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Ageing is a complex biological process with variations among individuals, leading to the development of ageing clocks to estimate biological age. Glycans, particularly in immunoglobulin G (IgG), have emerged as potential biomarkers of ageing, with changes in glycosylation patterns correlating with chronological age. For precision analysis, three different plasma pools were analysed over 26 days in tetraplicates, 312 samples in total. In short-term variability analysis, two cohorts were analysed: AstraZeneca MFO cohort of 26 healthy individuals (median age 20) and a cohort of 70 premenopausal Chinese women (median age 22.5) cohort monitored over 3 months. Long-term variability analysis involved two adult men aged 47 and 57, monitored for 5 and 10 years, respectively. Samples were collected every 3 months and 3 weeks, respectively. IgG N-glycan analysis followed a standardized approach by isolating IgG, its subsequent denaturation and deglycosylation followed by glycan cleanup and labelling. Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) and ultra-performance liquid chromatography analyses were employed for glycan profiling. Statistical analysis involved normalization, batch correction, and linear mixed models to assess time effects on derived glycan traits. The intermediate precision results consistently exhibited very low coefficient of variation values across all three test samples. This consistent pattern underscores the high level of precision inherent in the CGE method for analysing the glycan clock of ageing. The AstraZeneca MFO cohort did not show any statistically significant trends, whereas the menstrual cycle cohort exhibited statistically significant trends in digalactosylated (G2), agalactosylated (G0) and fucosylation (F). These trends were attributed to the effects of the menstrual cycle. Long-term stability analysis identified enduring age-related trends in both subjects, showing a positive time effect in G0 and bisected N-acetylglucosamine, as well as a negative time effect in G2 and sialylation, aligning with earlier findings. Time effects measured for monogalactosylation, and F remained substantially lower than ones observed for other traits. The study found that IgG N-glycome analysis using CGE-LIF exhibited remarkably high intermediate precision. Moreover, the study highlights the short- and long-term stability of IgG glycome composition, coupled with a notable capacity to adapt and respond to physiological changes and environmental influences such as hormonal changes, disease, and interventions. The discoveries from this study propel personalized medicine forward by deepening our understanding of how IgG glycome relates to age-related health concerns. This study underscores the reliability of glycans as a biomarker for tracking age-related changes and individual health paths. [ABSTRACT FROM AUTHOR]

Although single-cell and spatial sequencing methods enable simultaneous measurement of more than one biological modality, no technology can capture all modalities within the same cell. For current data integration methods, the feasibility of cross-modal integration relies on the existence of highly correlated, a priori 'linked' features. We describe matching X-modality via fuzzy smoothed embedding (MaxFuse), a cross-modal data integration method that, through iterative coembedding, data smoothing and cell matching, uses all information in each modality to obtain high-quality integration even when features are weakly linked. MaxFuse is modality-agnostic and demonstrates high robustness and accuracy in the weak linkage scenario, achieving 20~70% relative improvement over existing methods under key evaluation metrics on benchmarking datasets. A prototypical example of weak linkage is the integration of spatial proteomic data with single-cell sequencing data. On two example analyses of this type, MaxFuse enabled the spatial consolidation of proteomic, transcriptomic and epigenomic information at single-cell resolution on the same tissue section. MaxFuse enables data integration between modalities even when features are weakly correlated. [ABSTRACT FROM AUTHOR]

Purpose: Miscarriage, often resulting from a variety of genetic factors, is a common pregnancy outcome. Preconception genetic carrier screening (PGCS) identifies at-risk partners for newborn genetic disorders; however, PGCS panels currently lack miscarriage-related genes. In this study, we evaluated the potential impact of both known and candidate genes on prenatal lethality and the effectiveness of PGCS in diverse populations. Methods: We analyzed 125,748 human exome sequences and mouse and human gene function databases. Our goals were to identify genes crucial for human fetal survival (lethal genes), to find variants not present in a homozygous state in healthy humans, and to estimate carrier rates of known and candidate lethal genes in various populations and ethnic groups. Results: This study identified 138 genes in which heterozygous lethal variants are present in the general population with a frequency of 0.5% or greater. Screening for these 138 genes could identify 4.6% (in the Finnish population) to 39.8% (in the East Asian population) of couples at risk of miscarriage. This explains the cause of pregnancy loss in approximately 1.1–10% of cases affected by biallelic lethal variants. Conclusion: This study has identified a set of genes and variants potentially associated with lethality across different ethnic backgrounds. The variation of these genes across ethnic groups underscores the need for a comprehensive, pan-ethnic PGCS panel that includes genes related to miscarriage. [ABSTRACT FROM AUTHOR]

