Control of DNA replication timing in the 3D genome.

The 3D organization of mammalian chromatin was described more than 30 years ago by visualizing sites of DNA synthesis at different times during the S phase of the cell cycle. These early cytogenetic studies revealed structurally stable chromosome domains organized into subnuclear compartments. Active-gene-rich domains in the nuclear interior replicate early, whereas more condensed chromatin domains that are largely at the nuclear and nucleolar periphery replicate later. During the past decade, this spatiotemporal DNA replication programme has been mapped along the genome and found to correlate with epigenetic marks, transcriptional activity and features of 3D genome architecture such as chromosome compartments and topologically associated domains. But the causal relationship between these features and DNA replication timing and the regulatory mechanisms involved have remained an enigma. The recent identification of cis-acting elements regulating the replication time and 3D architecture of individual replication domains and of long non-coding RNAs that coordinate whole chromosome replication provide insights into such mechanisms.

Genome-wide mapping of human DNA replication by optical replication mapping supports a stochastic model of eukaryotic replication.

The heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation sites make mapping the location and timing of replication initiation in human cells difficult. To address this challenge, we have developed optical replication mapping (ORM), a high-throughput single-molecule approach, and used it to map early-initiation events in human cells. The single-molecule nature of our data and a total of >2,500-fold coverage of the human genome on 27 million fibers averaging ∼300 kb in length allow us to identify initiation sites and their firing probability with high confidence. We find that the distribution of human replication initiation is consistent with inefficient, stochastic activation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.

HiCRes: a computational method to estimate and predict the genomic resolution of Hi-C libraries.

Three-dimensional (3D) conformation of the chromatin is crucial to stringently regulate gene expression patterns and DNA replication in a cell-type specific manner. Hi-C is a key technique for measuring 3D chromatin interactions genome wide. Estimating and predicting the resolution of a library is an essential step in any Hi-C experimental design. Here, we present the mathematical concepts to estimate the resolution of a dataset and predict whether deeper sequencing would enhance the resolution. We have developed HiCRes, a docker pipeline, by applying these concepts to several Hi-C libraries.

Stabilization of the methyl-CpG binding protein ZBTB38 by the deubiquitinase USP9X limits the occurrence and toxicity of oxidative stress in human cells.

Reactive oxygen species (ROS) are a byproduct of cell metabolism, and can also arise from environmental sources, such as toxins or radiation. Depending on dose and context, ROS have both beneficial and deleterious roles in mammalian development and disease, therefore it is crucial to understand how these molecules are generated, sensed, and detoxified. The question of how oxidative stress connects to the epigenome, in particular, is important yet incompletely understood. Here we show that an epigenetic regulator, the methyl-CpG-binding protein ZBTB38, limits the basal cellular production of ROS, is induced by ROS, and is required to mount a proper response to oxidative stress. Molecularly, these functions depend on a deubiquitinase, USP9X, which interacts with ZBTB38, deubiquitinates it, and stabilizes it. We find that USP9X is itself stabilized by oxidative stress, and is required together with ZBTB38 to limit the basal generation of ROS, as well as the toxicity of an acute oxidative stress. Our data uncover a new nuclear target of USP9X, show that the USP9X/ZBTB38 axis limits, senses and detoxifies ROS, and provide a molecular link between oxidative stress and the epigenome.

Laboratory Findings, Compassionate Use of Favipiravir, and Outcome in Patients With Ebola Virus Disease, Guinea, 2015-A Retrospective Observational Study.

BACKGROUND: In 2015, the laboratory at the Ebola treatment center in Coyah, Guinea, confirmed Ebola virus disease (EVD) in 286 patients. The cycle threshold (Ct) of an Ebola virus-specific reverse transcription-polymerase chain reaction assay and 13 blood chemistry parameters were measured on admission and during hospitalization. Favipiravir treatment was offered to patients with EVD on a compassionate-use basis. METHODS: To reduce biases in the raw field data, we carefully selected 163 of 286 patients with EVD for a retrospective study to assess associations between potential risk factors, alterations in blood chemistry findings, favipiravir treatment, and outcome. RESULTS: The case-fatality rate in favipiravir-treated patients was lower than in untreated patients (42.5% [31 of 73] vs 57.8% [52 of 90]; P = .053 by univariate analysis). In multivariate regression analysis, a higher Ct and a younger age were associated with survival (P < .001), while favipiravir treatment showed no statistically significant effect (P = .11). However, Kaplan-Meier analysis indicated a longer survival time in the favipiravir-treated group (P = .015). The study also showed characteristic changes in blood chemistry findings in patients who died, compared with survivors. CONCLUSIONS: Consistent with the JIKI trial, this retrospective study revealed a trend toward improved survival in favipiravir- treated patients; however, the effect of treatment was not statistically significant, except for its influence on survival time.

