Human endogenous retroviruses (HERV) are relics of ancient retroviral infections in our genome. Most of them have lost their coding capacity, but proviral RNA or protein have been observed in several disease states . However, their clinical significance as well as their mechanisms of action have still remained elusive. As human aging is associated with several biological characteristics of these diseases, we now analyzed the aging-associated expression of the individual proviruses of two HERV families, HERV-K (91 proviruses) and HERV-W (213 proviruses) using genome-wide RNA-sequencing (RNA-seq). RNA was purified from blood cells derived from healthy young individuals (n = 7) and from nonagenarians (n = 7). The data indicated that in the case of HERV-K (HML-2) 33 proviruses had a detectable expression but in only 3 of those the expression levels were significantly different between the young and old individuals. In the HERV-W family expression was observed in 45 loci and only in one case the young/old difference was significant. However, applying hierarchical clustering on the HERV expression data resulted in the formation of two distinct clusters, one containing the young individuals and another the nonagenarians. This suggests, that even though the aging-associated differences in the expression levels of individual proviruses are minor, there seems to be some underlying aging-related pattern. These data indicate that aging does not have a strong effect on the expression of individual HERV proviruses, but instead several proviruses are affected moderately, leading to age-dependent expression profiles. During mammalian evolution, integration of retroviral RNA into a germ line cell may have led into a formation of a provirus that is transmitted vertically and inherited in a Mendelian manner. In humans, these endogenous retroviruses (HERV) comprise ca. 8% of our genome . While ide novo insertional mutagenesis nor with the formation of viral particles, it has been proposed, that potential pathogenicity of the HERVs could simply underlie in the presence of proviral DNA, acting as a transcriptional regulatory sequence, modifying the expression of neighboring and even more distant genes. HERVs can do this for example by acting as transcription factor binding sites. From this hypothesis it naturally follows, that potential effects of the HERVs would be restricted in some genomic window around the primary proviral insertion site. However, there is also evidence supporting more global mode of action as HERVs have been shown to activate immune and inflammatory responses of the body directly. For example, their RNA could be recognized as a pathogen-associated molecular pattern (PAMP) by Toll-like receptors and this would induce type I interferon production contributing to the pathogenesis of autoinflammatory diseases [Since the mechanism of action cannot be explained by diseases . Some HEdiseases –6. It hadiseases .As the diseases, where HERV-associations have been observed, demonstrate some of the fundamental and characteristic aspects of aging, e.g. increased level of inflammation and changes in the proportions of the various lymphocyte subsets ,10, we nThe most recent entrants to our genome are represented by HERV-K (HML-2) family (ca. 0.2–2 million years ago), of which Subramanian et al. have identified 91 full-length proviral sequences . HERV-W Two populations, representing young and elderly individuals, were used. The young ones consisted of healthy laboratory personnel, all female, aged 26 to 32 years  who did not have any medically diagnosed chronic illnesses, were non-smokers and had not had any infections or received any vaccinations within the two weeks prior to blood sample collection. The elderly individuals (n = 7) were selected among relatively healthy, community living, non-frail, nonagenarian females, without any severe aging-associated diseases, that were participants in The Vitality 90+ study. The nonagenarians were born in 1920 and the samples were collected in 2014. The recruitment and characterization of participants were performed as has been reported previously [Blood samples were collected by a trained laboratory technician in the laboratory facilities. All blood samples were drawn between 8 am and 12 am and collected into EDTA containing tubes. Samples were directly subjected to leucocyte separation on a Ficoll-Paque density gradient . The PBMC layer was collected and cells used for RNA extraction were suspended in 150 μl of RNAlater solution . Nonagenarian and control samples were collected at the same time.RNA used for RNA sequencing was purified using a miRNeasy mini kit  and the RNA used for PCR analysis using RNeasy mini kit  according to manufacturer’s protocol with on-column DNA digestion (Qiagen). The concentration and quality of the RNA was assessed with a NanoDrop ND-1000 spectrophotometer .Agilent Bioanalyzer RNA nano chips (Agilent) were used to evaluate the integrity of total RNA and Qubit RNA–kit (Life Technologies) to quantitate RNA in samples. 1 μg of total RNA was used for ScriptSeq Complete Gold System (Epicentre) to ribodeplete rRNA and further for RNA-seq library preparation. SPRI beads  were used for purification of RNAseq libraries. The library QC was evaluated on High Sensitivity chips by Agilent Bioanalyzer (Agilent). Paired-end sequencing of RNAseq libraries was done using Illumina HiSeq technology with a minimum of 60 million 2x100bp paired-end reads per sample.Raw reads were aligned to human genome reference build hg19 using TopHat v2.0.13  with theThe annotation data for HERV-K (HML-2) was from Subramanian et al.  and thatHierarchical clustering of the samples based on normalized read counts was done separately for both HERV-K (HML-2) and HERV-W. Spearman correlation was used as the distance metric, which is robust against outliers and non-Gaussian distributions, and can capture nonlinear relationships , 24. WarThe results of the RNA-seq analysis indicated that 33 HERV-K (HML-2) loci out of 91 had a detectable expression, but often at low level and not in all individuals. The expression levels of PBMCs derived from young and elderly individuals were generally similar. Only at three loci  the difference was statistically significant as shown in In the case of HERV-W, the results were similar, in 45 proviruses out of 213 the read count was >16 at least in one individual, and in the case of Xp11.21 the difference in expression levels between the young and old was significant as shown in Hierarchical clustering of the samples based on normalized provirus read counts was done to investigate expression patterns. Clustering of samples based on HERV-K HML-2) expression resulted in two groups separated along the age group lines . There w expressiBootstrap resampling of the clustering was done to quantify the certainty of the clustering. Both clusters have an approximately unbiased (AU) p-value of 97, which is the bias corrected percentage of resampling dendrogram variants where the specific cluster was observed. AU p-value of 97 is equivalent to a p-value of 0.03, indicating statistical significance. The same significant clusters resulted even if the significantly differentially expressed 1q22, 10p14 and 12q24.33 were excluded from clustering.HERV-W expression based clustering of samples resulted in one statistically significant cluster , which contains the same six nonagenarian samples that are grouped together in the HERV-K (HML-2) based clustering .The RNA levels of individual proviruses varied considerably between samples. It was not the case that some individuals would have been more active producers than others, but instead different proviruses seemed to be expressing non-systematically within and between individuals. A total of eight HERV-K proviruses and nine HERV-W proviruses were found to be expressed in all 14 samples and consequently these proviruses were expressed with highest RNA levels. This suggests that some individual proviruses could be less restricted in terms of their expression potential, that is brought by the regulation machinery of the cell. Several proviruses were expressed only in small part of individuals and it is tempting to think that these could be the ones behind potential adverse effects, especially if they would be mainly expressed in nonagenarians, as they are probably silenced for a reason. There were no proviruses that were expressed exclusively in nonagenarians, but for example HERV-K 8p23.1a was expressed in 6 nonagenarians and only in 1 young individual. Furthermore, only 4 aging-associated differentially expressed proviruses were identified (in HERV-K (HML-2) 1q22 and 10p14 having a higher and 12q24.33 a lower expression in the elderly and in HERV-W Xp11.21 a lower expression). Putting all this together, it seems to be the case, that aging has only moderate effect on the expression levels of individual proviruses.However, the hierarchical clustering of the expression data indicated that the expression profiles of the young and elderly subpopulations were different. The simplest way to achieve this kind of difference would be if, for example, all the proviruses were expressed systematically slightly up or down in one of the groups. This kind of behavior could be attributed to some kind of common regulator that has only one simple mode of action. However, this was not the case, as different proviruses were up- and downregulated equally in the nonagenarians. This requires more complex regulation and is possibly reflecting multilayered epigenetic regulation machinery involving, among other things, DNA methylation and histone modifications, and inducing distinguishable aging-associated expression profile. Due to Spearman correlation based distance metric in the clustering, each provirus has identical weight in the clustering result, regardless of level of expression. Therefore this result would indicate that there are differences between the age groups that are revealed when the proviral expression profiles are examined as a whole. The underlying cause behind observed expression profile difference thus has to affect the expression of many different proviruses. Understanding what causes this difference could increase knowledge of HERV expression associated disease states and of age-related decline. Since the same nonagenarian samples are clustered by both HERV-K (HML-2) and HERV-W expression, this phenomenon may not be limited to these families, and could be present in other HERV families as well. It is noteworthy, that our analysis is only limited to HERV-K (HML-2) and HERV-W families. Previous studies have indicated that upregulation of some other HERV subclasses might also have implications in tumor immunity –28. TherThere is a general agreement that the expression of HERVs should be under a strict control, i.e. allowing their expression in the germ line but silencing in most somatic cells, where their activity could disrupt normal gene expression or transcript processing. Several of these control mechanisms have been characterized in detail , 30. As The general expression profile of HERV-K (HML-2) in the resting blood cells used here, was dominated by a few loci, i.e. 3q12.3, 19q13.12b and 1q22., resembling the situation in in vitro pre-activated lymphocytes , suggestIn conclusion, transcriptional regulation of the proviruses belonging to HERV-K (HML-2) and HERV-W families appears to be two-dimensional in the PBMCs; a subset of HERVs are expressed constantly in age-independent manner having only slight aging-associated differences in the expression levels. These differences might be explained by a fine-tuning of transcriptional regulation that is brought by DNA methylation and is known to be heavily altered in aging. On the other hand, proviruses in another subset of HERVs were characterized by total lack of expression in some individuals. This could be the result of some more drastic mode of regulation such as that of H3K4me3, that is also known to be altered in aging . Aging-dThis finding might have some practical consequences. In the clinical studies demonstrating associations with HERV-expression the expression of only one or a few proviruses have been used as the indicator. In these studies, analysis of the whole HERV profile would help in finding the true pathogenic provirus. Small number of samples is a limitation of this study, and more comprehensive studies with bigger sample populations are needed for confident evaluation of the RNA levels.


