incorporated to account for the temporal association in the outcome. To investigate whether there was a trend in average length of stay, we fit an analogous linear regression model, assuming normality for the distribution of the outcome.All statistical analyses were performed using R version 2.11.1  and SAS 9.2 .Over the study period, the estimated number of hospitalized patients with sarcoidosis almost doubled from 37,516 cases in 1998 to 70,947 cases in 2008. Our time series analysis of the sarcoidosis incidence rate revealed a significant upward trend  remained in the final model, but the first-order seasonal moving average (p = 0.3195) and linear time trend p = 0.7259) were no longer significant  and patients with a primary diagnosis of sarcoidosis . A significant decrease in average length of stay was also observed for general sarcoidosis population , but not for patients with a primary diagnosis of sarcoidosis .Our results show a dramatic increase in the number of overall hospitalizations of sarcoidosis patients in the U.S. between 1998 and 2008. We found the highest increase in the rate of hospitalizations among African Americans, females, older patients, and those with insurance other than Medicaid. However, for those admitted with sarcoidosis listed as the primary diagnosis, the incidence was stable during the study period with no significant differences in trends between races, gender, or insurance status. Thus, it appears that this increasing trend is driven by admissions for people with sarcoidosis, rather than because of sarcoidosis.Although sarcoidosis was first described over 100 years ago, the cause is still unknown, treatment is still controversial, and epidemiologic data are difficult to collect due to the rarity of the disease and diverse clinical presentations. Our findings are important as there are few published data on the burden of hospitalizations in this disease in the past ten years. Our results clearly demonstrate that there is an increasing burden of inpatient medical care required by sarcoidosis patients, and that this trend is greater in older patients, women, and African Americans.With respect to race, gender, and age, our results are similar to those published by Foreman et al who reported finding hospitalizations more frequently among females, older patients, and African Americans . SarcoidIn contrast to previous work, however, we found that patients with insurance other than Medicaid actually had a higher increase in the rate of hospitalization compared to Medicaid patients. We speculate that this difference may be due to differential practice patterns or access to care/travel issues between patients with Medicaid and those with alternative insurance. For instance, those with insurance may be receiving more aggressive immunosuppression with corticosteroids, potentially more use of costly immunosuppressive alternatives to prednisone (with increased risky side effects), more diagnostic procedures, or may be able to more easily access care , thereby influencing treatment and monitoring decisions by practitioners. Advancing radiographic imaging technology has also made it easier to find potential sarcoidosis cases and identify more subtle lung findings, which may have in turn led to more aggressive treatment approaches than in times of monitoring only with chest x-rays. Thus, the use of more sensitive imaging in insured populations may contribute to more aggressive immunosuppression.Our hypothesis that the increase in hospitalizations is associated with more aggressive immunosuppressive treatments is further supported by the observation that hospitalizations with a primary sarcoidosis diagnosis are not increasing; increasing admission rates due to primary diagnoses other than sarcoidosis suggest that factors other than sarcoidosis itself are driving this trend. In addition, a recent retrospective study of healthcare utilization showed that sarcoidosis patients with greater corticosteroid use had a greater number of unscheduled emergency department visits . FurtherBecause other studies have suggested a seasonal peak of diagnosis of sarcoidosis cases in the early spring, we investigated the seasonality of hospital admissions among patients with a sarcoidosis diagnosis ,26. GeneThere are several limitations to our study. First, a major limitation is that we use that we use administrative data, and do not have access to laboratory, radiographic or medication data. Second, we cannot confirm our hypothesis that immunosuppressive drugs are driving the change in epidemiology, nor can the coded diagnosis of sarcoidosis be confirmed. We also cannot report the burden of repeated hospitalizations. Due to the sampling design in the AHRQ database, there are no individual identifiers that can link patients across multiple admissions or over multiple years. Third, it is possible that medical advances are sustaining patients for longer, leading to increased number of hospitalizations that are not necessarily related to the severity or treatment of sarcoidosis. Fourth, it is also possible that subgroups with limited access to outpatient care  may be admitted more frequently. Fifth, racial data in large databases may be missing or inaccurate. Last, it is possible that the increase in hospitalizations is due to an increase in the prevalence of sarcoidosis during the study period. However, hospitalizations specifically for a primary diagnosis have remained stable, suggesting that increased incidence of sarcoidosis is not the driving factor for hospitalization. The incidence in populations in the United Kingdom and Australia (both with similar incidence data to the United States) show that the diagnosis of sarcoidosis is not increasing over time ,28.This study highlights the need for heightened surveillance of sarcoidosis patients, as the requirement for inpatient care in this group is increasing. Our observations are important given the unknown implications of evolving diagnostic and treatment approaches over this period of time. Our results also point to a need for research investigating risk factors for hospitalization, including the effects of medications, co-morbidities, demographics, geographic patterns, socioeconomic status, and differing practice patterns.AHRQ: Agency for healthcare research and quality; ACF: Autocorrelation function; ICD-9-CM: International classification of diseases, 9th revision, clinical modification; SARIMA: Seasonal autoregressive integrated moving average; U.S.: United States.The authors declare that they have no financial or non-financial competing interests.AKG, the guarantor of the manuscript, contributed to the design of the study, was responsible for data collection and analyses, and contributed to the writing and revising of the first draft of the manuscript. MY was responsible for data analyses, and contributed to the writing and revising of the manuscript. FT was responsible for data analyses, and contributed to revising the manuscript. JEC contributed to the design of the study, data analyses, and to the writing and revising of the manuscript. PMP contributed to the design of the study, data collection and analyses, and to the writing and revising of the first draft of the manuscript. All authors read and approved the final manuscript.This work was supported by the National Institutes of Health, Grant 1KL2RR024980: Institute for Clinical and Translational Science, University of Iowa.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/12/19/prepub


G93A ALS mouse model and evaluated the impact of this mutation on motoneuron death, muscle weakness and disease progression. We found that TrkB.T1 deletion significantly slowed the onset of motor neuron degeneration. Moreover, it delayed the development of muscle weakness by 33 days. Although the life span of the animals was not affected we observed an overall improvement in the neurological score at the late stage of the disease. To investigate the effectiveness of strategies aimed at bypassing the TrkB.T1 limit to BDNF signaling we treated SOD1 mutant mice with the adenosine A2A receptor agonist CGS21680, which can activate motoneuron TrkB receptor signaling independent of neurotrophins. We found that CGS21680 treatment slowed the onset of motor neuron degeneration and muscle weakness similarly to TrkB.T1 removal. Together, our data provide evidence that endogenous TrkB.T1 limits motoneuron responsiveness to BDNF in vivo and suggest that new strategies such as Trk receptor transactivation may be used for therapeutic intervention in ALS or other neurodegenerative disorders.Brain Derived Neurotrophic Factor (BDNF) exerts strong pro-survival effects on developing and injured motoneurons. However, in clinical trials, BDNF has failed to benefit patients with amyotrophic lateral sclerosis (ALS). To date, the cause of this failure remains unclear. Motoneurons express the TrkB kinase receptor but also high levels of the truncated TrkB.T1 receptor isoform. Thus, we investigated whether the presence of this receptor may affect the response of diseased motoneurons to endogenous BDNF. We deleted TrkB.T1 in the hSOD1 Neurotrophins are potent regulators of survival and function of mammalian nervous system neurons Although TrkB or BDNF are not required for motoneuron survival during development, BDNF/TrkB kinase signaling is required for maintenance of the cholinergic phenotype of adult motor neurons and prevents embryonic and postnatal