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BackgroundRecent clinical trial data from Lynch Syndrome (LS) carriers demonstrated that naproxen administered for 6-months is a safe primary chemoprevention that promotes activation of different resident immune cell types without increasing lymphoid cellularity. While intriguing, the precise immune cell types enriched by naproxen remained unanswered. Here, we have utilized cutting-edge technology to elucidate the immune cell types activated by naproxen in mucosal tissue of LS patients.MethodsNormal colorectal mucosa samples (pre- and post-treatment) from a subset of patients enrolled in the randomized and placebo-controlled 'Naproxen Study' were obtained and subjected to a tissue microarray for image mass cytometry (IMC) analysis. IMC data was processed using tissue segmentation and functional markers to ascertain cell type abundance. Computational outputs were then used to quantitatively compare immune cell abundance in pre- and post-naproxen specimens.ResultsUsing data-driven exploration, unsupervised clustering identified four populations of immune cell types with statistically significant changes between treatment and control groups. These four populations collectively describe a unique cell population of proliferating lymphocytes within mucosal samples from LS patients exposed to naproxen.ConclusionsOur findings show that daily exposure of naproxen promotes T-cell proliferation in the colonic mucosa, which paves way for developing combination of immunoprevention strategies including naproxen for LS patients.

TCR stimulation triggers Ca2+ signals that are critical for T cell function and immunity. Several pore-forming α and auxiliary β subunits of voltage-gated Ca2+ channels (VGCC) were reported in T cells, but their mechanism of activation remains elusive and their contribution to Ca2+ signaling in T cells is controversial. We here identify CaVβ1, encoded by Cacnb1, as a regulator of T cell function. Cacnb1 deletion enhances apoptosis and impairs the clonal expansion of T cells after lymphocytic choriomeningitis virus (LCMV) infection. By contrast, Cacnb1 is dispensable for T cell proliferation, cytokine production and Ca2+ signaling. Using patch clamp electrophysiology and Ca2+ recordings, we are unable to detect voltage-gated Ca2+ currents or Ca2+ influx in human and mouse T cells upon depolarization with or without prior TCR stimulation. mRNAs of several VGCC α1 subunits are detectable in human (CaV3.3, CaV3.2) and mouse (CaV2.1) T cells, but they lack transcription of many 5' exons, likely resulting in N-terminally truncated and non-functional proteins. Our findings demonstrate that although CaVβ1 regulates T cell function, these effects are independent of VGCC channel activity.

Reversible modification of proteins with linkage-specific ubiquitin chains is critical for intracellular signaling. Information on physiological roles and underlying mechanisms of particular ubiquitin linkages during human development are limited. Here, relying on genomic constraint scores, we identify 10 patients with multiple congenital anomalies caused by hemizygous variants in OTUD5, encoding a K48/K63 linkage-specific deubiquitylase. By studying these mutations, we find that OTUD5 controls neuroectodermal differentiation through cleaving K48-linked ubiquitin chains to counteract degradation of select chromatin regulators (e.g., ARID1A/B, histone deacetylase 2, and HCF1), mutations of which underlie diseases that exhibit phenotypic overlap with OTUD5 patients. Loss of OTUD5 during differentiation leads to less accessible chromatin at neuroectodermal enhancers and aberrant gene expression. Our study describes a previously unidentified disorder we name LINKED (LINKage-specific deubiquitylation deficiency-induced Embryonic Defects) syndrome and reveals linkage-specific ubiquitin cleavage from chromatin remodelers as an essential signaling mode that coordinates chromatin remodeling during embryogenesis.

Use of low resolution single cell DNA FISH and population based high resolution chromosome conformation capture techniques have highlighted the importance of pairwise chromatin interactions in gene regulation. However, it is unlikely that associations involving regulatory elements act in isolation of other interacting partners that also influence their impact. Indeed, the influence of multi-loci interactions remains something of an enigma as beyond low-resolution DNA FISH we do not have the appropriate tools to analyze these. Here we present a method that uses standard 4C-seq data to identify multi-loci interactions from the same cell. We demonstrate the feasibility of our method using 4C-seq data sets that identify known pairwise and novel tri-loci interactions involving the Tcrb and Igk antigen receptor enhancers. We further show that the three Igk enhancers, MiEκ, 3'Eκ and Edκ, interact simultaneously in this super-enhancer cluster, which add to our previous findings showing that loss of one element decreases interactions between all three elements as well as reducing their transcriptional output. These findings underscore the functional importance of simultaneous interactions and provide new insight into the relationship between enhancer elements. Our method opens the door for studying multi-loci interactions and their impact on gene regulation in other biological settings.