The generalized additive model disclosed norepinephrine alone exerted a dose–dependent effect on 90-day mortality, while other vasoactive agents were not. (4) Conclusion: Using norepinephrine in septic AKI-D patients is associated with higher 90-day mortality and the effect is dose-dependent. Further study to explore the potential mechanism is needed.(1) Background: Norepinephrine (NE) is the first-line vasoactive agent used in septic shock patients; however, the effect of norepinephrine on dialysis-required septic acute kidney injury (AKI-D) patients is uncertain. (2) Methods: To evaluate the impact of NE on 90-day mortality and renal recovery in septic AKI-D patients, we enrolled patients in intensive care units from 30 hospitals in Taiwan. (3) Results: 372 patients were enrolled and were divided into norepinephrine users and non-users. After adjustment by Inverse probability of treatment weighted (IPTW), there was no significant difference of baseline comorbidities between the two groups. NE users had significantly higher 90-day mortality rate and using NE is a strong predictor of 90-day mortality in the multivariate Cox regression (HR = 1.497,  Sepsis is defined as a life-threatening organ dysfunction due to dysregulated host response to infection in accordance to recent Sepsis-3 consensus ; it is tSeptic shock, which is a condition of systemic vasodilatation and arterial hypotension, is now evidenced by a serum lactate level >2 mmol/L and vasopressor requirement to maintain a mean arterial pressure (MAP) of at least 65 mm Hg in the absence of hypovolemia . The useHowever, in AKI patients with septic shock, the different effects of NE, and other vasoactive agents has not been surveyed. In this study, we compare the impact of NE and other vasoactive agents on mortality and dialysis dependency in acute kidney injury patients who require dialysis (AKI-D).The nephrologists and intensivists in Taiwan have appealed for the development of a consortium to unite strength in the field of critical nephrology in Taiwan. The Consortium for Acute Kidney Injury and Renal Diseases (CAKs) and a division focusing on AKI (CAKs-AKI) were launched in the beginning of 2014. This study group has established a multicenter database since 2002 to improve the quality of care and the prognosis of AKI in critically ill patients and then set up a national registry program of AKI to prospectively enroll a large number of dialysis-requiring AKI (AKI-D) patients. This nationwide epidemiology and prognosis of AKI (NEP-AKI-D) requiring dialysis study is the first flagship study of CAKs-AKI, which aims to explore the epidemiology, risk factors, modality, dose, and frequency of renal replacement therapy (RRT), as well as prognoses of the patients with AKI-D, by using the established anonymous nationwide AKI database launched in the beginning of 2014. Up to January of 2016, 30 hospitals have joined this consortium. These hospitals are distributed widely through the four geographical regions  of Taiwan, and have a 1:1 ratio of medical centers to regional hospitals in each region . In the Sepsis is defined in Sepsis-3 as life-threatening organ dysfunction, which is known as an acute change in total SOFA score ≥2 points, caused by a dysregulated host response to infection. And septic shock is defined by a serum lactate level >2 mmol/L and persistent hypotension after fluid resuscitation; it also requires vasopressors to maintain MAP >65 mmHg ,4.Other organ failure is classified as the following ,13: (1) Disease severity was assessed by using the Acute Physiology and Chronic Health Evaluation II (APACHE II) score , the Seqp value > 0.05 one at a time until all regression coefficients were significantly different from 0 [Continuous variables between groups were compared using the Student t test. The chi-square test was applied for categorical variables with Yates’ correction where applicable. The inverse probability of treatment weighting (IPTW) using the propensity score was applied to correct the bias of the two groups in basic characteristics and outcomes . Applyint from 0 ,23.Forest plot was constructed for odds ratio of NE use (vs. other vasoactive agents) on 90-day mortality according to prior comorbidities and clinical conditions. The generalized additive model was used to analyze the dose-response relationship between vasoactive agents and the 90-day mortality ,25. Becap values < 0.05 were considered statistically significant.We used R software version 3.2.2  for the time-varying Cox model and Stata/MP version 14  for the competing risk analysis. Two-sided Approval of this prospective multi-center study follows the regulations of the National Research Program for Biopharmaceuticals (NRPB)-Institutional Review Board (IRB). All clinical trial consortiums have to fill in the application forms on the official website of the NRPB-IRB. Written t informed consent was obtained from all participants before inclusion. .p = 0.289) between the two groups initially, and other scoring systems including IE score and SOFA  were similar after adjustment. We record the etiology of acute kidney injury included with/without cardiorenal syndrome, drug, rhabdomyolysis, pigmentation (pigment nephropathy), hepatorenal syndrome, and contrast. And there was no significant difference in the etiologies of AKI between two groups. Besides, no significant difference was noted in major infection site which included respiratory tract, genitourinary tract, blood stream, or abdomen between NE user and non-user. Oliguria (81.9%) is the leading cause for RRT, followed by fluid overloaded (67.8%). The indications for RRT were similar except for oliguria (p = 0.009). Regarding the RRT modality, NE users tended to receive CVVH, and SLEDD (p = 0.034). The detail of these baseline characteristics is in the A total of 372 AKI-D patients fulfilled the criteria of septic shock at initialization of dialysis and 315 patients were NE users. The characteristics of patients are listed in p = 0.204). The number of dialysis days of the NE non-user groups was larger than that of the NE user group .A total of 302 (81.2%) patients died within 90 days of hospital discharge  and 260 p = 0.026).We further analyzed the outcomes of the use of NE; subgroup analysis using forest plots was performed to calculate IPTW adjusted HRs (HRs) of 90-day mortality. The detrimental effects of NE were consistent across the subgroups stratified by respiratory tract infection, congestive heart failure, diabetes mellitus, BUN with more than 64 mg/dL, and patients who did not have cerebrovascular disease .The quantity of NE was plotted against the log odds of predicting 90-day mortality by using generalized additive mode (GAM) after adjusting the risk factors in the final model . We sepap = 0.651).In the evaluation of the relation between the use of NE and dialysis dependency, we conducted a competing-risk regression model which took mortality as a competing risk . It reveIn this multi-center, observational study of AKI-D patients with septic shock at initialization of dialysis, we found that 81.2% of patients died within 90 days of hospital discharge. Our report first showed the high mortality of AKI-D patients with septic shock according to Sepsis-3 criteria. The use of NE was associated with a higher 90-day mortality rate than other vasoactive agents and that the detrimental effect is dose-dependence. We applied inverse probability of treatment weighting (IPTW), to minimize the effect of confounding in observational study . And impIn the general population, the use of NE is the first-line choice of vasoactive agents to restore organ perfusion and maintain the blood pressure in septic shock patients . The infChou et al . conductThus, differences between vasopressin and nor epinephrine-treated patient outcomes may be due to the beneficial effects of vasopressin or, alternatively, due to reduction in the detrimental effects of norepinephrine. This result is also consistent with the primary subgroup analysis of the VASST study in which vasopressin treatment was associated with decreased mortality in patients who had less severe shock and not in patients who had more severe shock .In a recent prospective cohort study , Passos Adequate fluid resuscitation is recommended and it is closely associated with blood pressure maintenance with the ultimate aim of maintaining tissue perfusion and oxygenation . HoweverIn our study we found worse outcomes when using NE in AKI-D patients with septic shock when compared to other vasoactive agents, even after adjustment for the disease severity. Besides, the disease severity was more severe in our study population in accordance with SOFA score and APACHEII score which might lead to higher mortality rate in our study. In previous study, the highest SOFA scores, higher than 11, were associated with a mortality rate greater than 80% . The meaIn general, the infusion of NE deceases the renal blood flow  and renaHowever, in acute endotoxemic status, the infusion of NE appears to improve renal blood flow, and had favorable effects on renal function in septic patients. One recent animal study  tried toThis is the first study evaluating NE in critical AKI-D patients with septic shock according to the sepsis-3 criteria from a nationwide cohort. We followed up patients with septic AKI on dialysis to 90 days after hospital discharge, and further evaluated the recovery from AKI promptly. However, our study still had some limitations. First, our study did not evaluate the catecholamine-sparing effect of NE, in addition to the different patient population; and we did not evaluate the vascular response to NE under critical dialysis. Secondly, the indication for the use of NE or other vasoactive agents is not standardized. However, due to the difficulty in the randomness of enrolling patients with septic shock, an observational study could provide valuable information. Third, there could be a selection bias in choosing vasoactive agents due to current guidelines showed that norepinephrine is the first choice for septic shock and low dose dopamine for prophylactic use is no longer recommended. Last but not the least, we did not record pre-dialysis data of hemodynamic status, such as central venous pressure or volume of fluid resuscitation completely. Therefore, we could not evaluate the impact of fluid status when dialysis-required septic AKI developed and the association with the vasoactive agent usage to mortality.The use of NE in septic AKI patients at the initialization of dialysis is associated with a higher 90-day mortality after we adjusted for severity by IPTW compared to other vasoactive agents, and the detrimental effect was dose-dependent. Therefore, consideration of early treatment to block septic AKI vicious cycle to stabilize the hemodynamic status should nonetheless precede increased doses of norepinephrine at dialysis initiation. Further study to explore the potential and possible mechanism is needed.


