Macrophage TNF-α mediates parathion-induced airway hyperreactivity in guinea pigs.

Organophosphorus pesticides (OPs) are implicated in human asthma. We previously demonstrated that, at concentrations that do not inhibit acetylcholinesterase activity, the OP parathion causes airway hyperreactivity in guinea pigs as a result of functional loss of inhibitory M2 muscarinic receptors on parasympathetic nerves. Because macrophages are associated with asthma, we investigated whether macrophages mediate parathion-induced M2 receptor dysfunction and airway hyperreactivity. Airway physiology was measured in guinea pigs 24 h after a subcutaneous injection of parathion. Pretreatment with liposome-encapsulated clodronate induced alveolar macrophage apoptosis and prevented parathion-induced airway hyperreactivity in response to electrical stimulation of the vagus nerves. As determined by qPCR, TNF-α and IL-1β mRNA levels were increased in alveolar macrophages isolated from parathion-treated guinea pigs. Parathion treatment of alveolar macrophages ex vivo did not significantly increase IL-1β and TNF-α mRNA but did significantly increase TNF-α protein release. Consistent with these data, pretreatment with the TNF-α inhibitor etanercept but not the IL-1β receptor inhibitor anakinra prevented parathion-induced airway hyperreactivity and protected M2 receptor function. These data suggest a novel mechanism of OP-induced airway hyperreactivity in which low-level parathion activates macrophages to release TNF-α-causing M2 receptor dysfunction and airway hyperreactivity. These observations have important implications regarding therapeutic approaches for treating respiratory disease associated with OP exposures

OVA: Integrating molecular and physical phenotype data from multiple biomedical domain ontologies with variant filtering for enhanced variant prioritization

Motivation: Exome sequencing has become a de facto standard method for Mendelian disease gene discovery in recent years, yet identifying disease-causing mutations among thousands of candidate variants remains a non-trivial task. Results: Here we describe a new variant prioritization tool, OVA (ontology variant analysis), in which user-provided phenotypic information is exploited to infer deeper biological context. OVA combines a knowledge-based approach with a variant-filtering framework. It reduces the number of candidate variants by considering genotype and predicted effect on protein sequence, and scores the remainder on biological relevance to the query phenotype. We take advantage of several ontologies in order to bridge knowledge across multiple biomedical domains and facilitate computational analysis of annotations pertaining to genes, diseases, phenotypes, tissues and pathways. In this way, OVA combines information regarding molecular and physical phenotypes and integrates both human and model organism data to effectively prioritize variants. By assessing performance on both known and novel disease mutations, we show that OVA performs biologically meaningful candidate variant prioritization and can be more accurate than another recently published candidate variant prioritization tool

Second asymptomatic carotid surgery trial (ACST-2): a randomised comparison of carotid artery stenting versus carotid endarterectomy

