High Resolution Detection and Analysis of CpG Dinucleotides Methylation Using MBD-Seq Technology

Methyl-CpG binding domain protein sequencing (MBD-seq) is widely used to survey DNA methylation patterns. However, the optimal experimental parameters for MBD-seq remain unclear and the data analysis remains challenging. In this study, we generated high depth MBD-seq data in MCF-7 cell and developed a bi-asymmetric-Laplace model (BALM) to perform data analysis. We found that optimal efficiency of MBD-seq experiments was achieved by sequencing ∼100 million unique mapped tags from a combination of 500 mM and 1000 mM salt concentration elution in MCF-7 cells. Clonal bisulfite sequencing results showed that the methylation status of each CpG dinucleotides in the tested regions was accurately detected with high resolution using the proposed model. These results demonstrated the combination of MBD-seq and BALM could serve as a useful tool to investigate DNA methylome due to its low cost, high specificity, efficiency and resolution

Characterization of FUS Mutations in Amyotrophic Lateral Sclerosis Using RNA-Seq

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting in severe muscle weakness and eventual death by respiratory failure. Although little is known about its pathogenesis, mutations in fused in sarcoma/translated in liposarcoma (FUS) are causative for familial ALS. FUS is a multifunctional protein that is involved in many aspects of RNA processing. To elucidate the role of FUS in ALS, we overexpressed wild-type and two mutant forms of FUS in HEK-293T cells, as well as knocked-down FUS expression. This was followed by RNA-Seq to identify genes which displayed differential expression or altered splicing patterns. Pathway analysis revealed that overexpression of wild-type FUS regulates ribosomal genes, whereas knock-down of FUS additionally affects expression of spliceosome related genes. Furthermore, cells expressing mutant FUS displayed global transcription patterns more similar to cells overexpressing wild-type FUS than to the knock-down condition. This observation suggests that FUS mutants do not contribute to the pathogenesis of ALS through a loss-of-function. Finally, our results demonstrate that the R521G and R522G mutations display differences in their influence on transcription and splicing. Taken together, these results provide additional insights into the function of FUS and how mutations contribute to the development of ALS.ALS Foundation NetherlandsAdessium FoundationSeventh Framework Programme (European Commission) (grant number 259867)Thierry Latran FoundationNational Institutes of Health (U.S.) (NIH/NINDS grant R01NS073873)National Institute of Neurological Disorders and Stroke (U.S.) (NIH/NINDS grant numbers 1R01NS065847

A distinct role for B1b lymphocytes in T cell-independent immunity

Pathogenesis of infectious disease is not only determined by the virulence of the microbe but also by the immune status of the host. Vaccination is the most effective means to control infectious diseases. A hallmark of the adaptive immune system is the generation of B cell memory, which provides a long-lasting protective antibody response that is central to the concept of vaccination. Recent studies revealed a distinct function for B1b lymphocytes, a minor subset of mature B cells that closely resembles that of memory B cells in a number of aspects. In contrast to the development of conventional B cell memory, which requires the formation of germinal centers and T cells, the development of B1b cell-mediated long-lasting antibody responses occurs independent of T cell help. T cell-independent (TI) antigens are important virulence factors expressed by a number of bacterial pathogens, including those associated with biological threats. TI antigens cannot be processed and presented to T cells and therefore are known to possess restricted T cell-dependent (TD) immunogenicity. Nevertheless, specific recognition of TI antigens by B1b cells and the highly protective antibody responses mounted by them clearly indicate a crucial role for this subset of B cells. Understanding the mechanisms of long-term immunity conferred by B1b cells may lead to improved vaccine efficacy for a variety of TI antigens

Pathogenic Huntingtin Repeat Expansions in Patients with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis.

We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40-64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington's disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered

Determinants of recovery from post-COVID-19 dyspnoea: analysis of UK prospective cohorts of hospitalised COVID-19 patients and community-based controls

Background The risk factors for recovery from COVID-19 dyspnoea are poorly understood. We investigated determinants of recovery from dyspnoea in adults with COVID-19 and compared these to determinants of recovery from non-COVID-19 dyspnoea. Methods We used data from two prospective cohort studies: PHOSP-COVID (patients hospitalised between March 2020 and April 2021 with COVID-19) and COVIDENCE UK (community cohort studied over the same time period). PHOSP-COVID data were collected during hospitalisation and at 5-month and 1-year follow-up visits. COVIDENCE UK data were obtained through baseline and monthly online questionnaires. Dyspnoea was measured in both cohorts with the Medical Research Council Dyspnoea Scale. We used multivariable logistic regression to identify determinants associated with a reduction in dyspnoea between 5-month and 1-year follow-up. Findings We included 990 PHOSP-COVID and 3309 COVIDENCE UK participants. We observed higher odds of improvement between 5-month and 1-year follow-up among PHOSP-COVID participants who were younger (odds ratio 1.02 per year, 95% CI 1.01–1.03), male (1.54, 1.16–2.04), neither obese nor severely obese (1.82, 1.06–3.13 and 4.19, 2.14–8.19, respectively), had no pre-existing anxiety or depression (1.56, 1.09–2.22) or cardiovascular disease (1.33, 1.00–1.79), and shorter hospital admission (1.01 per day, 1.00–1.02). Similar associations were found in those recovering from non-COVID-19 dyspnoea, excluding age (and length of hospital admission). Interpretation Factors associated with dyspnoea recovery at 1-year post-discharge among patients hospitalised with COVID-19 were similar to those among community controls without COVID-19. Funding PHOSP-COVID is supported by a grant from the MRC-UK Research and Innovation and the Department of Health and Social Care through the National Institute for Health Research (NIHR) rapid response panel to tackle COVID-19. The views expressed in the publication are those of the author(s) and not necessarily those of the National Health Service (NHS), the NIHR or the Department of Health and Social Care. COVIDENCE UK is supported by the UK Research and Innovation, the National Institute for Health Research, and Barts Charity. The views expressed are those of the authors and not necessarily those of the funders

What can whiskers tell us about mammalian evolution, behaviour, and ecology?

Most mammals have whiskers; however, nearly everything we know about whiskers derives from just a handful of species, including laboratory rats Rattus norvegicus and mice Mus musculus, as well as some species of pinniped and marsupial. We explore the extent to which the knowledge of the whisker system from a handful of species applies to mammals generally. This will help us understand whisker evolution and function, in order to gain more insights into mammalian behaviour and ecology. This review is structured around Tinbergen’s four questions, since this method is an established, comprehensive, and logical approach to studying behaviour. We ask: how do whiskers work, develop, and evolve? And what are they for? While whiskers are all slender, curved, tapered, keratinised hairs that transmit vibrotactile information, we show that there are marked differences between species with respect to whisker arrangement, numbers, length, musculature, development, and growth cycles. The conservation of form and a common muscle architecture in mammals suggests that early mammals had whiskers. Whiskers may have been functional even in therapsids. However, certain extant mammalian species are equipped with especially long and sensitive whiskers, in particular nocturnal, arboreal species, and aquatic species, which live in complex environments and hunt moving prey. Knowledge of whiskers and whisker use can guide us in developing conservation protocols and designing enriched enclosures for captive mammals. We suggest that further comparative studies, embracing a wider variety of mammalian species, are required before one can make large-scale predictions relating to evolution and function of whiskers. More research is needed to develop robust techniques to enhance the welfare and conservation of mammals