They used DNA methylation at CpG-sites with a variant...This study highlights dangers in over-interpreting fine-mapping results. Chundru  n = 1166). The choice of imputation reference panel had a strong effect on imputation accuracy, with the 1000 Genomes Project Phase 3 (1000G) reference panel (n = 2504 from 26 populations) giving a mean nonreference discordance rate between imputed and sequenced genotypes of 3.2% compared to 1.6% when using the Haplotype Reference Consortium (HRC) reference panel . These imputation errors had an impact on whether the CpG-SNP was included in the 95% credible set, with a difference of ∼23% and ∼7% between the WGS and the 1000G and HRC imputed datasets, respectively. All of the fine-mapping methods failed to reach the expected 95% coverage of the CpG-SNP. This is attributed to secondary cis genetic effects that are unable to be statistically separated from the CpG-SNP, and through a masking mechanism where the effect of the methylation disrupting allele at the CpG-SNP is hidden by the effect of a nearby SNP that has strong linkage disequilibrium with the CpG-SNP. The reduced accuracy in fine-mapping a known causal variant in a low-level biological trait with imputed genetic data has implications for the study of higher-order complex traits and disease.Genetic variants disrupting DNA methylation at CpG dinucleotides (CpG-SNP) provide a set of known causal variants to serve as models to test fine-mapping methodology. We use 1716 CpG-SNPs to test three fine-mapping approaches , assessing the impact of imputation errors and the choice of reference panel by using both whole-genome sequence (WGS), and genotype array data on the same individuals ( This asBecause of the small number of known causal variants, comparisons of fine-mapping methods need to be performed through simulation, and are often idealized and do not encompass the full range of experimental variation. However, high-throughput measurement of DNA methylation across the genome provides a potential model trait for testing fine-mapping methods. DNA methylation is an epigenetic modification that is influenced by both genetic and environmental factors, with an average heritability of 20%  , BayesiaThe Lothian birth cohorts of 1921 and 1936 (LBC)  or ScottFrom the LBC, 1370 individuals were whole-genome sequenced on a HiSeq X installation to an average coverage of 36× . All reads were mapped to the build 38 version of the reference genome using BWA  and variThe whole-genome sequence data were compared to array data for the same individuals using PLINK 1.90 , the SNPs with high missing rate (>5%), SNPs with Hardy–Weinberg exact test  Phase 3 . The impP < 0.01), and low-quality samples were removed. Individuals with low call rate  were removed. Probes on the X and Y chromosomes were removed, leaving 450,726 probes remaining. β Values were corrected for BeadChip, sample plate, hybridization date, white blood cell count, and sex.Details of the DNA methylation QC can be found elsewhere (We used the UK10K dataset [European Genome-phenome Archive (EGA) accession numbers: EGAS00001000108 and EGAS00001000090] for the simulations . The UK10K dataset (The Systems Genomics of Parkinson’s Disease (SGPD) cohort comprises 956 individuals with Parkinson’s disease, and 930 controls genotyped on the Illumina PsychArray-B.bpm. In our analyses we did not take disease status into account. The data were filtered to remove individuals with high missing rate, the SNPs with high missing rate (>5%), SNPs with Hardy–Weinberg exact test n server  and was n server . The impP < 0.01). The R meffil package was also used to perform sample QC using Illumina recommended thresholds. Samples were dropped if call rate was low , if predicted sex (based on XY probes) did not match reported sex, and if predicted median methylated signal was >3 SD from the expected. After these QC steps, methylation β values were quantile-normalized with respect to 20 principal components generated from the control matrix and the most variable probes. Additionally, normalization was adjusted for batch, slide, cohort, sentrix row/column, sex, and age. Of the 1716 probes in the LBC dataset, only 1678 remained after cleaning, thus the replication was only conducted on the respective probes.The DNA methylation data were measured using the Illumina HumanMethylation450 BeadChip array. Raw intensity data were background-corrected and normalized using internal controls, and methylation β values were generated using the R meffil package (n = 3781), and a subset of the UK10K dataset to match the sample size in the imputed LBC dataset (n = 1366). The causal variants were chosen at random from the genome, but restricted to have MAF > 0.05.Phenotypes similar to DNA methylation at CpG-SNPs were simulated using the GCTA software  because we use the coverage of the causal variant in the sets as a measure of fine-mapping accuracy. Both sets are designed so that 95% of the time the causal variant will be captured. For simplicity we will refer to both sets as credible sets.Rank the SNPs by strength of association, and add the most associated SNP to the credible set;Regress the most associated SNP against the phenotypeN = 2, regress the Nth best SNP against the phenotype with the fitted values from the regression in step 2 as a covariate:Starting at N, and repeat step 3. If If where y is the phenotype, i, and Nth best SNP. To construct a 95% credible set of causal variants, a set of SNPs with 95% probability to contain the causal variant, a Bonferroni-corrected significance of The J-test  is a simBIMBAM , which uBSLMM  method . The UK10K dataset is available from EGA (accession numbers: EGAS00001000108 and EGAS00001000090). The source code used to run the three fine-mapping methods is available on GitHub (https://github.com/chundruv/finemapping_GENETICS2019). Details of the simulation results, the discordance between sequence and genotyped data, and the SGPD consortium member list is provided in File S1. Supplemental material available at Figshare: https://doi.org/10.25386/genetics.7906109.The LBC methylation data are available at EGA under accession number EGAS00001000910. The LBC1921 and LBC36 genotype data are available on request for relevant research purposes (n = 2504 from 26 populations) and the HRC  reference panels . Fine-mapping was performed at 1716 DNA methylation sites previously identified to have a cis-mQTL We compare 95% credible sets  obtained from three fine-mapping approaches using DNA mQTL at a CpG-SNP in the 1166 individuals from the LBC , as well as the number of SNPs within each credible set. BIMBAM performed marginally better than both BSLMM and the J-test in terms of coverage of the CpG-SNP, with the trade-off of larger credible sets (Table S1). In the 672 cases where the CpG-SNP was not the most associated SNP (top SNP), the top SNP in the credible sets had a median distance of 2 kb to the CpG-SNP, with 95% of SNPs being within 34 kb. . While performing well on simulated data (see File S1), all three methods failed to reach the expected 95% coverage of the putatively causal CpG-SNP  using eivs. genotyped SNPs and overall coverage of 95% credible sets was replicated in an independent dataset of 1886 individuals imputed using the HRC reference panel  were included in 95% credible sets between 29 and 33% more often than imputed CpG-SNPs using the 1000 Genomes Project Phase 3 reference, and between 8 and 19% more often using the HRC reference dataset, with this being driven by differences in imputation accuracy (see File S1). The difference between imputed ce panel . The effce panel .cis-mQTL, with this being implicitly assumed in the construction of the 95% credible set for each of the methods. We performed two analyses to identify mQTL under the influence of multiple genetic variants: a standard forward selection approach and the CoJo stepwise selection model implemented in GCTA-CoJo . Only one independent signal was detected by both methods for 87% of the mQTL. However, when considering only those mQTL showing a single independent association for both methods, we see that the coverage is still below the expected 95% (The underlying assumption of our comparison of fine-mapping is the presence of a single causal variant underlying the cted 95% .For the mQTL with one independent association from the conditional analyses, and where the CpG-SNP was not the top SNP, we estimated LD between the top SNP and CpG-SNP. In all cases, the LD between the top SNP and CpG-SNP pairs had a hylation . As suchTo capture genuine biological complexity while assessing the performance of fine-mapping methodology, we examined the use of known genetic variation within DNA methylation CpG sites as a model trait. This identified limitations in fine-mapping with imputed sequence data and in statistically separating effects of closely linked variants.vs. sequencing. We have shown a dramatic reduction in the proportion of credible sets that actually contain the underlying causal variant when using imputed genotype data, particularly when using the 1000 Genomes Project Phase 3 reference panel for imputation. This imputation panel is still widely used, especially for GWAS meta-analysis combining populations with differing ancestry. In comparison, the more extensive HRC reference panel showed a great reduction in imputed genotype error rates, resulting in increased coverage of the causal variant. This highlights the need to continue the generation of large imputation reference panels across multiple ancestries. The HRC reference panel is ∼6.5 times larger than the African Genome Resource, which is currently by far the largest non-European imputation reference panel.Statistically minimizing the set of potential causal variants underlying the thousands of identified GWAS hits is essential for efficient experimental follow-up. However, we also need to ensure statistically derived sets of potential causal variants actually contain the underlying causal variant. While fine-mapping methods implicitly assume all potential causal variants are available, GWAS generally use imputed genotypes because of large sample size requirements and the relative cost of genotyping arrays cis region surrounding the CpG site for 11% of probes. It is likely that a much higher proportion of probes would be identified as having multiple genetic effects with a greater sample size. In addition, we identified SNPs that effectively masked the effect of the CpG-SNP; these variants had an effect on the methylation levels, and the methylation disrupting allele of these variants were in high LD Although common CpG-SNPs will have a very large effect on the DNA methylation, we were unable to reach the expected 95% coverage of the putatively causal CpG-SNP in our credible sets even when using whole-genome sequenced genotypes. We detected multiple statistically independent genetic associations in the The difficulties in fine-mapping a known causal variant in a low-level biological trait have implications for the study of higher-order complex traits and disease. For example,