motoneuron cell death in a variety of experimental paradigms  mice were acquired from The Jackson Laboratory  and crossed to TrkB.T1 knockout mice backcrossed on C57/B16J background for at least 10 generations SOD1th section  was collected for analysis. Motoneurons were visualized by immunofluorescent staining for Choline acetyltransferase  and by Cresyl Violet staining and counted by the stereological modified optical fractionator method multiplying the number of counted neurons per section with the sampling fraction.Mice were anesthetized and perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde. The lumbar region of the spinal cord (L1–L5) was removed and 50-µm transverse sections were cut on a cryostat. Every 10Muscle strength and coordination were assessed by an accelerating rota-rod  that rotates from 4 to 40 RPM over 5 min To monitor the progression of the disease we used two different parameters: early progression of disease and end-stage disease in the animals. Starting at 8 weeks of age, animals were weighed three times weekly. Given that animals start losing weight with disease progression, we defined the early progression of disease as the duration between the time of peak weight and the time when animals lose 10% of peak weight due to muscle atrophy. End-stage was defined as the age of actual death of the animal or the age at which paralysis rendered the animal unable to right itself within 20 seconds when placed on its side Animals were scored for disease signs and symptoms at 20 weeks based on the following scale 0 =  normal functions, no sign of disease onset.1 =  distal muscle (paw) weakness of one limb.2 =  distal muscle weakness of two limbs, slight wobble when walking.3 =  weakness of proximal muscle (leg) of one limb plus weakness in two distal muscles, clear wobble when walking.4 =  both hind limbs paralyzed with dragging of hind limbs.5 =  animal is immobile.The adenosine A2A receptor agonist CGS21680 was purchased from Tocris (USA) or custom synthesized from ChemieTek . CGS21680 was dissolved in 5% DMSO prior to use. Starting at 8 weeks of age animals received a single daily intraperitoneal injection of CGS21680 or vehicle (5% DMSO) and were sacrificed at different time points depending on the experiments. The dose of CGS21680 (5mg/kg) was determined on the basis of our pilot study and other studies GraphPad software (InStat and Prism) was used for all statistical analysis. Comparisons of motor neuron numbers and rota-rod performance between the groups at different ages was done by ANOVA followed by post-hoc Tukey-Kramer Multiple comparison test or Dunn test. Survival analysis  was subjected to Kaplan-Meier Statistics. A Log rank P value was calculated to compare the survival curves. Neurological scores were compared by Mann-Whitney test. Data are represented as Mean ± SEM.


The bone and cartilage destruction seen inrheumatoid arthritis (RA) is caused by synovial pannus formation, which is characterized by aberrant proliferation of synovial fibroblasts. Inhibition of synovial proliferation has recently been reported to be a promising therapeutic strategy for RA.However, the specific mechanism underlyingdysregulated proliferation of synovial fibroblasts remains unclear.We aimed toidentify and characterize genesthat are involved in the aberrant proliferation of synovial fibroblasts.Microarray analysiswas performed to identifythe genes that had upregulated expression inmice with collagen-induced arthritis (CIA). The effect of candidate genes on the proliferation of synovial fibroblasts was screened using antisense oligodeoxynucleotides and small interfering RNAs (siRNAs).SPACIA1/SAAL1 (synoviocyte proliferation-associated in CIA 1/serum amyloid A-like 1)that was associated with aberrant proliferation of synovial fibroblasts. Immunohistochemicalanalysis indicated that SPACIA1/SAAL1 was strongly expressed in the foot joints of mice with CIA and in the thickened synovial lining of the human RA synovium. Transfection of siRNA targeting SPACIA1/SAAL1into RA synovial fibroblastscould inhibit tumor necrosis factor (TNF)α-induced proliferation more effectively thanit could inhibit serum-induced proliferation.In addition,the antiproliferative effect of SPACIA1/SAAL1 siRNA was caused byinhibition of cell cycle progression and not by induction of apoptosis.We established transgenic (Tg) mice that overexpressed SPACIA1/SAAL1. These Tg mice did not spontaneously develop arthritis or cancer. However,inducing CIA causedgreatersynovial proliferation and worse diseasein Tg mice thanin wild-type mice.We identified a novel gene named SPACIA1/SAAL1 plays an important role in the aberrant proliferation of synovial fibroblasts under inflammatory conditions.