Background: Among asymptomatic patients with severe carotid artery stenosis but no recent stroke or transient cerebral ischaemia, either carotid artery stenting (CAS) or carotid endarterectomy (CEA) can restore patency and reduce long-term stroke risks. However, from recent national registry data, each option causes about 1% procedural risk of disabling stroke or death. Comparison of their long-term protective effects requires large-scale randomised evidence. Methods: ACST-2 is an international multicentre randomised trial of CAS versus CEA among asymptomatic patients with severe stenosis thought to require intervention, interpreted with all other relevant trials. Patients were eligible if they had severe unilateral or bilateral carotid artery stenosis and both doctor and patient agreed that a carotid procedure should be undertaken, but they were substantially uncertain which one to choose. Patients were randomly allocated to CAS or CEA and followed up at 1 month and then annually, for a mean 5 years. Procedural events were those within 30 days of the intervention. Intention-to-treat analyses are provided. Analyses including procedural hazards use tabular methods. Analyses and meta-analyses of non-procedural strokes use Kaplan-Meier and log-rank methods. The trial is registered with the ISRCTN registry, ISRCTN21144362. Findings: Between Jan 15, 2008, and Dec 31, 2020, 3625 patients in 130 centres were randomly allocated, 1811 to CAS and 1814 to CEA, with good compliance, good medical therapy and a mean 5 years of follow-up. Overall, 1% had disabling stroke or death procedurally (15 allocated to CAS and 18 to CEA) and 2% had non-disabling procedural stroke (48 allocated to CAS and 29 to CEA). Kaplan-Meier estimates of 5-year non-procedural stroke were 2·5% in each group for fatal or disabling stroke, and 5·3% with CAS versus 4·5% with CEA for any stroke (rate ratio [RR] 1·16, 95% CI 0·86–1·57; p=0·33). Combining RRs for any non-procedural stroke in all CAS versus CEA trials, the RR was similar in symptomatic and asymptomatic patients (overall RR 1·11, 95% CI 0·91–1·32; p=0·21). Interpretation: Serious complications are similarly uncommon after competent CAS and CEA, and the long-term effects of these two carotid artery procedures on fatal or disabling stroke are comparable. Funding: UK Medical Research Council and Health Technology Assessment Programme

An alternative to array-based diagnostics: a prospectively recruited cohort, comparing arrayCGH to next-generation sequencing to evaluate foetal structural abnormalities

Molecular diagnostic investigations, following the identification of foetal abnormalities, are routinely performed using array comparative genomic hybridisation (aCGH). Despite the utility of this technique, contemporary approaches for the detection of copy number variation are typically based on next-generation sequencing (NGS). We sought to compare an in-house NGS-based workflow (CNVseq) with aCGH, for invasively obtained foetal samples from pregnancies complicated by foetal structural abnormality. DNA from 40 foetuses was screened using both 8 × 60 K aCGH oligoarrays and low-coverage whole genome sequencing. Sequencer-compatible libraries were combined in a ten–sample multiplex and sequenced using an Illumina HiSeq2500. The mean resolution of CNVseq was 29 kb, compared to 60 kb for aCGH analyses. Four clinically significant, concordant, copy number imbalances were detected using both techniques, however, genomic breakpoints were more precisely defined by CNVseq. This data indicates CNVseq is a robust and sensitive alternative to aCGH, for the prenatal investigation of foetuses with structural abnormalities. • Impact statement • What is already known about this subject? Copy number variant analysis using next-generation sequencing has been successfully applied to investigations of tumour specimens and patients with developmental delays. The application of our approach, to a prospective prenatal diagnosis cohort, has not hitherto been assessed. • What do the results of this study add? Next-generation sequencing has a comparable turnaround time and assay sensitivity to copy number variant analysis performed using array CGH. We demonstrate that having established a next-generation sequencing facility, high-throughput CNVseq sample processing and analysis can be undertaken within the framework of a regional diagnostic service. • What are the implications of these findings for clinical practice and/or further research? Array CGH is a legacy technology which is likely to be superseded by low-coverage whole genome sequencing, for the detection of copy number variants, in the prenatal diagnosis of structural abnormalities

m6aViewer: software for the detection, analysis and visualization of N6-methyl-adenosine peaks from m6A-seq/ME-RIP sequencing data.

Recent methods for transcriptome-wide N6-methyl-adenosine(m6A) profiling have facilitated investigations into the RNA methylome and established m6A as a dynamic modification that has critical regulatory roles in gene expression and may play a role in human disease. However, bioinformatics resources available for the analysis of m6A sequencing data are still limited. Here, we describe m6aViewer - a cross-platform application for analysis and visualisation of m6A peaks from sequencing data. m6aViewer implements a novel m6A peak-calling algorithm that identifies high-confidence methylated residues with more precision than previously described approaches. The application enables data analysis through a graphical user interface, and thus, in contrast to other currently available tools, does not require the user to be skilled in computer programming. m6aViewer and test data can be downloaded here: http://dna2.leeds.ac.uk/m6a