The effectiveness of lifestyle interventions in reducing caloric intake and increasing physical activity for preventing Type 2 Diabetes (T2D) has been previously demonstrated. The use of modern technologies can potentially further improve the success of these interventions, promote metabolic health, and prevent T2D at scale. To test this concept, we built a remote program that uses continuous glucose monitoring (CGM) and wearables to make lifestyle recommendations that improve health. We enrolled 2,217 participants with varying degrees of glucose levels (normal range, and prediabetes and T2D ranges), using continuous glucose monitoring (CGM) over 28 days to capture glucose patterns. Participants logged food intake, physical activity, and body weight via a smartphone app that integrated wearables data and provided daily insights, including overlaying glucose patterns with activity and food intake, macronutrient breakdown, glycemic index (GI), glycemic load (GL), and activity measures. The app furthermore provided personalized recommendations based on users' preferences, goals, and observed glycemic patterns. Users could interact with the app for an additional 2 months without CGM. Here we report significant improvements in hyperglycemia, glucose variability, and hypoglycemia, particularly in those who were not diabetic at baseline. Body weight decreased in all groups, especially those who were overweight or obese. Healthy eating habits improved significantly, with reduced daily caloric intake and carbohydrate-to-calorie ratio and increased intake of protein, fiber, and healthy fats relative to calories. These findings suggest that lifestyle recommendations, in addition to behavior logging and CGM data integration within a mobile app, can enhance the metabolic health of both nondiabetic and T2D individuals, leading to healthier lifestyle choices. This technology can be a valuable tool for T2D prevention and treatment. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension (PAH) is a progressive disease in which pulmonary arterial (PA) endothelial cell (EC) dysfunction is associated with unrepaired DNA damage. BMPR2 is the most common genetic cause of PAH. We report that human PAEC with reduced BMPR2 have persistent DNA damage in room air after hypoxia (reoxygenation), as do mice with EC-specific deletion of Bmpr2 (EC-Bmpr2-/-) and persistent pulmonary hypertension. Similar findings are observed in PAEC with loss of the DNA damage sensor ATM, and in mice with Atm deleted in EC (EC-Atm-/-). Gene expression analysis of EC-Atm-/- and EC-Bmpr2-/- lung EC reveals reduced Foxf1, a transcription factor with selectivity for lung EC. Reducing FOXF1 in control PAEC induces DNA damage and impaired angiogenesis whereas transfection of FOXF1 in PAH PAEC repairs DNA damage and restores angiogenesis. Lung EC targeted delivery of Foxf1 to reoxygenated EC-Bmpr2-/- mice repairs DNA damage, induces angiogenesis and reverses pulmonary hypertension. It is unknown whether unrepaired DNA damage in lung endothelial cells causes persistent pulmonary arterial hypertension. Here, the authors combine oxidative stress with impaired BMPR2 signaling to link a reduction in FOXF1 to unrepaired DNA damage and impaired regeneration of normal endothelium. [ABSTRACT FROM AUTHOR]

The Human BioMolecular Atlas Program (HuBMAP) aims to compile a Human Reference Atlas (HRA) for the healthy adult body at the cellular level. Functional tissue units (FTUs), relevant for HRA construction, are of pathobiological significance. Manual segmentation of FTUs does not scale; highly accurate and performant, open-source machine-learning algorithms are needed. We designed and hosted a Kaggle competition that focused on development of such algorithms and 1200 teams from 60 countries participated. We present the competition outcomes and an expanded analysis of the winning algorithms on additional kidney and colon tissue data, and conduct a pilot study to understand spatial location and density of FTUs across the kidney. The top algorithm from the competition, Tom, outperforms other algorithms in the expanded study, while using fewer computational resources. Tom was added to the HuBMAP infrastructure to run kidney FTU segmentation at scale—showcasing the value of Kaggle competitions for advancing research. Results from a Kaggle competition and expanded analysis of the winning algorithms are presented for segmentation of functional tissue units as part of the Human BioMolecular Atlas Program (HuBMAP). [ABSTRACT FROM AUTHOR]