Tgr from 1600 to 2000°C). For all the considered growth temperatures, few layers of graphene (FLG) conformally covering the 100 to 200-nm wide terraces of the SiC surface have been observed by high-resolution cross-sectional transmission electron microscopy (HR-XTEM). Tapping mode atomic force microscopy (t-AFM) showed the formation of wrinkles with approx. 1 to 2 nm height and 10 to 20 nm width in the FLG film, as a result of the release of the compressive strain, which builds up in FLG during the sample cooling due to the thermal expansion coefficients mismatch between graphene and SiC. While for EG grown on on-axis 4H-SiC an isotropic mesh-like network of wrinkles interconnected into nodes is commonly reported, in the present case of a vicinal SiC surface, wrinkles are preferentially oriented in the direction perpendicular to the step edges of the SiC terraces. For each Tgr, the number of graphene layers was determined on very small sample areas by HR-XTEM and, with high statistics and on several sample positions, by measuring the depth of selectively etched trenches in FLG by t-AFM. Both the density of wrinkles and the number of graphene layers are found to increase almost linearly as a function of the growth temperature in the considered temperature range.In this work, we present a nanometer resolution structural characterization of epitaxial graphene (EG) layers grown on 4H-SiC (0001) 8° off-axis, by annealing in inert gas ambient (Ar) in a wide temperature range ( Graphene has attracted the interest of the scientific community due to its excellent electronic transport properties ,2, whichTgr, ramp rate, pressure in the chamber) and on the initial surface conditions of SiC wafers, e.g., the termination (Si- or C-face), the miscut angle, the defectivity. It has been shown that the rate of Si evaporation can be strongly reduced by performing the annealing in inert gas ambient at atmospheric pressure instead than in vacuum [Tgr ~1300°C in vacuum to Tgr > 1600°C at atmospheric pressure [The latter method is the strongest candidate for graphene based electronics, owing to the fact that few layers of graphene (FLG) can be grown on a semiconductor substrate without any need to transfer . Graphene such produced is known as epitaxial graphene (EG). Graphene growth during annealing of SiC relies on the interplay of two different mechanisms: (i) the preferential Si sublimation from SiC surface, which leaves an excess of C atoms in the near surface region, and (ii) the diffusion of these C atoms on the SiC surface and their reorganization in the two-dimensional graphene lattice structure. Both mechanisms depend in a complex way both on the annealing conditions . The modifications in the surface morphology after thermal treatments at the different temperatures were studied by tapping mode atomic force microscopy (t-AFM), showing the increase in the density of peculiar corrugations (wrinkles) in the EG multilayers with the growth temperature. High-resolution cross-sectional transmission electron microscopy (HRXTEM) was used to determine the number of graphene layers on very small sample areas. An alternative method to estimate the EG thickness on larger areas, based on etching out trenches selectively in graphitized SiC and measurements of the depth of those trenches by AFM, is also presented. The accuracy of this method is discussed.In this work, we present a structural characterization of EG growth on the Si face of 8° off-axis 4H-SiC  by anneaTgr from 1600 to 1700°C, or in an inductively heated reactor at Tgr = 2000°C. The substrates used for graphene growth were lowly doped (approx. 1014 cm-3) epitaxially grown SiC layers on top of highly doped, 8° off-axis 4H-SiC (0001).Graphene growth was carried out in argon (Ar) ambient in a commercial furnace by Centrotherm Thermal Solutions GmbH + Co. KG, Blaubeuren, Germany at temperatures k = 20 to 80 N/m, oscillation frequency 332 to 375 kHz in high-amplitude mode were employed.t-AFM measurements were carried out with Veeco DI3100 atomic force microscope fitted with Nanoscope V controller. Commercial silicon probes with spring constant Cross-sectional observations of the few layers of graphene grown on SiC were carried out using a JEOL JEM 2010F transmission electron microscope with a Schottky field emission gun operating at an acceleration voltage of 200 kV. Thin specimens for TEM observation were prepared by Ar-ion thinning by the following procedure: the graphene-on-SiC samples were mechanically ground to 70 μm thick, and the center of the sample was thinned by dimple grinding, followed by Ar-ion beam thinning under a low angle of 8° at 5 kV.2 plasma (25 sccm) treatment for 16 min in an Roth & Rau Microsys 400 ICP-RIE (inductively coupled plasma) equipment. The step height between etched and not etched regions was determined by t-AFM. Before each t-AFM map, we acquired amplitude-distance curves both on not-etched and etched regions and the amplitude