Telomerecat: A ploidy-agnostic method for estimating telomere length from whole genome sequencing data.

Telomere length is a risk factor in disease and the dynamics of telomere length are crucial to our understanding of cell replication and vitality. The proliferation of whole genome sequencing represents an unprecedented opportunity to glean new insights into telomere biology on a previously unimaginable scale. To this end, a number of approaches for estimating telomere length from whole-genome sequencing data have been proposed. Here we present Telomerecat, a novel approach to the estimation of telomere length. Previous methods have been dependent on the number of telomeres present in a cell being known, which may be problematic when analysing aneuploid cancer data and non-human samples. Telomerecat is designed to be agnostic to the number of telomeres present, making it suited for the purpose of estimating telomere length in cancer studies. Telomerecat also accounts for interstitial telomeric reads and presents a novel approach to dealing with sequencing errors. We show that Telomerecat performs well at telomere length estimation when compared to leading experimental and computational methods. Furthermore, we show that it detects expected patterns in longitudinal data, repeated measurements, and cross-species comparisons. We also apply the method to a cancer cell data, uncovering an interesting relationship with the underlying telomerase genotype

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Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Transcriptional Profiling of Quiescent Muscle Stem Cells In Vivo

Summary: Muscle stem cells (MuSCs) persist in a quiescent state and activate in response to specific stimuli. The quiescent state is both actively maintained and dynamically regulated. However, analyses of quiescence have come primarily from cells removed from their niche. Although these cells are still quiescent, biochemical changes certainly occur during the isolation process. Here, we analyze the transcriptome of MuSCs in vivo utilizing MuSC-specific labeling of RNA. Notably, labeling transcripts during the isolation procedure revealed very active transcription of specific subsets of genes. However, using the transcription inhibitor α-amanitin, we show that the ex vivo transcriptome remains largely reflective of the in vivo transcriptome. Together, these data provide perspective on the molecular regulation of the quiescent state at the transcriptional level, demonstrate the utility of these tools for probing transcriptional dynamics in vivo, and provide an invaluable resource for understanding stem cell state transitions. : van Velthoven et al. use metabolic labeling of RNA to examine the quiescent transcriptome of MuSCs in vivo and during early activation. They find dynamic regulation of specific subsets of transcripts in quiescent and activating MuSCs, and they demonstrate the utility of these tools for probing transcriptional dynamics in vivo. Keywords: satellite cells, quiescence, nascent transcriptio

Dynamics of SARS-CoV-2 immunity after vaccination and breakthrough infection in rituximab-treated rheumatoid arthritis patients: a prospective cohort study

BackgroundSARS-CoV-2 vaccination in rheumatoid arthritis (RA) patients treated with B cell-depleting drugs induced limited seroconversion but robust cellular response. We aimed to document specific T and B cell immunity in response to vaccine booster doses and breakthrough infection (BTI).MethodsWe included 76 RA patients treated with rituximab who received up to four SARS-CoV-2 vaccine doses or three doses plus BTI, in addition to vaccinated healthy donors (HD) and control patients treated with tumor necrosis factor inhibitor (TNFi). We quantified anti-SARS-CoV-2 receptor-binding domain (RBD) Spike IgG, anti-nucleocapsid (NC) IgG, 92 circulating inflammatory proteins, Spike-binding B cells, and Spike-specific T cells along with comprehensive high-dimensional phenotyping and functional assays.FindingsThe time since the last rituximab infusion, persistent inflammation, and age were associated with the anti-SARS-CoV-2 RBD IgG seroconversion. The vaccine-elicited serological response was accompanied by an incomplete induction of peripheral Spike-specific memory B cells but occurred independently of T cell responses. Vaccine- and BTI-elicited cellular immunity was similar between RA and HD ex vivo in terms of frequency or phenotype of Spike-specific cytotoxic T cells and in vitro in terms of the functionality and differentiation profile of Spike-specific T cells.InterpretationSARS-CoV-2 vaccination in RA can induce persistent effector T-cell responses that are reactivated by BTI. Paused rituximab medication allowed serological responses after a booster dose (D4), especially in RA with lower inflammation, enabling efficient humoral and cellular immunity after BTI, and contributed overall to the development of potential durable immunity