Dysfunction of lipid sensor GPR120 leads to obesity in both mouse and human.

Free fatty acids provide an important energy source as nutrients, and act as signalling molecules in various cellular processes. Several G-protein-coupled receptors have been identified as free-fatty-acid receptors important in physiology as well as in several diseases. GPR120 (also known as O3FAR1) functions as a receptor for unsaturated long-chain free fatty acids and has a critical role in various physiological homeostasis mechanisms such as adipogenesis, regulation of appetite and food preference. Here we show that GPR120-deficient mice fed a high-fat diet develop obesity, glucose intolerance and fatty liver with decreased adipocyte differentiation and lipogenesis and enhanced hepatic lipogenesis. Insulin resistance in such mice is associated with reduced insulin signalling and enhanced inflammation in adipose tissue. In human, we show that GPR120 expression in adipose tissue is significantly higher in obese individuals than in lean controls. GPR120 exon sequencing in obese subjects reveals a deleterious non-synonymous mutation (p.R270H) that inhibits GPR120 signalling activity. Furthermore, the p.R270H variant increases the risk of obesity in European populations. Overall, this study demonstrates that the lipid sensor GPR120 has a key role in sensing dietary fat and, therefore, in the control of energy balance in both humans and rodents.

Relationship between salivary/pancreatic amylase and body mass index: a systems biology approach.

BACKGROUND: Salivary (AMY1) and pancreatic (AMY2) amylases hydrolyze starch. Copy number of AMY1A (encoding AMY1) was reported to be higher in populations with a high-starch diet and reduced in obese people. These results based on quantitative PCR have been challenged recently. We aimed to re-assess the relationship between amylase and adiposity using a systems biology approach. METHODS: We assessed the association between plasma enzymatic activity of AMY1 or AMY2, and several metabolic traits in almost 4000 French individuals from D.E.S.I.R. longitudinal study. The effect of the number of copies of AMY1A (encoding AMY1) or AMY2A (encoding AMY2) measured through droplet digital PCR was then analyzed on the same parameters in the same study. A Mendelian randomization analysis was also performed. We subsequently assessed the association between AMY1A copy number and obesity risk in two case-control studies (5000 samples in total). Finally, we assessed the association between body mass index (BMI)-related plasma metabolites and AMY1 or AMY2 activity. RESULTS: We evidenced strong associations between AMY1 or AMY2 activity and lower BMI. However, we found a modest contribution of AMY1A copy number to lower BMI. Mendelian randomization identified a causal negative effect of BMI on AMY1 and AMY2 activities. Yet, we also found a significant negative contribution of AMY1 activity at baseline to the change in BMI during the 9-year follow-up, and a significant contribution of AMY1A copy number to lower obesity risk in children, suggesting a bidirectional relationship between AMY1 activity and adiposity. Metabonomics identified a BMI-independent association between AMY1 activity and lactate, a product of complex carbohydrate fermentation. CONCLUSIONS: These findings provide new insights into the involvement of amylase in adiposity and starch metabolism.

A central role for GRB10 in regulation of islet function in man.

Variants in the growth factor receptor-bound protein 10 (GRB10) gene were in a GWAS meta-analysis associated with reduced glucose-stimulated insulin secretion and increased risk of type 2 diabetes (T2D) if inherited from the father, but inexplicably reduced fasting glucose when inherited from the mother. GRB10 is a negative regulator of insulin signaling and imprinted in a parent-of-origin fashion in different tissues. GRB10 knock-down in human pancreatic islets showed reduced insulin and glucagon secretion, which together with changes in insulin sensitivity may explain the paradoxical reduction of glucose despite a decrease in insulin secretion. Together, these findings suggest that tissue-specific methylation and possibly imprinting of GRB10 can influence glucose metabolism and contribute to T2D pathogenesis. The data also emphasize the need in genetic studies to consider whether risk alleles are inherited from the mother or the father.