An epoxy chain extender was used during preparation of the blends and nanocomposites to enhance the mechanical properties of the products. Different methods such as scanning electron microscopy, X-ray diffraction and adsorption/desorption analyses were used to characterize the prepared nanoparticles and their localization in the blends. Dynamic oscillatory shear behavior, elongational viscosity and mechanical characteristics of the nanocomposites of PLA and the blends were evaluated. The results obtained for nanocomposites filled with unmodified SFD-MMT were compared with those obtained when the filler was a commercial organically modified montmorillonite nanoclay  quaternary ammonium chloride) (C30B), which was not spray freeze-dried.Nanoparticles based on cellulose nanocrystals (CNC) and montmorillonite clay (MMT) were prepared using spray freeze-drying. The nanoparticles were then used as reinforcement to prepare nanocomposites with poly(lactic acid) (PLA) as the polymer matrix. The effect of spray freeze-dried CNC (SFD-CNC) and spray freeze-dried MMT (SFD-MMT) on the rheological and mechanical properties of PLA and its blends with poly[(butylene succinate)- Biosource, biodegradable and ecofriendly thermoplastic polymers are of interest in the fabrication of sustainable nanocomposites . Poly, and it exhibits poor toughness with less than 10% elongation at break, poor thermal stability, low-heat distortion temperature and weak barrier properties ,6. Thus,co-adipate)] (PBSA) as well as their nanocomposites with nanoclay  with mass density of 1.23 g/cm3  was obtained from Showa Denko K.K., Tokyo, Japan. PBSA is a random copolyester which contains highly flexible macromolecules with excellent impact strength. It has relatively low production cost and good mechanical properties and processability compared to polyolefins  was investigated by performing small amplitude oscillatory shear measurements. The effect of spray freeze-dried cellulose nanocrystals (SFD-CNC) and montmorillonite (SFD-MMT) on the rheological properties of PLA nanocomposites and their corresponding blends with poly[(butylene succinate)-The Newtonian region at low shear rates and the pronounced shear thinning were observed for these samples; the second Newtonian region cannot be observed. The Carreau–Yasuda model (1) was fit quite well to the experimental data. The calculated zero-shear viscosity and pseudo-plasticity index values are presented in All PLA nanocomposites had higher zero-shear viscosities and more pronounced shear thinning behavior when compared to the PLA and chain extended PLA (CPLA). Among the PLA nanocomposites, pseudo-plasticity was more pronounced for the samples incorporating SFD-CNC (pseudo-plasticity index of 0.65). The use of Cloisite 30B (C30B) is more effective than chemically unmodified spray freeze-dried nanoparticles (SFD-CNC and SFD-MMT) in changing the rheological properties of CPLA-PBSA ternary nanocomposites b which iω, also provides insight about the morphological features of the materials and their rheological response. As seen in ω2 at low frequencies for PLA, which is the typical behavior for linear polymers. Chain extension reaction caused a slight deviation from terminal behavior for CPLA. Higher storage modulus and smaller slope were observed in the low frequency region by incorporation of nanoparticles, as compared to PLA and CPLA. Higher elasticity in the low frequency region and lower slope in the terminal zone were found for the PLA nanocomposites prepared based on spray freeze-dried nanoparticles as compared to the chemically modified MMT (C30B). The deviation from the terminal zone behavior was more pronounced for SFD-MMT-CPLA due to the improved dispersion of SFD-MMT nanoparticles which contributes to higher reinforcement of the PLA matrix.Storage modulus as a function of angular frequency, The stiffness of C30B is about 3 times that of CNC . HoweverThe storage modulus of C30B-CPLA-PBSA ternary nanocomposite was higher than those of CPLA-PBSA ternary nanocomposites prepared based on spray freeze-dried nanoparticles because of the higher extent of chain extension reaction. The barrier role of C30B layers that controls the local concentration of chain extender in the PLA phase and its effect on the extent of chain extension reaction was discussed in detail in a previous study . Furthermore, The Cole–Cole plot b and modThe rheological responses of the CPLA-PBSA ternary nanocomposites were quite different. The characteristic of the Cole–Cole plot for homogeneous polymeric blends is the existence of only one circular arc. After the phase temperature transition of a system, a tail develops and forms a second arc on the right-hand side of the first arc . The morUnmodified PLA exhibits low elongational viscosity and melt strength which has limited its use in many important applications such as film blowing, blow molding, thermoforming, and foaming. Different approaches have been reported to enhance the elongational viscosity and melt strength of PLA systems such as the use of chain extender in reactive extrusion process , the useAs mentioned before, the localization of the nanoparticles due to the surface energy considerations and their shapes affect the efficiency of the chain extender. Therefore, the higher melt strength of the C30B-CPLA-PBSA and SFD-CNC-CPLA-PBSA, as compared to that of SFD-MMT-CPLA-PBSA, is attributed to the barrier effect of montmorillonite layers at the interface between PLA and PBSA. It increases the local concentration of the chain extender within the PLA phase and leads to longer chains/branches.c-onset and the heat of crystallization ΔHC during cooling at 10 °C/min, the onset of cold-crystallization temperature Tcc-onset, the heat of fusion ΔHm and the heat of cold-crystallization resulting from the re-arrangement of macromolecules during the second heating ΔHcc in J/(g of PLA) are shown in C is obtained by dividing the value of ΔHm of each sample by the value of 106 J/g for ΔHm* of 100% PLA as reported by Sarasua and co-workers [The values of onset of crystallization temperature T-workers . All samcc-onset) in both CPLA and CPLA-PBSA, followed by SFD-CNC and SFD-MMT. C30B present in C30B-CPLA also crystallized to form lamellae that exhibited only a predominant melting peak at 166.25 °C  in CPLA, while the CPLA showed a predominant melting peak at 163.25 °C . Only a single melting peak around 166 °C was observed for the CPLA-PBSA series.C30B seems to provide more effective nucleation sites .CPLA with 0.5% SFD-MMT and 0.5% SFD-CNC showed heat of fusion of 37.7 J/g with double Tcc-onset, while that of C30B in CPLA and CPLA-PBSA were the lowest. Thus, the CPLA-C30B was expected to show the smallest PLA spherulites during the cold crystallization. SFD-MMT and SFD-CNC composites with PBSA showed some hindrance effects on the cold crystallization of the PLA resulting in a much lower value of ΔHcc (66.3–83% of ΔHm), compared to the PLA without PBSA (86.7–95%).The nanoparticles of MMT and CNC are well known to provide heterogeneous sites for the nucleation of polymers . SFD-CNCWhile the particles and clusters can be observed under parallel polarizers at room temperature, they could be best seen at 200 °C under crossed polarized light, when the matrix is completely melted . This isThe particle size distribution for each specimen was obtained using ImageJ . The preIt is well known that a good quality of dispersion and/or exfoliation of nanofillers in the polymer matrix is required to enhance the mechanical properties. Different approaches have been taken to enhance the dispersion quality of nanoparticles, and consequently the mechanical properties of the nanocomposites, such as the use of chemical modifiers for montmorillonite layers  and grafPLA as a biosource, biocompatible and biodegradable polymer is a likely candidate for use in many applications. However, significant shortcomings in its processing behavior and mechanical properties limit its suitability in a variety of applications. Thus, a variety of approaches have been used to overcome some of these limitations. Typically, blending with other biopolymers, such as PBSA, the use of chain extenders, and the incorporation of nanoparticles, have been used individually or in combination for this purpose. The present study evaluates the effects of some of these modifications. Emphasis is placed on the following factors, both individually and in combination: the use of chain extender, the blending with PBSA, the use of montmorillonite clay (modified or unmodified) and cellulose nanocrystals (CNC) as fillers to form nanocomposites.The results indicate that the comprehensive approach utilizing the nanocomposites of the chain extended PLA-PBSA blend yields significant improvement. Obviously, it would be desirable for many applications to employ CNC, in order to maintain the biofriendly character of the nanocomposites. In this case, it is shown that permeable spray freeze-dried cellulose nanocrystal (SFD-CNC) particles provide satisfactory enhancements in rheological and mechanical properties. Similarly, it is shown that spray freeze-dried unmodified clay yields results comparable to those of chemically modified clay. Thus, spray freeze drying of clay is a viable approach to producing nanoparticles suitable for the production of nanocomposites without chemical modifications.