setpoint as well as the free amplitude were chosen accordingly, in order to operate in the repulsive interaction regime [HRXTEM images allow an extremely precise determination of the number of grown layers, but on very small areas. An alternative method to determine the thickness of the grown FLG film on larger areas has been also developed. Stripes were patterned onto graphitized SiC substrates by optical lithography. Graphene was then selectively removed from these stripes with an On regime  on both The etch rates of graphene and bare SiC were calibrated independently. To this aim, graphene flakes were exfoliated from HOPG on SiC and their thickness was measured after different etching times by AFM. A patterned virgin SiC sample was etched under identical conditions and the etched depth was also measured by AFM. For the used plasma conditions, the etch rate of graphene is much higher than that of bare SiC.A morphological AFM image of the virgin 4H-SiC (0001) surface is reported in Figure A root mean square (RMS) roughness of 0.2 nm is obtained from this surface analysis.The morphological transformation of SiC surface after annealing is described in the following. AFM images of the surface morphology for the samples annealed at 1600, 1700, and 2000°C are reported in Figure From the linescan in the direction parallel to the steps (d), it can be observed that the features in the morphology map are some peculiar corrugations with 1 to 2 nm typical height. These features appear on the phase linescan as a peak surrounded by two valleys. It is worth noting that these peculiar corrugations run preferentially in direction orthogonal to the step edges of the substrate.In order to get further insight on the structural properties of the graphene films grown at the different temperature and to clarify the nature of these peculiar corrugations, HRXTEM analyses were carried out.As an example, in Figure From Figure These peculiar defects have been observed also by other authors in the case of few layers of graphene grown on the C face  or on th2) as a function of Tgr, as obtained by a statistical analysis of a large number of AFM images collected each sample, is reported in Figure Comparing the morphology and phase images in Figure 2 plasma. This plasma treatment is known to remove efficiently carbonaceous species through a chemical reaction leading to the formation of CO2. In Figure t0 ~2 nm of SiC is etched during the plasma treatment, due to the physical action of the plasma. This depth must be subtracted while evaluating the number of layers on graphitized SiC. Hence the number of layers can be estimated according to the relation n = (D - D0)/Dgr, being Dgr the interlayer separation between two stacked graphene layers (Dgr ≈ 0.35 nm). The average number of grown layers as a function of Tgr is reported in Figure In the following, we will study the increase in the average number of graphene layers as a function of the growth temperature. HRXTEM images on the samples annealed at 1600, 1700 and 2000°C are reported in Figure Few-layers of graphene grown on 4H-SiC (0001) 8° off-axis, by annealing in inert gas ambient (Ar) in a wide temperature range (from 1600 to 2000°C) have been structurally characterized by atomic force microscopy and HR-XTEM. For all the considered growth temperatures, FLG conformally covering the 100 to 200-nm wide terraces of the SiC surface have been observed. The formation of wrinkles (approx. 1 to 2 nm high and 10 to 20 nm wide) preferentially oriented in the direction perpendicular to the step edges of the SiC terraces has been observed in the FLG film, as a result of the release of the compressive strain accumulated in FLG during the sample cooling due to the thermal expansion coefficients mismatch between graphene and SiC. This parallel orientation of wrinkles is peculiar of EG grown a vicinal SiC surface, whereas for EG grown on on-axis 4H-SiC an isotropic mesh-like network of wrinkles interconnected into nodes is commonly reported. The observed phenomenon deserves further investigations. Both the density of wrinkles and the number of graphene layers are found to increase almost linearly as a function of the growth temperature in the considered temperature range.CVD: chemical vapor deposition; EG: epitaxial graphene; FLG: few layers of graphene; HR-TEM: high-resolution cross-sectional transmission electron microscopy; HOPG: highly oriented pyrolithic graphite; RMS: root mean square; t-AFM: tapping mode atomic force microscopyThe authors declare that they have no competing interests.FG and VR conceived the study. FG coordinated the experiment and participated to the analysis of the data. FG, MR and RY worked on graphene growth. CV and SS carried out the t-AFM measurements and the analysis of the data. CV prepared the sample cross-sections for HR-TEM analyses and CB carried out the HR-TEM measurements. FG and CV wrote the article. All the authors read and approved the manuscript.