Mass surveillance testing can help control outbreaks of infectious diseases such as COVID-19. However, diagnostic test shortages are prevalent globally and continue to occur in the US with the onset of new COVID-19 variants and emerging diseases like monkeypox, demonstrating an unprecedented need for improving our current methods for mass surveillance testing. By targeting surveillance testing toward individuals who are most likely to be infected and, thus, increasing the testing positivity rate (i.e., percent positive in the surveillance group), fewer tests are needed to capture the same number of positive cases. Here, we developed an Intelligent Testing Allocation (ITA) method by leveraging data from the CovIdentify study (6765 participants) and the MyPHD study (8580 participants), including smartwatch data from 1265 individuals of whom 126 tested positive for COVID-19. Our rigorous model and parameter search uncovered the optimal time periods and aggregate metrics for monitoring continuous digital biomarkers to increase the positivity rate of COVID-19 diagnostic testing. We found that resting heart rate (RHR) features distinguished between COVID-19-positive and -negative cases earlier in the course of the infection than steps features, as early as 10 and 5 days prior to the diagnostic test, respectively. We also found that including steps features increased the area under the receiver operating characteristic curve (AUC-ROC) by 7–11% when compared with RHR features alone, while including RHR features improved the AUC of the ITA model's precision-recall curve (AUC-PR) by 38–50% when compared with steps features alone. The best AUC-ROC (0.73 ± 0.14 and 0.77 on the cross-validated training set and independent test set, respectively) and AUC-PR (0.55 ± 0.21 and 0.24) were achieved by using data from a single device type (Fitbit) with high-resolution (minute-level) data. Finally, we show that ITA generates up to a 6.5-fold increase in the positivity rate in the cross-validated training set and up to a 4.5-fold increase in the positivity rate in the independent test set, including both symptomatic and asymptomatic (up to 27%) individuals. Our findings suggest that, if deployed on a large scale and without needing self-reported symptoms, the ITA method could improve the allocation of diagnostic testing resources and reduce the burden of test shortages. [ABSTRACT FROM AUTHOR]

Physiologic laminar shear stress (LSS) induces an endothelial gene expression profile that is vasculo-protective. In this report, we delineate how LSS mediates changes in the epigenetic landscape to promote this beneficial response. We show that under LSS, KLF4 interacts with the SWI/SNF nucleosome remodeling complex to increase accessibility at enhancer sites that promote the expression of homeostatic endothelial genes. By combining molecular and computational approaches we discover enhancers that loop to promoters of KLF4- and LSS-responsive genes that stabilize endothelial cells and suppress inflammation, such as BMPR2, SMAD5, and DUSP5. By linking enhancers to genes that they regulate under physiologic LSS, our work establishes a foundation for interpreting how non-coding DNA variants in these regions might disrupt protective gene expression to influence vascular disease. Here the authors studied pulmonary arterial endothelial cells (PAEC) under laminar shear stress and show that this physiologic condition markedly changes chromatin accessibility at regulatory regions, when compared to cells grown in a static state. They find that KLF4 organizes chromatin by interacting with the SWI/SNF nucleosome remodeling complex to regulate vasculo-protective gene expression. [ABSTRACT FROM AUTHOR]

Reproducibility, traceability, and transparency have been long-standing issues for metabolomics data analysis. Multiple tools have been developed, but limitations still exist. Here, we present the tidyMass project (https://www.tidymass.org/), a comprehensive R-based computational framework that can achieve the traceable, shareable, and reproducible workflow needs of data processing and analysis for LC-MS-based untargeted metabolomics. TidyMass is an ecosystem of R packages that share an underlying design philosophy, grammar, and data structure, which provides a comprehensive, reproducible, and object-oriented computational framework. The modular architecture makes tidyMass a highly flexible and extensible tool, which other users can improve and integrate with other tools to customize their own pipeline. Reproducibility, traceability, and transparency have been long-standing issues in metabolomics data analysis. Here, the authors present tidyMass, an R-based computational framework that allows designing traceable, shareable, and reproducible data processing and analysis workflows for untargeted metabolomics. [ABSTRACT FROM AUTHOR]