Furthermore, a series of uniaxial tension tests under different relative humidity (RH) were carried out on cured EPI samples before and after post-curing treatment, and the corresponding chemical structure, as well as the microstructure of polymers, was investigated in detail. In addition, a constitutive equation was formulated to calculate the viscoelastic characteristics of the adhesive layer. The results indicate that the EPI films reveal various kinds of intrinsic deformation as RH increases, and the tensile rupture stress and stiffness would obviously decrease, even at cross-linker weight ratios of up to 20%. Furthermore, the moisture resistance could be markedly improved by increasing the isocyanate content and post-cure. Importantly, EMDI-cross-linked film not only exhibits much better mechanical properties than that containing p-MDI at 0–80% RH, but is also more sensitive to post-cure. Finally, the derived viscoelastic model could efficiently track moisture-dependent stress-strain curves of EPI films, and the obtained relaxation time further reveals the influence mechanism of isocyanate and post-cure on the mechanical response of the cured polymer under moist conditions.Knowledge of the mechanical behaviors of polymer film in humid environments is of great significance for predicting the long-term performance of emulsion polymer isocyanate (EPI) as a high-performance wood adhesive. A tri-copolymer latex was cross-linked by the general polymeric methylene diisocyanate ( Emulsion polymer isocyanates (EPI) are composed of a water-based polymer emulsion and an isocyanate cross-linker. There is a remarkably strong interest in the application of EPI adhesives in solid wood processing and wood-based panel production for furniture manufacturing. The outstanding characteristics  make EPI one of the most promising choices for adhesives. It should also be noted that EPI-bonded wood products comprised of structural-composite lumber and cross-laminated timber are commonly used for buildings and bridges in Europe and North America . As suchp-MDI) respectively. It was found that aqueous emulsified isocyanate (EMDI) exhibits better cross-linking performance, followed by p-MDI mixed with organic solvent. The compression shear strength and percent wood failure are improved as the polyvinyl alcohol (PVOH) content increases, but excess PVOH impairs resistance to warm/boiling water [3 to a whey protein isolate solution, Guo et al. developed a novel EPI adhesive which has wet compression shear strength which surpass the JIS K6806 standards [To improve the bonding performance of EPI in different humid conditions, considerable efforts have been made to optimize the adhesive formulation. For example, Hu et al. investigated the warm and boiling water resistances of EPI adhesives for bonding rose wood, in which vinyl acetate homopolymer and copolymer emulsion were cross-linked by three types of polymeric methylene diisocyanate , chemical structure, and microstructure of polymers were checked using DMA RH scans, Fourier transform infrared spectroscopy (FTIR) and optical microscopy respectively. Moreover, a constitutive equation was formulated to quantitatively describe the moisture-effected stress-strain curves of EPI films, and the obtained relaxation time was used to unveil the effect mechanism of cross-linker and post-cure on the mechanical properties of the polymer in humid environments.In this study, we focus on exploring the role of moisture on the mechanical response of EPI film, aiming to provide a simple and accurate basis for theoretical modeling and applications of EPI-bonded wood products. A tri-copolymer latex was cross-linked by the polyisocyanate at different types and loadings for preparing EPI. In addition, a series of uniaxial tension tests under different RH were performed to determine the mechanical properties of EPI cured samples before and after post-cure treatment, and the glass transition relative humidity (RH(1)3, as well as 10% (wt.) of PVOH as a protective colloid and the main provider of hydroxyl groups. The aqueous vinyl latex was synthesized by Gclock Adhesive Company  and was used as received. It had a solid content of 52.5%, viscosity of 7500 mPa·s (at 25 °C), and pH of 6.3.Main component: vinyl acetate/butyl acrylate/hydroxypropyl methacrylate tri-copolymer latex , containing approximately 30% (wt.) of CaCO(2)p-MDI (Rubinate 5005), supplied by Huntsman Polyurethanes (China) Ltd. , were employed as received. The characteristics of both cross-linkers are shown in Cross-linkers: EMDI (Rubinate 9259) and EPI adhesives were prepared by adding the cross-linker to the main component at cross-linker weight ratios of 0%, 5%, 10%, 15%, and 20%, respectively. The two components, after metering, were mixed for 30 min by an electric agitator, and degassed for 30 s. The films were prepared on Teflon-coated glass plates and dried in a constant temperature and humidity chamber at 40 °C for 48 h to evaporate the water. Subsequently, some of the samples were placed in the chamber at 140 °C for 24 h to further remove residual isocyanate groups in EPI. Finally, all the cured films, with dimensions of 20 mm × 5 mm × 0.04 mm, were kept in a desiccator with freshly dried silica gel at ambient temperature as samples for testing. 2 and a saturated water vapor mixture of different proportions. The humidity accuracy was ±3% at 0–90% RH and ±5% RH above 90% RH.The mechanical measurements were carried out by a film/fiber tension clamp on a DMA  with a DMA-RH Accessory, where the RH regulation of the sample chamber was realized by controlling pure NThe mechanical tests were conducted in different RHs at ambient temperature. The samples were first equilibrated at 0% RH for 60 min, and then the relative humidity in the testing chamber was adjusted to fixed values of 0%, 20%, 40%, 60%, and 80% RH. After 90 min equilibration, uniaxial tension was carried out at a strain rate of 2% under these various constant RHs.DMA tests were carried out under strain mode at a frequency of 1 Hz and amplitude of 20 m. For the RH scans under a controlled temperature, specimens were quickly dried in a measuring chamber at 0% RH, 25 °C for about 60 min, after which RH scans were performed isothermally at the rate of 1.0% RH/min, with the humidity ranging from 0% RH to 90% RH.−1 between 400 and 4000 cm−1. The obtained spectra were normalized, with the peak height of the C–H stretching band at approximately 2922 cm−1.FTIR spectra were measured with an Avatar Nicolet 380 spectrometer  using the KBr tablet method. Each sample was scanned 16 times with a resolution of 4 cmThe surface morphology of EPI films was observed on an optical microscope , and the eyepiece used for image acquisition tasks was PlanApo D 5x/0.3 FWD 30 mm. These image acquisition tasks were carried out by graphic stitching under free examination modes. Mixed illumination was also used in the tests and the magnification 200×. The obtained optical microscopy images were then transformed into binary images using a MATLAB program based on the region growing method.The mechanical behaviors of the bonding layer are crucial to the comprehensive understanding of the complicated deformation response of EPI adhesive in a moist environment. Therefore, a series of uniaxial tension tests under different RHs  were carried out on the EPI cured samples, which were cross-linked by EMDI or p-MDI with 0%, 5%, 10%, 15% and 20% of loadings, respectively. p-MDI are 240% and 350% at 80% RH, respectively, which are separately 12 times and 15 times of those when the compounds are in a dry state. However, within the range of higher cross-linker loading (15–20%), the polymers are neither rubber-like nor glassy, where the stress steeply goes up to the fracture point corresponding to a strain of 18–34%. It is much less than the value (240–500%) at low isocyanate addition levels (0–5%). At an intermediate cross-linker loading (10%), the specimen cross-linked by p-MDI bears a notably hyperelastic material with Under high humidity conditions, due to the penetration of water molecules, scissioning of chain segments and the destruction of cross-links occur, resulting in the deterioration of the mechanical properties of the polymer. Thus, the tensile strength of the EPI films is greatly weakened, and the fracture strain increases considerably, as shown in 2O is evaporated into the environment and reacts with the isocyanate groups as shown in Equation (1), the water content in the adhesive layer decreases, and tiny particles of polymer gradually form a film; while –OH cross-links with –NCO, the result forms the urethane bond [p-MDI compared to the neat xPVAc sample. As the weight ratio of cross-linker increases to 20%, the relative difference in p-MDI content, the strength of dry film continuously increases. This is different to the effect of polymeric isocyanate on the bonding performance of xPVAc-based EPI at ambient temperature, which first rises then descends as the weight ratio of cross-linker increases from 0 to 20.0% [After EPI is applied on the substrate, as Hto 20.0% , but alsWhen water-based polymer film is subjected to moisture, because of the strong attraction of the water molecules to the amorphous chains of aqueous polymer, the electrostatic forces create a mutually exclusive system, forcing molecular chains to become a looser framework, and leaving extra free volume. Therefore, the small molecules can penetrate into the non-crystalline zone and reduce the local density of the system, which results in the strength of the cured film decreasing through an increase in the relative humidity at isothermal conditions see . As antiAs seen in p-MDI films with the same isocyanate, and that the difference tends to become more obvious as the cross-linker amount increases. This result may be interpreted as follows: On the one hand, as shown in p-MDI films throughout the cross-linker content range tested. Moreover, as the cross-linker addition increases, the gap in cross-link density between the two types of films gradually widens. Therefore, the water resistance of xPVAc/EMDI films is better. On the other hand, as the more non-polar cross-linker, p-MDI in the form of small particles is not easy to disperse into the polar aqueous main polymer component, and this situation is further exacerbated at higher isocyanate ratios. In EMDI, where the polymeric isocyanate is modified with non-ionic surfactants, the interface between the hydrophilic and lyophilic groups in the EPI adhesive is optimized. Thus, EMDI will facilitate the dispersion of the isocyanate into xPVAc and PVOH, and thus, enhance the contact probability and cross-linking reaction between the –OH of the aqueous polymer and –NCO in the cross-linker. Consequently, the mechanical properties of the cured film are improved by the higher crosslink density through its effect on the molecular mobility and the glass transition relative humidity.