DataSheet_1_Dynamics of SARS-CoV-2 immunity after vaccination and breakthrough infection in rituximab-treated rheumatoid arthritis patients: a prospective cohort study.pdf

BackgroundSARS-CoV-2 vaccination in rheumatoid arthritis (RA) patients treated with B cell-depleting drugs induced limited seroconversion but robust cellular response. We aimed to document specific T and B cell immunity in response to vaccine booster doses and breakthrough infection (BTI).MethodsWe included 76 RA patients treated with rituximab who received up to four SARS-CoV-2 vaccine doses or three doses plus BTI, in addition to vaccinated healthy donors (HD) and control patients treated with tumor necrosis factor inhibitor (TNFi). We quantified anti-SARS-CoV-2 receptor-binding domain (RBD) Spike IgG, anti-nucleocapsid (NC) IgG, 92 circulating inflammatory proteins, Spike-binding B cells, and Spike-specific T cells along with comprehensive high-dimensional phenotyping and functional assays.FindingsThe time since the last rituximab infusion, persistent inflammation, and age were associated with the anti-SARS-CoV-2 RBD IgG seroconversion. The vaccine-elicited serological response was accompanied by an incomplete induction of peripheral Spike-specific memory B cells but occurred independently of T cell responses. Vaccine- and BTI-elicited cellular immunity was similar between RA and HD ex vivo in terms of frequency or phenotype of Spike-specific cytotoxic T cells and in vitro in terms of the functionality and differentiation profile of Spike-specific T cells.InterpretationSARS-CoV-2 vaccination in RA can induce persistent effector T-cell responses that are reactivated by BTI. Paused rituximab medication allowed serological responses after a booster dose (D4), especially in RA with lower inflammation, enabling efficient humoral and cellular immunity after BTI, and contributed overall to the development of potential durable immunity.</p

Arachidonic acid supplementation transiently augments the acute inflammatory response to resistance exercise in trained men

Strenuous exercise can result in skeletal muscle damage, leading to the systemic mobilization, activation and intramuscular accumulation of blood leukocytes. Eicosanoid metabolites of arachidonic acid (ARA) are potent inflammatory mediators, but whether changes in dietary ARA intake influence exercise-induced inflammation is not known. This study investigated the effect of 4 weeks of dietary supplementation with 1.5 g/day ARA (n=9, 24 {plus minus} 1.5 years) or corn-soy oil placebo (n=10, 26 {plus minus} 1.3 years) on systemic and intramuscular inflammatory responses to an acute bout of resistance exercise (8 sets each of leg press and extension at 80% 1RM) in previously trained men. Whole EDTA blood, serum, peripheral blood mononuclear cells (PMBCs), and skeletal muscle biopsies were collected prior to exercise, immediately post-exercise and at 2, 4 and 48 h of recovery. ARA supplementation resulted in higher exercise-stimulated serum creatine kinase activity (incremental area under the curve (iAUC) p=0.046) and blood leukocyte counts (iAUC for total white cells p<0.001, neutrophils p=0.007, monocytes p=0.015). The exercise-induced fold change in PBMC mRNA expression of interleukin-1 beta (IL1B), CD11b (ITGAM) and neutrophil elastase (ELANE), as well as muscle mRNA expression of the chemokines interleukin-8 (CXCL8) and monocyte chemoattractant protein 1 (CCL2) was also greater in the ARA group than placebo. Despite this, ARA supplementation did not influence the histological presence of leukocytes within muscle, perceived muscle soreness or the extent and duration of muscle force loss. These data show that ARA supplementation transiently increased the inflammatory response to acute resistance exercise, but did not impair recovery