RNA polymerase III (Pol III) includes two alternate isoforms, defined by mutually exclusive incorporation of subunit POLR3G (RPC7α) or POLR3GL (RPC7β), in mammals. The contributions of POLR3G and POLR3GL to transcription potential has remained poorly defined. Here, we discover that loss of subunit POLR3G is accompanied by a restricted repertoire of genes transcribed by Pol III. Particularly sensitive is snaR-A, a small noncoding RNA implicated in cancer proliferation and metastasis. Analysis of Pol III isoform biases and downstream chromatin features identifies loss of POLR3G and snaR-A during differentiation, and conversely, re-establishment of POLR3G gene expression and SNAR-A gene features in cancer contexts. Our results support a model in which Pol III identity functions as an important transcriptional regulatory mechanism. Upregulation of POLR3G, which is driven by MYC, identifies a subgroup of patients with unfavorable survival outcomes in specific cancers, further implicating the POLR3G-enhanced transcription repertoire as a potential disease factor. RNA polymerase III changes its subunit composition during mammalian development. Here the authors report that loss of subunit POLR3G, which re-emerges in cancer, promotes expression of small NF90-associated RNA (snaR-A), a noncoding RNA implicated in cell proliferation and metastasis. [ABSTRACT FROM AUTHOR]

In this study, two long-read sequencing (LRS) techniques, MinION from Oxford Nanopore Technologies and Sequel from the Pacific Biosciences, were used for the transcriptional characterization of a prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus. LRS is able to read full-length RNA molecules, and thereby distinguish between transcript isoforms, mono- and polycistronic RNAs, and overlapping transcripts. Altogether, we detected 875 transcript species, of which 759 were novel and 116 were annotated previously. These RNA molecules include 41 novel putative protein coding transcripts [each containing 5′-truncated in-frame open reading frames (ORFs), 14 monocistronic transcripts, 99 polygenic RNAs, 101 non-coding RNAs, and 504 untranslated region isoforms. This work also identified novel replication origin-associated transcripts, upstream ORFs, cis-regulatory sequences and poly(A) sites. We also detected RNA methylation in 99 viral genes and RNA hyper-editing in the longer 5′-UTR transcript isoform of the canonical ORF 19 transcript. [ABSTRACT FROM AUTHOR]

Although alterations in chromatin structure are known to exist in tumors, how these alterations relate to molecular phenotypes in cancer remains to be demonstrated. Multi-omics profiling of human tumors can provide insight into how alterations in chromatin structure are propagated through the pathway of gene expression to result in malignant protein expression. We applied multi-omics profiling of chromatin accessibility, RNA abundance, and protein abundance to 36 human thyroid cancer primary tumors, metastases, and patient-match normal tissue. Through quantification of chromatin accessibility associated with active transcription units and global protein expression, we identify a local chromatin structure that is highly correlated with coordinated RNA and protein expression. In particular, we identify enhancers located within gene-bodies as predictive of correlated RNA and protein expression, that is independent of overall transcriptional activity. To demonstrate the generalizability of these findings we also identify similar results in an independent cohort of human breast cancers. Taken together, these analyses suggest that local enhancers, rather than distal enhancers, are likely most predictive of cancer gene expression phenotypes. This allows for identification of potential targets for cancer therapeutic approaches and reinforces the utility of multi-omics profiling as a methodology to understand human disease. Studies show the cancer transcriptome correlates poorly with the cancer proteome, questioning the role of chromatin regulation. Here the authors demonstrate proximal-gene-body chromatin elements and transcription predict abundances of differentially expressed proteins in thyroid and breast cancers. [ABSTRACT FROM AUTHOR]

Long-read sequencing (LRS), a powerful novel approach, is able to read full-length transcripts and confers a major advantage over the earlier gold standard short-read sequencing in the efficiency of identifying for example polycistronic transcripts and transcript isoforms, including transcript length- and splice variants. In this work, we profile the human cytomegalovirus transcriptome using two third-generation LRS platforms: the Sequel from Pacific BioSciences, and MinION from Oxford Nanopore Technologies. We carried out both cDNA and direct RNA sequencing, and applied the LoRTIA software, developed in our laboratory, for the transcript annotations. This study identified a large number of novel transcript variants, including splice isoforms and transcript start and end site isoforms, as well as putative mRNAs with truncated in-frame ORFs (located within the larger ORFs of the canonical mRNAs), which potentially encode N-terminally truncated polypeptides. Our work also disclosed a highly complex meshwork of transcriptional read-throughs and overlaps. [ABSTRACT FROM AUTHOR]