4C-Seq has proven to be a powerful technique to identify genome-wide interactions with a single locus of interest (or "bait") that can be important for gene regulation. However, analysis of 4C-Seq data is complicated by the many biases inherent to the technique. An important consideration when dealing with 4C-Seq data is the differences in resolution of signal across the genome that result from differences in 3D distance separation from the bait. This leads to the highest signal in the region immediately surrounding the bait and increasingly lower signals in far-cis and trans. Another important aspect of 4C-Seq experiments is the resolution, which is greatly influenced by the choice of restriction enzyme and the frequency at which it can cut the genome. Thus, it is important that a 4C-Seq analysis method is flexible enough to analyze data generated using different enzymes and to identify interactions across the entire genome. Current methods for 4C-Seq analysis only identify interactions in regions near the bait or in regions located in far-cis and trans, but no method comprehensively analyzes 4C signals of different length scales. In addition, some methods also fail in experiments where chromatin fragments are generated using frequent cutter restriction enzymes. Here, we describe 4C-ker, a Hidden-Markov Model based pipeline that identifies regions throughout the genome that interact with the 4C bait locus. In addition, we incorporate methods for the identification of differential interactions in multiple 4C-seq datasets collected from different genotypes or experimental conditions. Adaptive window sizes are used to correct for differences in signal coverage in near-bait regions, far-cis and trans chromosomes. Using several datasets, we demonstrate that 4C-ker outperforms all existing 4C-Seq pipelines in its ability to reproducibly identify interaction domains at all genomic ranges with different resolution enzymes.

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OBJECTIVE: To describe cases, deaths, and hospital mortality from covid-19 in children and adolescents in Brazil, according to age group, during the evolving phases of the pandemic in 2020 and 2021. METHODS: Census of patients aged up to 19 committed with severe acute respiratory syndrome, due to covid-19 or unspecified, notified to the Brazilian Influenza Epidemiological Surveillance Information System, from January 1, 2020, to December 31, 2021. The two years were divided into six phases, covering the spread of the disease--first, second and third wave--as well as the impact of vaccination. The pediatric population was categorized into infants, preschoolers, schoolchildren, and adolescents. Hospital mortality was assessed by pandemic phase and age group. RESULTS: A total of 144,041 patients were recorded in the two years, 18.2% of whom had confirmed cases of covid-19. Children under 5 years old (infants and preschoolers) accounted for 62.8% of those hospitalized. A total of 4,471 patients died, representing about 6.1 deaths per day. Infants were the ones who most progressed to the intensive care unit (24.7%) and had the highest gross number of deaths (n = 2,012), but mortality was higher among adolescents (5.7%), reaching 9.8% in phase 1. The first peak of deaths occurred in phase 1 (May/2020), and two other peaks occurred in phase 4 (March/2021 and May/2021). There was an increase in cases and deaths for younger ages since phase 4. Hospital mortality in the pediatric population was higher in phases 1, 4, and 6, following the phenomena of dissemination/interiorization of the virus in the country, beginning of the second wave and beginning of the third wave, respectively. CONCLUSION: The absolute number of cases of covid-19 in children and adolescents is significant. Although complete vaccination in descending order of age provided a natural deviation in age range, there was a greater gap between the curve of new hospitalized cases and the curve of deaths, indicating the positive impact of immunization. DESCRIPTORS: COVID-19, epidemiology. Severe Acute Respiratory Syndrome. Child. Hospital Mortality. [ABSTRACT FROM AUTHOR]

The objective of this work is to evaluate the effects of mean stress on the fatigue behaviour of an All Aluminium Conductor (AAC Orchid), Aluminium Conductor Steel Reinforced (ACSR Tern), and an Aluminium Conductor Alloy Reinforced (ACAR 750 MCM). In this sense, 72 fatigue tests on overhead conductors were performed using different values of H/w parameter. Based on the experimental results, the parameters which describe the fatigue behaviour of the conductors were determined after generating theirs S-N curves. In the assessment of the mean stress effects on the fatigue life, Goodman and Gerber’s relations were fitted to evaluate the use of such models for the conductors. It was observed that the evaluation of the mean stress effect on the overhead conductor could be made by using the fatigue relations.