Contribution of the low-frequency, loss-of-function p.R270H mutation in FFAR4 (GPR120) to increased fasting plasma glucose levels.

BACKGROUND: We previously reported that the low-frequency, loss-of-function variant p.R270H in FFAR4 encoding the lipid sensor GPR120 was associated with obesity. Gpr120-deficient mice develop obesity and both impaired fasting glucose and glucose intolerance under a high-fat diet. We aimed to assess the contribution of p.R270H to type 2 diabetes (T2D) risk and the variation of glucose-related traits. METHODS: We genotyped p.R270H in 8996 non-diabetic individuals (among whom 4523 had an oral glucose tolerance test (OGTT)) and in a T2D case-control study including 4725 cases and 4339 controls. The regression models were adjusted for age, sex and body mass index (BMI). RESULTS: We found a significant association between p.R270H and increased fasting glucose levels (ß=0.092±0.05 mmol/L; p=4.13×10(-4)). Furthermore, p.R270H nominally contributed to decreased homeostasis model of pancreatic ß-cell function (HOMA-B; ß=-0.090±0.06; p=6.01×10(-3)). Despite a high statistical power, we did not find any significant association between p.R270H and T2D risk or the variation of fasting insulin levels, the homeostasis model of insulin resistance or OGTT-derived indices. CONCLUSIONS: These results suggest that the low-frequency p.R270H variant which inhibits GPR120 activity might influence fasting glucose levels in a normal physiological range. This study does not exclude that other coding mutations in FFAR4 with stronger functional effect than p.R270H may be associated with T2D.

Variation in FTO contributes to childhood obesity and severe adult obesity.

We identified a set of SNPs in the first intron of the FTO (fat mass and obesity associated) gene on chromosome 16q12.2 that is consistently strongly associated with early-onset and severe obesity in both adults and children of European ancestry with an experiment-wise P value of 1.67 x 10(-26) in 2,900 affected individuals and 5,100 controls. The at-risk haplotype yields a proportion of attributable risk of 22% for common obesity. We conclude that FTO contributes to human obesity and hence may be a target for subsequent functional analyses.

Emerging concept in DNA methylation: role of transcription factors in shaping DNA methylation patterns.

DNA methylation in mammals is a key epigenetic modification essential to normal genome regulation and development. DNA methylation patterns are established during early embryonic development, and subsequently maintained during cell divisions. Yet, discrete site-specific de novo DNA methylation or DNA demethylation events play a fundamental role in a number of physiological and pathological contexts, leading to critical changes in the transcriptional status of genes such as differentiation, tumor suppressor or imprinted genes. How the DNA methylation machinery targets specific regions of the genome during early embryogenesis and in adult tissues remains poorly understood. Here, we report advances being made in the field with a particular emphasis on the implication of transcription factors in establishing and in editing DNA methylation profiles.

Promoting the place of the allied health professions in clinical research.

Clinical research is of major importance to today's society, as scientific evidence is increasingly demanded as a basis for progress, whether this involves developing new healthcare products, improving clinical practice and care protocols or progress in prevention. Clinical research therefore requires professionals who are both experienced and increasingly well trained. Against this background, allied health professionals are becoming involved more and more, both as team members supporting clinical research projects and as managers or coordinators of projects in their own field. Clinical research activities provide an ideal opportunity for continuing professional development. All of this means that the professional skills of the allied health professions and clinical research support professions must be enhanced, their role promoted in the context of lecturer status and in the longer term, their status recognised by the supervisory authorities.

Two new Loci for body-weight regulation identified in a joint analysis of genome-wide association studies for early-onset extreme obesity in French and german study groups.