Introduction: While continuous glucose monitoring (CGM) has been shown to decrease both hyper- and hypoglycemia in insulin-treated diabetes, its value in non-insulin-treated type 2 diabetes (T2D) and prediabetes is unclear. Studies examining the reduction in hyperglycemia with the use of CGM in non-insulin-treated T2D are limited. Methods: We investigated the potential benefit of CGM combined with a mobile app that links each individual's glucose tracing to meal composition, heart rate, and physical activity in a cohort of 1022 individuals, ranging from nondiabetic to non-insulin-treated T2D, spanning a wide range of demographic, geographic, and socioeconomic characteristics. The primary endpoint was the change in time in range (TIR), defined as 54–140 mg/dL for healthy and prediabetes, and 54–180 mg/dL for T2D, from the beginning to end of a 10-day period of use of the Freestyle Libre CGM. Logged food intake, physical activity, continuous glucose, and heart rate data were captured by a smartphone-based app that continuously provided feedback to participants, overlaying daily glucose patterns with activity and food intake, including macronutrient breakdown, glycemic load (GL), and glycemic index (GI). Results: A total of 665 participants meeting eligibility and data requirements were included in the final analysis. Among self-reported nondiabetic participants, CGM identified glucose excursions in the diabetic range among 15% of healthy and 36% of those with prediabetes. In the group as a whole, TIR improved significantly (p < 0.001). Among the 51.4% of participants who improved, TIR increased by an average of 6.4% (p < 0.001). Of those with poor baseline TIR, defined as TIR below comparable A1c thresholds for T2D and prediabetes, 58.3% of T2D and 91.7% of healthy/prediabetes participants improved their TIR by an average of 22.7% and 23.2%, respectively. Predictors of improved response included no prior diagnosis of T2D and lower BMI. Conclusions: These results indicate that 10-day use of CGM as a part of multimodal data collection, with synthesis and feedback to participants provided by a mobile health app, can significantly reduce hyperglycemia in non-insulin-treated individuals, including those with early stages of glucose dysregulation. [ABSTRACT FROM AUTHOR]

Obesity is associated with chronic peripheral inflammation, is a risk factor for stroke, and causes increased infarct sizes. To characterize how obesity increases infarct size, we fed a high-fat diet to wild-type C57BL/6J mice for either 6 weeks or 15 weeks and then induced distal middle cerebral artery strokes. We found that infarct expansion happened late after stroke. There were no differences in cortical neuroinflammation (astrogliosis, microgliosis, or pro-inflammatory cytokines) either prior to or 10 h after stroke, and also no differences in stroke size at 10 h. However, by 3 days after stroke, animals fed a high-fat diet had a dramatic increase in microgliosis and astrogliosis that was associated with larger strokes and worsened functional recovery. RNA sequencing revealed a dramatic increase in inflammatory genes in the high-fat diet-fed animals 3 days after stroke that were not present prior to stroke. Genetic pathways unique to diet-induced obesity were primarily related to adaptive immunity, extracellular matrix components, cell migration, and vasculogenesis. The late appearance of neuroinflammation and infarct expansion indicates that there may be a therapeutic window between 10 and 36 h after stroke where inflammation and obesity-specific transcriptional programs could be targeted to improve outcomes in people with obesity and stroke. [ABSTRACT FROM AUTHOR]

Preterm birth is the leading cause of mortality in children under the age of five worldwide. Despite major efforts, we still lack the ability to accurately predict and effectively prevent preterm birth. While multiple factors contribute to preterm labor, dysregulations of immunological adaptations required for the maintenance of a healthy pregnancy is at its pathophysiological core. Consequently, a precise understanding of these chronologically paced immune adaptations and of the biological pacemakers that synchronize the pregnancy "immune clock" is a critical first step towards identifying deviations that are hallmarks of peterm birth. Here, we will review key elements of the fetal, placental, and maternal pacemakers that program the immune clock of pregnancy. We will then emphasize multiomic studies that enable a more integrated view of pregnancy-related immune adaptations. Such multiomic assessments can strengthen the biological plausibility of immunological findings and increase the power of biological signatures predictive of preterm birth [ABSTRACT FROM AUTHOR]