δ, and decreased the peak height, suggesting that the molecular weight increases and the molecular weight distribution broadens. It can be clearly observed that there exists the critical relative humidity denoted as RHg, corresponding to the peak of loss tangent. Admittedly, the addition of polyisocyanate was helpful in increasing the glass transition relative humidity, causing a greater constraint on chain movement and a higher crosslink degree. Accordingly, the more isocyanate added, the higher the RHg. As anticipated, the RHg value of xPVAc/EMDI is higher than that of xPVAc/p-MDI at the same cross-linker content.Analogous to the glass transition induced by temperature change, external relative humidity variation and the resulting change in water content can also lead to a remarked viscoelastic state change of the EPI film, as seen in p-MDI. The value of p-MDI shows a similar trend. All the EPI cured films with various cross-link densities exhibit almost the same profile of evolution As stated above, experimental results show that an increase in relative humidity would lead to not only a decrease of mechanical properties, such as yield and rupture stress, but also of the glass transition of adhesive film. One may wonder whether there exists some connection between plastic behavior and the segmental mobility process. Inspired by the work of the Halary group ,16,17,18−1 was still detected in the spectrum of an EPI film stored for 60 days at ambient temperature [2) gas arising from this reaction, as shown in Equation (1). Therefore, to improve the durability performance of EPI-bonded structures under operating conditions, reducing the amount of unreacted isocyanate in the glue-line is essential. Importantly, the post-curing treatment, i.e., by heating at 120–180 °C, could decrease the content of residual NCO groups in an EPI film sample, thereby contributing to the higher dynamic storage modulus [Although the NCO groups can be so reactive that they react with almost all polar substances in the wood and aqueous polymers such as water and hydroxyl groups, the isocyanate absorption band at 2270 cmperature . As a re modulus . How theTo decrease the content of residual NCO groups in polymer film, EPI specimens were heated in an oven at 140 °C for 24 h, and then subjected to the same uniaxial tensile test. The representative engineering stress-strain curves are shown in p-MDI also show the similar trend, but relatively less improvement. This observation indicates that the treatment effectively promotes the cross-linking reaction between the –OH and the functional groups in the main component, and may obtain additional intramolecular crosslinking, as indicated in Equation (4). As stated by Wei and Haag [As shown in and Haag , the intp-MDI are increased by approximately 80% and 60% respectively, compared to their untreated specimens. It was also found that the treatment is more effective for the film cross-linked by EMDI than p-MDI at other loadings . Due to plasticization effect of moisture, the rupture stress of the post-cured films also decreases as the relative humidity increases. As anticipated, heating treatment can significantly increase moisture resistance of EPI specimens. Certainly, the degree of improvement depends upon the water absorption in the polymer films. At 80% RH, the treated specimens with 5–10% cross-linker show increases by 20–30% in p-MDI, before and after post-curing treatment at various isocyanate loadings.To determine the effect mechanism of the post-cure on the mechanical behavior of EPI films, it is necessary to systematically analyze the changes in the structure and composition of the polymer. As the cross-linked structure is an effective barrier to water molecule infiltration, the higher the crosslink density, the more difficult the diffusion by water molecules. The first aim of the following discussion was the clarification of the changes in the cross-link density , the micropore magnitude on the specimen surface will be considerably changed due to the released carbon dioxide gas. Therefore, the change in micropore distribution may reflect the extent of this reaction. Based on this hypothesis, we used a 3D optical microscope to observe the morphologies of the micropores on the surface of the EPI specimens with different cross-linkers before and after heat treatment, as shown in In the previous section, EPI films with different cross-linkers were systematically subjected to quasi-static tensile tests in a variety of humid environments, and the corresponding stress-strain curves were obtained. However, the experiments could not encompass all relative humidity, making it necessary to establish a theoretical model that can predict the mechanical behavior of adhesive films. This section offers a constitutive model to quantitatively describe the moisture-dependent stress-strain curves of cured films; calculated relaxation time is used to determine the effect mechanism of cross-linker and post-curing on the water resistance of EPI.As shown in p-MDI. The representative results are shown in Equation (5) was used to describe all the uniaxial tensile test curves of the dry and wet specimen cross-linked by EMDI or The relaxation time (τ) against RH for EPI film with different crosslinkers before and after the post-curing treatment is shown in p-MDI of the same cross-linker ratio, owing to the higher crosslinking capacity of emulsified polyisocyanate. Meanwhile, the relaxation times of all EPI specimens can be also greatly increased by post-curing.The change in relaxation time With increasing relative humidity, typical plasticization still occurs for EPI with high isocyanate content, leading to a clear decrease in the tensile rupture stress and Young’s modulus of cured adhesive layer at ambient temperature.(2)p-MDI, it exhibits much better mechanical properties and cross-linking for the whole RH range, because aqueous emulsified isocyanate has led to a more efficient chemical reaction with the functional groups available in/on the emulsion polymer compared to the general polyisocyanate cross-linker.The moisture resistance of EPI film can be significantly enhanced not only by increasing the isocyanate content, but also by the post-curing treatment. Although xPVAc/EMDI has a higher amount of residual NCO groups in the cured film than xPVAc/(3)g value of xPVAc/EMDI is higher than that of xPVAc/p-MDI at the same crosslinker loading due to a greater constraint on chain movement. Moreover, at a given The introduction of isocyanate groups contributes to an increase in the glass transition relative humidity of EPI film, and the RH(4)p-MDI.Post-cure is found to efficiently reduce the residual NCO groups in cured film, resulting in a significant increase in the crosslink density and also the mechanical behavior of EPI. Notably, the post-curing effect on EMDI cross-linked films is stronger than on those containing (5)The derived viscoelastic constitutive equation has quantitatively characterized the tensile stress-strain curves of EPI films under variable relative humidity conditions, and the related relaxation time can clarify the influence mechanism of the isocyanate type and loading, as well as post-curing treatment on the moisture resistance of the cured polymer.These results may shed light on the complicated mechanical behaviors of emulsion polymer isocyanate film in humid environments, which is of great significance for predicting the long-term performance of EPI-bonded structures. This study also provides insights into the role of EMDI and post-curing treatment, facilitating proper engineering designs and applications for high-performance adhesives.


We calibrate and validate the model using an in vitro retinal preparation. An RGC model based on accurate biophysics and realistic representation of cell morphology, was used to investigate how RGCs respond to HFS. The model was able to closely replicate the stimulus-strength-dependent suppression of RGC action potentials observed experimentally. Our results suggest that spike inhibition during HFS is due to local membrane hyperpolarization caused by outward membrane currents near the stimulus electrode. In addition, the extent of HFS-induced inhibition can be largely altered by the intrinsic properties of the inward sodium current. Finally, stimulus-strength-dependent suppression can be modulated by a wide range of stimulation frequencies, under generalized electrode placement conditions. In vitro experiments verified the computational modeling data. This modeling and experimental approach can be extended to further our understanding on the effects of novel stimulus strategies by simulating RGC stimulus-response profiles over a wider range of stimulation frequencies and electrode locations than have previously been explored.Recent retinal studies have directed more attention to sophisticated stimulation strategies based on high-frequency (>1.0 kHz) electrical stimulation (HFS). In these studies, each retinal ganglion cell (RGC) type demonstrated a characteristic stimulus-strength-dependent response to HFS, offering the intriguing possibility of focally targeting retinal neurons to provide useful visual information by retinal prosthetics. Ionic mechanisms are known to affect the responses of electrogenic cells during electrical stimulation. However, how these mechanisms affect RGC responses is not well understood at present, particularly when applying HFS. Here, we investigate this issue via an  Extracellular electrical stimulation is extensively used in electro-neural interfaces for the central and peripheral nervous systems . In partConsiderable research into high-frequency (defined as being higher than 1.0 kHz) electrical stimulation (HFS) is underway to understand the extent to which neuronal activity can be quantitatively controlled with greater spatiotemporal precision, in order to improve the performance of neuroprostheses. For example, HFS ranging from 2.0 to 20 kHz has been extensively used to block unwanted or unregulated generation of nerve impulses in many disabling conditions . In addiIn the retina, recent studies suggest that epiretinal HFS (1.0–6.25 kHz) is able to differentially activate functionally-different retinal ganglion cell (RGC) types . The RGCin silico investigations to gain insights into the mechanisms underlying experimentally-observed non-monotonic stimulus-response profiles during HFS. The model included accurate 3D morphological reconstruction of a single RGC, and its electrical response behavior was optimized against multiple whole-cell recordings from the same cell for accurate biophysics  location, a limitation of previous HFS work on RGCs , R is the radius of the disk (R = 15 μm), oI is the stimulus current, and ρe is the extracellular resistivity as described by in vitro methods for more details of stimulation parameters).where mV with a 3rd-order Butterworth filter with 20 Hz 3-dB cut-off frequency to remove spiking activities and the stimulus artifacts , or online within NEURON.To examine the effects of HFS on the (non-spiking) RGC membrane polarization over time, we lowpass-filtered the membrane potential in vitro experiments. Details on our whole-mount preparation and patch clamp recording with HFS can be found in All procedures were approved by the UNSW Animal Care and Ethics Committee and were carried out in compliance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Wild-type C57BL/6 female or male mice aged 4–8 weeks , were used in the in vitro experiments, each pulse amplitude was delivered three times. The mean spike-stimulus curve was calculated for each cell. For each RGC, we defined a 3D Cartesian  coordinate system, with the soma as the origin, such that the upper surface of the RGC dendritic field was aligned in the x-y plane and the RGC axon was aligned with the y-axis. A platinum-iridium stimulating electrode of 12.5-μm radius was placed at location 0, 0, and -40 μm.We delivered HFS using a STG 4002 stimulator , with a stimulus duration of 300 ms. We began by stimulating the RGCs with conventional 2-kHz extracellular biphasic pulse trains to investigate RGC stimulus-response profiles. Electrical stimulation waveforms were adapted from + reversal potentials on RGC responses to electrical stimulation, extracellular concentrations were custom-made (instead of using Ames’ solution) in order to adjust the Na+ concentration. There are three different [Na+] solutions. Chemical components and concentrations of each solution are listed in + concentrations and the calculated reversal potentials. In particular, the extracellular Na+ concentration in the first and second low Na+ solutions were adjusted to achieve custom Na+ reversal potentials. Na+ reversal potentials were calculated by the Goldman equation with [Na+]i of 19.5 mM in our internal solution recipe.To characterize the effects of NaNaE is the reversal potential of Na+, R is the ideal gas constant (8.314 J mol-1 K-1), T is the temperature in kelvin (307 K), F is Faraday’s constant (96485 C mol-1). The sequence in which the three solutions were applied was randomly set to avoid possible ordering effects.where in vitro studies  2-kHz extracellular biphasic pulse trains see . The amp studies .in vitro experiments.To investigate possible mechanisms underlying the non-monotonic extracellular response, and how this could be influenced by neuronal biophysical properties, we performed several sets of follow-up simulations and In another set of simulations, time-dependent membrane behavior was examined when the RGC was stimulated by 2-kHz HFS pulse trains of 10, 50, 62, or 80 μA amplitude. The slow potentials across the entire cell were plotted at 20, 30, and 40 ms time points (at which steady state was reached) after stimulation onset . When 10eV (extracellular voltage) and mV  are recorded below the stimulus electrode as a function of stimulus amplitude. Given low-amplitude stimuli (<10 μA), the intracellular membrane potential (iV) is the main contributor to mV due to the low magnitude of eV. However, with stimulus currents in excess of 20 μA, the magnitude of eV becomes more dominant and starts exceeding the sodium reversal potential, resulting in the mV range increasing in proportion to the increasing stimulus amplitude.In mV spanned from -40 to +30 mV at the peaks of the cathodic and anodic phases, respectively. Because mV is always below the reversal potential of sodium (NaV) activation of sodium channels by the stimuli therefore causes depolarization, and subsequently, action potentials. At stimulus amplitudes >20 μA, mV begins to exceed NaV  and inactivation (h) gating variables start to passively following the extracellular voltage changes with increasing HFS amplitude. At the same time, the outward sodium current becomes increasingly stronger with higher HFS amplitudes (bottom right panel).In Our simulations shown in NaV, we progressively altered the sodium . We modified HFS waveforms to generate stimulus frequencies up to 9-kHz. Cathodic-first, charge-balanced biphasic stimuli with a pulse width of 40 μs per phase were used  of stimulation were used to show the stimulus-frequency-dependent RGC response. N = 6) stimulus-dependent response curves from 1.0 to 8.33 kHz. Each pulse train was delivered three times. The mean spike-stimulus curve was calculated for each cell and the overall mean was calculated again across all RGCs (N = 6). The standard error of mean (SEM) was calculated to estimate the variability of the estimated mean of population-based RGC spike rates. For comparison, in vitro data suggested frequency-dependent inhibition which highly agrees with model predictions  in the spike-stimulus curve. In summary, both our modeling and in vitro results suggested that other than stimulus-strength dependency, HFS-induced spike suppression is also highly frequency-dependent and can be maximized by modulating stimulation frequencies.In in vitro patch-clamp recording and a spherical model of the soma. In their studies, somatic responses were inhibited when the monophasic stimulation pulse was above a certain amplitude. They suggested that sodium current reversal was the primary reason for the inhibition. in silico investigations using a dendrite-soma-axon computational model to propose an anodal block phenomenon, in which an anodic surround of the focal cathodic pulse caused the nerve membrane on the outer wall of a pipette to become hyperpolarized due to current converging toward the tip of the electrode. This upper threshold phenomenon was further studied using charge-balanced biphasic pulses of various amplitude and phase duration  localized membrane hyperpolarization is generated near the stimulus electrode and subsequently propagates to other neurites to suppress RGC excitation; (2) influence of sodium channel kinetics can strongly alter the shape of the stimulus-strength-dependent response profile; suggesting the important role of sodium channel properties in shaping RGC responses to HFS; (3) the inhibitory effect induced by electric stimulation can be maximized with sufficient stimulation rate. In addition, our results indicate that the non-monotonic RGC response to HFS does not arise through synaptic circuitry, since all in vitro results were observed after application of synaptic blockade.The non-monotonic stimulus-strength-dependent response has been previously reported in several retinal studies. duration , suggestStudies in the peripheral nervous system suggest that strong electrical stimulation may induce tonic membrane depolarization, keeping the channels in the inactivated state, thereby increasing activation threshold and preventing further spiking . Bianchiin vitro results shown in The second hypothesis is the inability of neurons to fully recover from their refractory period during HFS . This wain vitro and modeling studies indicated that appropriate HFS neuromodulation may elicit preferential excitation of different RGCs in a manner similar to RGC responses to light in a healthy retina, i.e., ON and OFF RGCs which respond with an increase in neural spiking activity to an increase or decrease in light intensity, respectively (in vitro and modeling studies are required to validate the reliability and generalizability of the non-monotonic nature of population-based RGC responses. Moreover, current modeling results are largely limited to somatic simulations, due to the lack of experimental data recorded in other neural processes. Recent modeling studies suggest that inhibition induced by electrical stimulation in the soma may not necessarily occur in the axon (in vivo studies indicate that high-amplitude inhibition in the retina could occur at higher visual processing centers (Without knowing how best to stimulate the retina, the vision quality elicited by retinal prosthetic devices will remain poor and unnatural. Previous ectively . In thesthe axon . However centers . In our In this study, using previously optimized ionic channel distributions and kinetic parameters for each cellular region and incorporating detailed cell morphology, we were able to predict RGC strength-dependent stimulus response patterns observed experimentally. Our computational modeling approaches allowed us to investigate a wide range of biophysical properties and stimulation settings beyond those recorded in the initial biological dataset, guiding further experimental design. The electric field can be accurately described by mathematical formalisms, and the neurons can be “probed” at resolutions well beyond those achievable by today’s state-of-the-art biological techniques, furthering our understanding of the effects of novel stimulus strategies by simulating RGC stimulus-response profiles over a larger stimulation parameter space than have previously been explored.All procedures were approved by the UNSW Animal Care and Ethics Committee and were carried out in compliance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.in vitro experiments. TG and DT performed the computational simulations. TG, DT, AAA, GS, JM, PT, SF, and NL analyzed the data. All the authors drafted the manuscript and read and approved of the final manuscript.TG, DT, SF, SD, and NL conceived and designed the study. CY, TG, and DT performed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study aimed to build a radiomics signature based on 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) for noninvasive measurement of the MGMT promoter methylation status in glioma.The methylation status of the On = 71) or validation cohort (n = 36). The MGMT promoter methylation status was measured by pyrosequencing. A total of 1561 radiomics features were extracted from the three-dimensional region of interest (ROI) on the standard uptake value (SUV) maps that were generated from the original 18F-FDG PET data. A radiomics signature, a clinical signature and a fusion signature that combined the clinical and radiomics features together were generated. The performance of the three signatures was evaluated by receiver operating characteristic (ROC) curve analysis, and the patient prognosis was stratified based on the MGMT promoter methylation status and the signature with the best performance.One hundred and seven pathology-confirmed primary diffuse glioma patients were retrospectively included and randomly assigned to the primary (p = 0.04).Five radiomics features were selected to construct the radiomics signature, and displayed the best performance with area under the receiver operating characteristic (ROC) curve (AUC) reaching 0.94 and 0.86 in the primary and validation cohorts, respectively, which outweigh the performances of clinical signature and fusion signature. With a median follow-up time of 32.4 months, the radiomics signature stratified the glioma patients into two risk groups with significantly different prognoses (18F-FDG-PET-based radiomics is a promising approach for preoperatively evaluating the MGMT promoter methylation status in glioma and predicting the prognosis of glioma patients noninvasively. This men glioma –5 that in glioma , and theRadiomics, a recently emerging technique for quantifying tumor characteristics with high-throughput radiomics features, allows prediction of the tumor phenotype through mathematic models that are built with selected radiomics features . Current18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) is an alternative molecular imaging technique that has been applied to tumor grading [18F-FDG uptake [18F-FDG-PET results and the MGMT promoter methylation status in glioma. However, to the best of our knowledge, no studies have focused on predicting the MGMT promoter methylation status using an 18F-FDG-PET-based radiomics approach. Since the MGMT promoter methylation status has been proven to be an independent prognostic and predictive marker in glioma regardless of the WHO classification or chemotherapy regimen [18F-FDG-PET radiomics may have great clinical potential. grading , surgica grading , and pro grading  in gliom grading . In addiG uptake , suggest regimen , 23, 24,18F-FDG-PET to build a conceivable model for predicting the MGMT promoter methylation status and patient prognosis noninvasively.This study retrospectively investigated the radiomics characteristics of gliomas by 18F-FDG-PET/CT examination between March 2010 and May 2018 at Peking Union Medical College Hospital were retrospectively reviewed. The inclusion criteria included the following: 1) adults with histopathologically confirmed WHO grade II-IV primary diffuse glioma without a previous history of CNS tumors; 2) preoperative 18F-FDG PET/CT examination of the brain; 3) sufficient paraffin-embedded tumor tissue for measurement of the MGMT promoter methylation status; and 4) no chemotherapy or radiotherapy delivered before 18F-FDG PET/CT acquisition and surgery. The study design was approved by the Institutional Review Board, and all patients provided informed consent. A total of 107 patients met the inclusion criteria and were randomly assigned to the primary cohort (n = 71) or the validation cohort (n = 36). The patient recruitment pathway is displayed