Functional analysis of the a-defensin disulfide array in mouse cryptdin-4

The alpha-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines alpha-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell alpha-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and proCrp4 molecules to degradation by MMP-7. Although native Crp4 and the alpha-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining alpha-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase

Rattusin, an intestinal a-defensin-related peptide in rats with a unique cysteine spacing pattern and salt-insensitive antibacterial activities

Cationic antimicrobial peptides are essential components of the innate immune system. As a major family of mammalian antimicrobial peptides, defensins are expressed mainly by mucosal epithelial cells and promyelocytes. Despite the capacity to kill a broad spectrum of bacteria through physical disruption of membranes, most defensins show substantially reduced antibacterial activities in the presence of monovalent and divalent cations, thereby limiting their therapeutic potential, particularly for the treatment of systemic infections. Genome-wide computational screening of the rat genome led to the identification of the gene for a novel a-defensin-related peptide that we termed rattusin. Rattusin shares a highly conserved signal and prosequence with mammalian a-defensins, but instead of the canonical a-defensin six-cysteine motif, rattusin consists of five cysteines with a distinctive spacing pattern. Furthermore, rattusin is preferentially expressed in Paneth cells of the distal small intestine with potent antibacterial activity against a broad range of Gram-negative and Gram-positive bacteria, including antibiotic-resistant strains. The MICs were mostly in the range of 2 to 4 uM, with no appreciable toxicity to mammalian cells at up to 100 uM. In contrast to classical a- and B-defensins, rattusin retained its activity in the presence of physiological concentrations of NaCl and Mg2+, making it an attractive antimicrobial candidate for both topical and systemic applications.Peer reviewedAnimal Scienc

Examination of psychological risk factors for chronic pain following cardiac surgery: protocol for a prospective observational study

© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. INTRODUCTION: Approximately 400 000 Americans and 36 000 Canadians undergo cardiac surgery annually, and up to 56% will develop chronic postsurgical pain (CPSP). The primary aim of this study is to explore the association of pain-related beliefs and gender-based pain expectations on the development of CPSP. Secondary goals are to: (A) explore risk factors for poor functional status and patient-level cost of illness from a societal perspective up to 12 months following cardiac surgery; and (B) determine the impact of CPSP on quality-adjusted life years (QALYs) borne by cardiac surgery, in addition to the incremental cost for one additional QALY gained, among those who develop CPSP compared with those who do not. METHODS AND ANALYSES: In this prospective cohort study, 1250 adults undergoing cardiac surgery, including coronary artery bypass grafting and open-heart procedures, will be recruited over a 3-year period. Putative risk factors for CPSP will be captured prior to surgery, at postoperative day 3 (in hospital) and day 30 (at home). Outcome data will be collected via telephone interview at 6-month and 12-month follow-up. We will employ generalised estimating equations to model the primary (CPSP) and secondary outcomes (function and cost) while adjusting for prespecified model covariates. QALYs will be estimated by converting data from the Short Form-12 (version 2) to a utility score. ETHICS AND DISSEMINATION: This protocol has been approved by the responsible bodies at each of the hospital sites, and study enrolment began May 2015. We will disseminate our results through CardiacPain.Net, a web-based knowledge dissemination platform, presentation at international conferences and publications in scientific journals. TRIAL REGISTRATION NUMBER: NCT01842568

High-coverage whole-genome analysis of 1220 cancers reveals hundreds of genes deregulated by rearrangement-mediated cis-regulatory alterations.

The impact of somatic structural variants (SVs) on gene expression in cancer is largely unknown. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole-genome sequencing data and RNA sequencing from a common set of 1220 cancer cases, we report hundreds of genes for which the presence within 100 kb of an SV breakpoint associates with altered expression. For the majority of these genes, expression increases rather than decreases with corresponding breakpoint events. Up-regulated cancer-associated genes impacted by this phenomenon include TERT, MDM2, CDK4, ERBB2, CD274, PDCD1LG2, and IGF2. TERT-associated breakpoints involve ~3% of cases, most frequently in liver biliary, melanoma, sarcoma, stomach, and kidney cancers. SVs associated with up-regulation of PD1 and PDL1 genes involve ~1% of non-amplified cases. For many genes, SVs are significantly associated with increased numbers or greater proximity of enhancer regulatory elements near the gene. DNA methylation near the promoter is often increased with nearby SV breakpoint, which may involve inactivation of repressor elements

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Differential Effects on Human Immunodeficiency Virus Type 1 Replication by α-Defensins with Comparable Bactericidal Activities

In addition to their antibacterial activities, certain antimicrobial peptides inactivate enveloped viruses, including the human immunodeficiency virus (HIV). To determine whether peptide bactericidal activities are predictive of antiviral activity, the anti-HIV properties of recombinant human α-defensin 5, mouse α-defensins, cryptdins (Crp) 3 and 4, and rhesus macaque myeloid α-defensins (RMADs) 3 and 4 were determined in vitro. The peptides, purified to homogeneity, had equivalent bactericidal activities that were similar to those of the native molecules. Nuclear magnetic resonance spectroscopy showed RMAD-4 and Crp3 had characteristic α-defensin tridisulfide arrays. Of the peptides analyzed, only RMAD-4 inhibited HIV infectivity at 150 μg/ml, and Crp3 unexpectedly increased HIV replication. Quantitative real-time PCRs for minus-strand strong stop DNA and complete viral cDNA synthesis were used to distinguish between preentry and postentry anti-HIV effects by RMAD-4. Viral exposure to RMAD-4 for 1 h prior to infection reduced HIV minus-strand strong stop DNA and HIV cDNA by 4- to 20-fold during the first round of replication, showing that RMAD-4-exposed virions were not entering cells during the first 24 h. On the other hand, when RMAD-4 was added coincident with HIV inoculation, no anti-HIV activity was detected. Viral exposure to Crp3 resulted in a threefold increase in both HIV minus-strand strong stop DNA and HIV cDNA over the first round of replication. Therefore, two α-defensins, RMAD-4 and Crp3, inhibit or augment HIV replication, respectively, by mechanisms that precede reverse transcription