Physical interactions between distal regulatory elements have a key role in regulating gene expression, but the extent to which these interactions vary between cell types and contribute to cell-type-specific gene expression remains unclear. Here, to address these questions as part of phase III of the Encyclopedia of DNA Elements (ENCODE), we mapped cohesin-mediated chromatin loops, using chromatin interaction analysis by paired-end tag sequencing (ChIA-PET), and analysed gene expression in 24 diverse human cell types, including core ENCODE cell lines. Twenty-eight per cent of all chromatin loops vary across cell types; these variations modestly correlate with changes in gene expression and are effective at grouping cell types according to their tissue of origin. The connectivity of genes corresponds to different functional classes, with housekeeping genes having few contacts, and dosage-sensitive genes being more connected to enhancer elements. This atlas of chromatin loops complements the diverse maps of regulatory architecture that comprise the ENCODE Encyclopedia, and will help to support emerging analyses of genome structure and function. A map of cohesin-mediated chromatin loops in 24 types of human cells identifies loops that show cell-type-specific variation, indicating that chromatin loops may help to specify cell-specific gene expression programs and functions. [ABSTRACT FROM AUTHOR]

In this meta-analysis, we re-analysed and compared herpes simplex virus type 1 transcriptomic data generated by eight studies using various short- and long-read sequencing techniques and different library preparation methods. We identified a large number of novel mRNAs, non-coding RNAs and transcript isoforms, and validated many previously published transcripts. Here, we present the most complete HSV-1 transcriptome to date. Furthermore, we also demonstrate that various sequencing techniques, including both cDNA and direct RNA sequencing approaches, are error-prone, which can be circumvented by using integrated approaches. This work draws attention to the need for using multiple sequencing approaches and meta-analyses in transcriptome profiling studies to obtain reliable results. [ABSTRACT FROM AUTHOR]

Environmental and epigenetic factors often play an important role in polygenic disorders. However, how such factors affect disease-specific tissues at the molecular level remains to be understood. Here, we address this in pulmonary arterial hypertension (PAH). We obtain pulmonary arterial endothelial cells (PAECs) from lungs of patients and controls (n = 19), and perform chromatin, transcriptomic and interaction profiling. Overall, we observe extensive remodeling at active enhancers in PAH PAECs and identify hundreds of differentially active TFs, yet find very little transcriptomic changes in steady-state. We devise a disease-specific enhancer-gene regulatory network and predict that primed enhancers in PAH PAECs are activated by the differentially active TFs, resulting in an aberrant response to endothelial signals, which could lead to disturbed angiogenesis and endothelial-to-mesenchymal-transition. We validate these predictions for a selection of target genes in PAECs stimulated with TGF-β, VEGF or serotonin. Our study highlights the role of chromatin state and enhancers in disease-relevant cell types of PAH. Pulmonary arterial hypertension (PAH) is a heterogeneous disease, causing severe breathing problems and cardiac morbidity. Here, the authors study chromatin marks in pulmonary arterial endothelial cells from PAH patients and controls and find changes in transcription factor and enhancer activity that suggest an aberrant response to signalling in PAH. [ABSTRACT FROM AUTHOR]

Age-associated chronic inflammation (inflammageing) is a central hallmark of ageing1, but its influence on specific cells remains largely unknown. Fibroblasts are present in most tissues and contribute to wound healing2,3. They are also the most widely used cell type for reprogramming to induced pluripotent stem (iPS) cells, a process that has implications for regenerative medicine and rejuvenation strategies4. Here we show that fibroblast cultures from old mice secrete inflammatory cytokines and exhibit increased variability in the efficiency of iPS cell reprogramming between mice. Variability between individuals is emerging as a feature of old age5–8, but the underlying mechanisms remain unknown. To identify drivers of this variability, we performed multi-omics profiling of fibroblast cultures from young and old mice that have different reprogramming efficiencies. This approach revealed that fibroblast cultures from old mice contain 'activated fibroblasts' that secrete inflammatory cytokines, and that the proportion of activated fibroblasts in a culture correlates with the reprogramming efficiency of that culture. Experiments in which conditioned medium was swapped between cultures showed that extrinsic factors secreted by activated fibroblasts underlie part of the variability between mice in reprogramming efficiency, and we have identified inflammatory cytokines, including TNF, as key contributors. Notably, old mice also exhibited variability in wound healing rate in vivo. Single-cell RNA-sequencing analysis identified distinct subpopulations of fibroblasts with different cytokine expression and signalling in the wounds of old mice with slow versus fast healing rates. Hence, a shift in fibroblast composition, and the ratio of inflammatory cytokines that they secrete, may drive the variability between mice in reprogramming in vitro and influence wound healing rate in vivo. This variability may reflect distinct stochastic ageing trajectories between individuals, and could help in developing personalized strategies to improve iPS cell generation and wound healing in elderly individuals. Fibroblasts from old mice are heterogeneous, which affects the ability of these fibroblasts to reprogram into induced pluripotent stem cells in vitro and influences wound healing rate in vivo. [ABSTRACT FROM AUTHOR]