in Fig. Patients who were pathologically diagnosed with primary glioma and underwent an The methylation status of the MGMT promoter was measured by pyrosequencing, as previously described . Briefly18F-FDG was produced using an RDS-111 Cyclotron . A dose of 5.55 MBq (0.15 mCi) 18F-FDG per kilogram of body weight was intravenously administered after the patient had fasted at least 4 h and their blood glucose level was determined not to exceed the normal limit (6.4 mM). The patient underwent 18F-FDG-PET/CT on a Biograph 64 TruePoint TrueV PET/CT system  after a 40–60 min time lag in standardized conditions , and acquired 148 axial slides with an interslice spacing of 3 mm.18F-FDG-PET data on the merged PET/CT images using ITK-SNAP software (http://www.itksnap.org/pmwiki/pmwiki.php), with patients’ contrast-enhanced T1-weighted images (for contrast-enhanced tumors) and T2-weighted fluid attenuated inversion recovery (FLAIR) images (for non-contrast-enhanced tumors) as anatomical reference. The ROIs were subsequently reviewed by a senior nuclear medical scientist blinded to the patients’ information. If there was a discrepancy of less than 5% between the ROIs placed by the two neurosurgeons, the final ROI was defined as the region of overlap, and if the discrepancy was greater than or equal to 5%, the nuclear medical scientist made the final decision.The three-dimensional region of interest (ROI) was segmented by two experienced neurosurgeons for the 18F-FDG-PET DICOM data using MATLAB version R2015b . A total of 1561 radiomics features, including 13 shape and size features, 18 first-order features, 68 texture features, 688 wavelet features and 680 further filtered  features were extracted using PyRadiomics (https://github.com/Radiomics/pyradiomics) [Standard uptake value (SUV) maps were generated from the original diomics) . The radThe radiomics features were reduced and selected through sequential application of the Wilcoxon rank-sum test and multivariate linear logistic regression with the L1 penalty.18F-FDG update but significant low central radioactivity, and solid metabolic tumor was defined as a lesion without a significant low metabolic necrosis or cysts inside the ROI [Five clinical features, respectively, age, sex, metabolic pattern (cystic or solid), SUVmax and SUVmean, were also evaluated. Cystic metabolic tumor was defined as a lesion with visible marginal  the ROI , 28. SUVThree predictive signatures, namely, a radiomics signature, clinical signature, and fusion signature, were constructed. The radiomics signature was generated with the radiomics features that were previously selected with a support vector machine (SVM). The clinical signature was generated with 5 clinical features using the logistic regression after selection by the Akaike information criterion (AIC). The selected clinical features and selected radiomics features were combined to generate the fusion signature using the logistic regression. The 3 signatures were independently validated in the validation cohort.The signatures were evaluated in terms of the area under the receiver operating characteristic (ROC) curve (AUC), accuracy, sensitivity, specificity, and positive and negative predictive values. Decision curve analysis was applied to reflect the clinical utility of the model , 30, andThe overall survival (OS) of patients was evaluated up to May 31, 2018. Kaplan-Meier curves were plotted based on the MGMT promoter methylation status and the signature with the best performance in stratifying the OS of patients. The log-rank test was utilized to determine differences in survival between the groups.https://www.r-project.org/). Statistically significant differences were defined by a two-tailed threshold of p < 0.05.Statistical analysis was performed with SPSS Statistics software, version 18.0  and R software, version 3.4.1 (p = 0.11–0.84). However, tumors with MGMT promoter methylation tend to have a higher rate for cystic metabolic pattern, and the difference of metabolic pattern for MGMT methylated and MGMT unmethylated patients reached statistical significance in the validation cohort .The clinical characteristics of patients in the primary and validation cohorts are summarized in Table Among the 1561 extracted radiomics features, 1543 redundant features were reduced through the Wilcoxon rank-sum test, and 5 final features were selected by logistic regression with the L1 penalty to build the radiomics signature. Only the metabolic pattern was selected by the AIC to build the clinical signature, and the fusion signature was built based on the radiomics signature and metabolic pattern. The selected radiomics features are shown in Table p < 0.0001 and p = 0.036, respectively), but the differences in the validation cohort were not significant  due to the limited number of patients. The decision curve reflecting the benefit of the radiomics signature showed a net benefit outweighing both schemes at any threshold probability in the primary cohort. The performance of the radiomics, clinical and fusion signatures is summarized in Table The radiomics signature performed the best among the three signatures in predicting the MGMT promoter methylation status, reaching an AUC of 0.94 in the primary cohort and 0.86 in the validation cohort. The clinical signature demonstrated a moderate predictive value and reached an AUC of 0.64 and 0.69 in the primary and validation cohorts, respectively. The fusion signature performed better than the clinical signature but poorer than the radiomics signature, with an AUC of 0.85 both in the primary and validation cohorts. The Delong test demonstrated that the radiomics signature performed significantly better than the clinical and fusion signatures in the primary cohort , and the differences within the high- and low-risk groups did not reach statistical significance. The Kaplan-Meier curves are shown in Fig. Among the 107 included patients, 100 patients who were known to survive to the closing date or to have an exact time of death were included in the prognosis analysis, and the median follow-up time is 32.4 months. Both the MGMT promoter methylation status and the radiomics signature stratified the glioma patients into a high-risk group and a low-risk group , in which the MGMT promoter status also has prognostic and predictive value [18F-FDG-PET-based radiomics signature can predict the MGMT promoter methylation status regardless of the WHO grade  or pathological information , suggesting the capability of noninvasive prediction without previous knowledge based on tumor samples.Previous studies on radiological evaluation of the MGMT promoter methylation status have mainly focused on the visual features, quantitative parameters or high-throughput radiomics features , 31–34 ove value , 23, 24.18F-FDG, a glucose analogue, can be taken up by cells but not further catabolized through glycolysis, making it a reliable radiotracer for measuring cancer cell metabolism. Considering the relationship between the glucose metabolism and oncogenic reprogramming [18F-FDG-PET may reflect certain molecular processes through imaging data, which is the theoretical basis of our study. However, compared with anatomical imaging modalities , 18F-FDG-PET has a relatively low spatial resolution, which limits the stability and accuracy of certain features, especially in lesions with a relatively small volume [Unlike MRI, which displays the structural characteristics of tumors, PET is a highly sensitive molecular imaging technique that reflects the altered tumor metabolism that is ubiquitous among cancer cells. Malignant brain tumors usually exhibit an altered glucose metabolism, in which glucose is converted to pyruvate and further into lactate instead of entering mitochondria and the citric acid cycle . 18F-FDGgramming , radiogel volume .Feature selection is a core step in radiomics studies since most features have little relevance to the MGMT promoter methylation status and may overwhelm the distinguishable features if they cannot be effectively reduced. The number of final selected features also needs to be balanced according to the patient cohort size because the addition of relevant features may increase performance in the primary cohort but may also result in overfitting of the radiomics signature. In our study, the Wilcoxon rank-sum test removed 1543 of the 1561 radiomics features that were irrelevant to the MGMT promoter methylation status, and logistic regression with the L1 penalty diluted the weights, allowing selection of the final 5 radiomics features to construct the radiomics signature. Although the selected radiomics features are not visually available to nuclear medicine physicians , the radiomics signature did provide additional assistance to physicians in the noninvasive molecular diagnosis of glioma , and the fusion signature was built with the 5 selected radiomics features and the metabolic pattern. However, the radiomics signature demonstrated the best performance and outweighed the clinical signature in both the primary and validation cohorts, suggesting that the selected radiomics features are more reliable than the clinically assessed imaging features in differentiating tumors based on the MGMT promotor methylation status. Objective clinical features  and the most frequently used quantitative imaging parameters  were excluded by the AIC when building the clinical signature, although some of these features are the only references for physicians in noninvasively evaluating the MGMT promoter methylation status without radiomics. Moreover, the addition of the clinical feature  to the set of radiomics features decreased the AUC of the prediction model, indicating a potential disturbance to the signature with the addition of features with less relevance. Thus, clinical features may not be integrated into the noninvasive radiomics evaluation of the MGMT promoter methylation status.The MGMT promoter has proven to be a strong prognostic biomarker in glioma. The retrospective investigation of the EORTC 26981/22981 trial demonstrated that the MGMT promoter methylation status is a favorable independent prognostic biomarker in glioblastoma , 6; the 18F-FDG-PET examination was not mandatorily performed. The necessity of differential diagnosis of the intracranial lesion or the evaluation of the extracranial situation were the major consideration to suggest an 18F-FDG-PET scan. Third, the radiomics model was constructed without subclassification of the metabolic pattern  and therefore may not include distinguishable features for determining the MGMT promoter methylation status in each subclassification. Fourth, more than half of the patients did not reach the endpoint of the prognosis analysis, which may have introduced bias to the prognosis data. Further studies with long-term follow-up periods may be needed to eliminate such imbalances. Finally, in addition to 18F-FDG-PET data, multimodality imaging data  may be further integrated into the radiomics model for predicting the MGMT promoter methylation status in glioma.The current study has several limitations. First, this was a single-center, retrospective study with a limited sample size, and the validation cohort is particularly restricted. Further prospective, multicenter studies with large patient cohorts may be essential for improving the generality and performance of the prediction model. Second, there may be a selection bias of the included patients since 18F-FDG-PET-based radiomics is a promising method for preoperatively evaluating the MGMT promoter methylation status in glioma and has the potential to guide the treatment and predict the prognosis of glioma patients noninvasively.