Meta-analyses of population-based genome-wide association studies (GWAS) in adults have recently led to the detection of new genetic loci for obesity. Here we aimed to discover additional obesity loci in extremely obese children and adolescents. We also investigated if these results generalize by estimating the effects of these obesity loci in adults and in population-based samples including both children and adults. We jointly analysed two GWAS of 2,258 individuals and followed-up the best, according to lowest p-values, 44 single nucleotide polymorphisms (SNP) from 21 genomic regions in 3,141 individuals. After this DISCOVERY step, we explored if the findings derived from the extremely obese children and adolescents (10 SNPs from 5 genomic regions) generalized to (i) the population level and (ii) to adults by genotyping another 31,182 individuals (GENERALIZATION step). Apart from previously identified FTO, MC4R, and TMEM18, we detected two new loci for obesity: one in SDCCAG8 (serologically defined colon cancer antigen 8 gene; p = 1.85x10(-8) in the DISCOVERY step) and one between TNKS (tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase gene) and MSRA (methionine sulfoxide reductase A gene; p = 4.84x10(-7)), the latter finding being limited to children and adolescents as demonstrated in the GENERALIZATION step. The odds ratios for early-onset obesity were estimated at approximately 1.10 per risk allele for both loci. Interestingly, the TNKS/MSRA locus has recently been found to be associated with adult waist circumference. In summary, we have completed a meta-analysis of two GWAS which both focus on extremely obese children and adolescents and replicated our findings in a large followed-up data set. We observed that genetic variants in or near FTO, MC4R, TMEM18, SDCCAG8, and TNKS/MSRA were robustly associated with early-onset obesity. We conclude that the currently known major common variants related to obesity overlap to a substantial degree between children and adults.

Stage-specific dynamic reorganization of genome topology shapes transcriptional neighborhoods in developing human retinal organoids.

We have generated a high-resolution Hi-C map of developing human retinal organoids to elucidate spatiotemporal dynamics of genomic architecture and its relationship with gene expression patterns. We demonstrate progressive stage-specific alterations in DNA topology and correlate these changes with transcription of cell-type-restricted gene markers during retinal differentiation. Temporal Hi-C reveals a shift toward A compartment for protein-coding genes and B compartment for non-coding RNAs, displaying high and low expression, respectively. Notably, retina-enriched genes are clustered near lost boundaries of topologically associated domains (TADs), and higher-order assemblages (i.e., TAD cliques) localize in active chromatin regions with binding sites for eye-field transcription factors. These genes gain chromatin contacts at their transcription start site as organoid differentiation proceeds. Our study provides a global view of chromatin architecture dynamics associated with diversification of cell types during retinal development and serves as a foundational resource for in-depth functional investigations of retinal developmental traits.

Prediction of childhood obesity by infancy weight gain: an individual-level meta-analysis.

To assess the predictive ability of infant weight gain on subsequent obesity we performed a meta-analysis of individual-level data on 47,661 participants from 10 cohort studies from the UK, France, Finland, Sweden, the US and Seychelles. For each individual, weight SD scores at birth and age 1 year were calculated using the same external reference (British 1990). Childhood obesity was defined by International Obesity Task Force criteria. Each +1 unit increase in weight SD scores between 0 and 1 year conferred a twofold higher risk of childhood obesity (odds ratio = 1.97 [95% confidence interval (CI) 1.83, 2.12]), and a 23% higher risk of adult obesity (odds ratio = 1.23 [1.16, 1.30]), adjusted for sex, age and birthweight. There was little heterogeneity between studies. A risk score for childhood obesity comprising weight gain 0-1 year, mother's body mass index, birthweight and sex was generated in a random 50% selection of individuals from general population cohorts with available information (n = 8236); this score showed moderate predictive ability in the remaining 50% sample (area under receiving operating curve = 77% [95% CI 74, 80%]). A separate risk score for childhood overweight showed similar predictive ability (area under receiving operating curve = 76% [73, 79%]). In conclusion, infant weight gain showed a consistent positive association with subsequent obesity. A risk score combining birthweight and infant weight gain (or simply infant weight), together with mother's body mass index and sex may allow early stratification of infants at risk of childhood obesity.

Context-dependent CpG methylation directs cell-specific binding of transcription factor ZBTB38.

DNA methylation on CpGs regulates transcription in mammals, both by decreasing the binding of methylation-repelled factors and by increasing the binding of methylation-attracted factors. Among the latter, zinc finger proteins have the potential to bind methylated CpGs in a sequence-specific context. The protein ZBTB38 is unique in that it has two independent sets of zinc fingers, which recognize two different methylated consensus sequences in vitro. Here, we identify the binding sites of ZBTB38 in a human cell line, and show that they contain the two methylated consensus sequences identified in vitro. In addition, we show that the distribution of ZBTB38 sites is highly unusual: while 10% of the ZBTB38 sites are also bound by CTCF, the other 90% of sites reside in closed chromatin and are not bound by any of the other factors mapped in our model cell line. Finally, a third of ZBTB38 sites are found upstream of long and active CpG islands. Our work therefore validates ZBTB38 as a methyl-DNA binder in vivo and identifies its unique distribution in the genome.