Transformative technologies are enabling the construction of three-dimensional maps of tissues with unprecedented spatial and molecular resolution. Over the next seven years, the NIH Common Fund Human Biomolecular Atlas Program (HuBMAP) intends to develop a widely accessible framework for comprehensively mapping the human body at single-cell resolution by supporting technology development, data acquisition, and detailed spatial mapping. HuBMAP will integrate its efforts with other funding agencies, programs, consortia, and the biomedical research community at large towards the shared vision of a comprehensive, accessible three-dimensional molecular and cellular atlas of the human body, in health and under various disease conditions. HuBMAP supports technology development, data acquisition, and spatial analyses to generate comprehensive molecular and cellular three-dimensional tissue maps. [ABSTRACT FROM AUTHOR]

The human genome is generally organized into stable chromosomes, and only tumor cells are known to accumulate kilobase (kb)-sized extrachromosomal circular DNA elements (eccDNAs). However, it must be expected that kb eccDNAs exist in normal cells as a result of mutations. Here, we purify and sequence eccDNAs from muscle and blood samples from 16 healthy men, detecting ~100,000 unique eccDNA types from 16 million nuclei. Half of these structures carry genes or gene fragments and the majority are smaller than 25 kb. Transcription from eccDNAs suggests that eccDNAs reside in nuclei and recurrence of certain eccDNAs in several individuals implies DNA circularization hotspots. Gene-rich chromosomes contribute to more eccDNAs per megabase and the most transcribed protein-coding gene in muscle, TTN (titin), provides the most eccDNAs per gene. Thus, somatic genomes are rich in chromosome-derived eccDNAs that may influence phenotypes through altered gene copy numbers and transcription of full-length or truncated genes. [ABSTRACT FROM AUTHOR]

Pseudorabies virus (PRV) is an animal alphaherpesvirus with a wide host range. PRV has 67 protein-coding genes and several non-coding RNA molecules, which can be classified into three temporal groups, immediate early, early and late classes. The ul54 gene of PRV and its homolog icp27 of herpes simplex virus have a multitude of functions, including the regulation of viral DNA synthesis and the control of the gene expression. Therefore, abrogation of PRV ul54 function was expected to exert a significant effect on the global transcriptome and on DNA replication. Real-time PCR and real-time RT-PCR platforms were used to investigate these presumed effects. Our analyses revealed a drastic impact of the ul54 mutation on the genome-wide expression of PRV genes, especially on the transcription of the true late genes. A more than two hour delay was observed in the onset of DNA replication, and the amount of synthesized DNA molecules was significantly decreased in comparison to the wild-type virus. Furthermore, in this work, we were able to successfully demonstrate the utility of long-read SMRT sequencing for genotyping of mutant viruses. [ABSTRACT FROM AUTHOR]

RNA-sequencing (RNA-seq) is an essential technique for transcriptome studies, hundreds of analysis tools have been developed since it was debuted. Although recent efforts have attempted to assess the latest available tools, they have not evaluated the analysis workflows comprehensively to unleash the power within RNA-seq. Here we conduct an extensive study analysing a broad spectrum of RNA-seq workflows. Surpassing the expression analysis scope, our work also includes assessment of RNA variant-calling, RNA editing and RNA fusion detection techniques. Specifically, we examine both short- and long-read RNA-seq technologies, 39 analysis tools resulting in ~120 combinations, and ~490 analyses involving 15 samples with a variety of germline, cancer and stem cell data sets. We report the performance and propose a comprehensive RNA-seq analysis protocol, named RNACocktail, along with a computational pipeline achieving high accuracy. Validation on different samples reveals that our proposed protocol could help researchers extract more biologically relevant predictions by broad analysis of the transcriptome. [ABSTRACT FROM AUTHOR]

Alopecia with mental retardation syndrome (APMR) is a very rare autosomal recessive condition that is associated with total or partial absence of hair from the scalp and other parts of the body as well as variable intellectual disability. Here we present whole-exome sequencing results of a large consanguineous family segregating APMR syndrome with seven affected family members. Our study revealed a novel predicted pathogenic, homozygous missense mutation in the AHSG (OMIM 138680) gene (AHSG: NM_001622:exon7:c.950G>A:p.Arg317His). The variant is predicted to affect a region of the protein required for protein processing and disrupts a phosphorylation motif. In addition, the altered protein migrates with an aberrant size relative to healthy individuals. Consistent with the phenotype, AHSG maps within APMR linkage region 1 (APMR 1) as reported before, and falls within runs of homozygosity (ROH). Previous families with APMR syndrome have been studied through linkage analyses and the linkage resolution did not allow pointing out to a single gene candidate. Our study is the first report to identify a homozygous missense mutation for APMR syndrome through whole-exome sequencing. [ABSTRACT FROM AUTHOR]

Mycosis fungoides and Sézary syndrome comprise the majority of cutaneous T cell lymphomas (CTCLs), disorders notable for their clinical heterogeneity that can present in skin or peripheral blood. Effective treatment options for CTCL are limited, and the genetic basis of these T cell lymphomas remains incompletely characterized. Here we report recurrent point mutations and genomic gains of TNFRSF1B, encoding the tumor necrosis factor receptor TNFR2, in 18% of patients with mycosis fungoides and Sézary syndrome. Expression of the recurrent TNFR2 Thr377Ile mutant in T cells leads to enhanced non-canonical NF-κB signaling that is sensitive to the proteasome inhibitor bortezomib. Using an integrative genomic approach, we additionally discovered a recurrent CTLA4-CD28 fusion, as well as mutations in downstream signaling mediators of these receptors. [ABSTRACT FROM AUTHOR]

Cell-to-cell variation is a universal feature of life that affects a wide range of biological phenomena, from developmental plasticity to tumour heterogeneity. Although recent advances have improved our ability to document cellular phenotypic variation, the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of mammalian DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells by assay for transposase-accessible chromatin using sequencing (ATAC-seq) integrated into a programmable microfluidics platform. Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provide insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types. Targeted perturbations of cell cycle or transcription factor signalling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome compartments de novo, linking single-cell accessibility variation to three-dimensional genome organization. Single-cell analysis of DNA accessibility provides new insight into cellular variation of the 'regulome'. [ABSTRACT FROM AUTHOR]

Aberrant regulation of gene expression in cancer can promote survival and proliferation of cancer cells. Here we integrate whole-genome sequencing data from The Cancer Genome Atlas (TCGA) for 436 patients from 8 cancer subtypes with ENCODE and other regulatory annotations to identify point mutations in regulatory regions. We find evidence for positive selection of mutations in transcription factor binding sites, consistent with these sites regulating important cancer cell functions. Using a new method that adjusts for sample- and genomic locus-specific mutation rates, we identify recurrently mutated sites across individuals with cancer. Mutated regulatory sites include known sites in the TERT promoter and many new sites, including a subset in proximity to cancer-related genes. In reporter assays, two new sites display decreased enhancer activity upon mutation. These data demonstrate that many regulatory regions contain mutations under selective pressure and suggest a greater role for regulatory mutations in cancer than previously appreciated. [ABSTRACT FROM AUTHOR]

Alternative splicing shapes mammalian transcriptomes, with many RNA molecules undergoing multiple distant alternative splicing events. Comprehensive transcriptome analysis, including analysis of exon co-association in the same molecule, requires deep, long-read sequencing. Here we introduce an RNA sequencing method, synthetic long-read RNA sequencing (SLR-RNA-seq), in which small pools (≤1,000 molecules/pool, ≤1 molecule/gene for most genes) of full-length cDNAs are amplified, fragmented and short-read-sequenced. We demonstrate that these RNA sequences reconstructed from the short reads from each of the pools are mostly close to full length and contain few insertion and deletion errors. We report many previously undescribed isoforms (human brain: ∼13,800 affected genes, 14.5% of molecules; mouse brain ∼8,600 genes, 18% of molecules) and up to 165 human distant molecularly associated exon pairs (dMAPs) and distant molecularly and mutually exclusive pairs (dMEPs). Of 16 associated pairs detected in the mouse brain, 9 are conserved in human. Our results indicate conserved mechanisms that can produce distant but phased features on transcript and proteome isoforms. [ABSTRACT FROM AUTHOR]