High-resolution genome topology of human retina uncovers super enhancer-promoter interactions at tissue-specific and multifactorial disease loci.

Chromatin organization and enhancer-promoter contacts establish unique spatiotemporal gene expression patterns in distinct cell types. Non-coding genetic variants can influence cellular phenotypes by modifying higher-order transcriptional hubs and consequently gene expression. To elucidate genomic regulation in human retina, we mapped chromatin contacts at high resolution and integrated with super-enhancers (SEs), histone marks, binding of CTCF and select transcription factors. We show that topologically associated domains (TADs) with central SEs exhibit stronger insulation and augmented contact with retinal genes relative to TADs with edge SEs. Merging genome-wide expression quantitative trait loci (eQTLs) with topology map reveals physical links between 100 eQTLs and corresponding eGenes associated with retinal neurodegeneration. Additionally, we uncover candidate genes for susceptibility variants linked to age-related macular degeneration and glaucoma. Our study of high-resolution genomic architecture of human retina provides insights into genetic control of tissue-specific functions, suggests paradigms for missing heritability, and enables the dissection of common blinding disease phenotypes.

Antialarmin effect of tick saliva during the transmission of Lyme disease.

Tick saliva has potent immunomodulatory properties. In arthropod-borne diseases, this effect is largely used by microorganisms to increase their pathogenicity and to evade host immune responses. We show that in Lyme borreliosis, tick salivary gland extract and a tick saliva protein, Salp15, inhibit in vitro keratinocyte inflammation induced by Borrelia burgdorferi sensu stricto or by the major outer surface lipoprotein of Borrelia, OspC. Chemokines (interleukin-8 [IL-8] and monocyte chemoattractant protein 1 [MCP-1]) and several antimicrobial peptides (defensins, cathelicidin, psoriasin, and RNase 7) were downregulated. Interestingly, antimicrobial peptides (AMPs) transiently inhibited bacterial motility but did not kill the organisms when tested in vitro. We conclude that tick saliva affects the chemotactic properties of chemokines and AMPs on immune cells and has an antialarmin effect on human primary keratinocytes. Alarmins are mediators that mobilize and activate antigen-presenting cells. Inhibition of cutaneous innate immunity and of the migration of immune cells to the site of the tick bite ensures a favorable environment for Borrelia. The bacterium can then multiply locally and, subsequently, disseminate to the target organs, including joints, heart, and the central nervous system.

European survey on national harmonization in clinical research.

BACKGROUND: Clinical trials remain key to the development of evidence-based medical practice. However, they are becoming increasingly complex, mainly in a multinational setting. To address these challenges, the European Union (EU) adopted the Clinical Trial Regulation EU No. 536/2014 (CTR). Once in force, the CTR will lead to more consistent rules and simplification of procedures for conducting clinical trials throughout the EU. Existing harmonization initiatives and "research infrastructures" for clinical trials may facilitate this process. This publication offers a snapshot of the current level of harmonization activities in academic clinical research in Europe. METHODS: A survey was performed among the member and observer countries of the European Clinical Research Infrastructure Network (ECRIN), using a standardized questionnaire. Three rounds of data collection were performed to maximize completeness and comparability of the received answers. The survey aimed to describe the harmonization of academic clinical research processes at national level, to facilitate the exchange of expertise and experience among countries, and to identify new fields of action. RESULTS: Most scientific partners already have in place various working groups and harmonization activities at national level. Furthermore, they are involved in and open to sharing their know-how and documents. Since harmonization was mainly a bottom-up approach up until now, the extent and topics dealt with are diverse and there is only little cross-networking and cross-country exchange so far. CONCLUSIONS: Currently, the ECRIN member countries offer a very solid base and collaborative spirit for further aligning processes and exchanging best practices for clinical research in Europe. They can support a smooth implementation of the EU CTR and may act as single contact with consolidated expertise in a country.