Single-nucleotide polymorphisms in the Toll-like receptor pathway increase susceptibility to infections in severely injured trauma patients

Background: Sepsis and subsequent multiple-organ failure are the predominant causes of late mortality in trauma patients. Susceptibility and response to infection is, in part, heritable. Single-nucleotide polymorphisms (SNPs) in Toll-like receptor (TLR) and cluster of differentiation 14 (CD14) genes of innate immunity may play a key role. The aim of this study was to assess if SNPs in TLR/CD14 predisposed trauma patients to infection. Methods: A prospective cohort of trauma patients (age 18-80 years; injury severity score [ISS] ≥ 16) admitted to a Level I trauma center between January 2008 and April 2011 was genotyped for SNPs in TLR2 (T-16934A and R753Q), TLR4 (D299G and T399I), TLR9 (T-1486C and T-1237C), and CD14 (C-159T) using high-resolution melting analysis. Association of genotype with prevalence of positive culture findings (gram positive, gram negative, fungi), systemic inflammatory response syndrome (SIRS), sepsis, septic shock, and mortality was tested with χ2and logistic regression analysis. Results: Genotyping was performed for 219 patients, of whom 51% developed positive culture findings in sputum, wounds, blood

Carriage of three plasmids in a single human clinical isolate of Clostridioides difficile

A subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential importance, C. difficile plasmids remain poorly characterized. Here, we provide the complete genome sequence of a human clinical isolate that carries three high-copy number plasmids from three different plasmid families that are therefore compatible. For two of these, we identify a region capable of sustaining plasmid replication in C. difficile that is also compatible with the plasmid pCD630 that is found in many laboratory strains. Together, our data advance our understanding of C. difficile plasmid biology.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Clostridioides difficile infection with isolates of cryptic clade C-II: a genomic analysis of polymerase chain reaction ribotype 151

Objectives: We report a patient case of pseudomembranous colitis associated with a monotoxinproducing Clostridioides difficile belonging to the very rarely diagnosed polymerase chain reaction (PCR) ribotype (RT) 151. To understand why this isolate was not identified using a routine commercial test, we performed a genomic analysis of RT151. Methods: Illumina short-read sequencing was performed on n = 11 RT151s from various geographical regions to study their genomic characteristics and relatedness. Subsequently, we used PacBio circular consensus sequencing to determine the complete genome sequence of isolates belonging to cryptic clades CeI and C-II, which includes the patient isolate. Results: We found that 1) RT151s are polyphyletic with isolates falling into clades 1 and cryptic clades C eI and C-II; 2) RT151 contains both nontoxigenic and toxigenic isolates and 3) RT151 C-II isolates contained monotoxin pathogenicity loci. The isolate from our patient case report contains a novelpathogenicity loci insertion site, lacked tcdA and had a divergent tcdB sequence that might explain the failure of the diagnostic test. Discussion: This study shows that RT151 encompasses both typical and cryptic clades and provides conclusive evidence for C. difficile infection due to clade C-II isolates that was hitherto lacking. Vigilance towards C. difficile infection as a result of cryptic clade isolates is warranted. Quinten R. Ducarmon, Clin Microbiol Infect 2023;29:538.e1-538.e6 (c) 2022 The Author(s). Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Combined vascular endothelial growth factor and TP53 status predicts poor response to tamoxifen therapy in estrogen receptor-positive advanced breast cancer

PURPOSE: In recent studies, we showed that TP53 gene mutation or high levels of cytosolic vascular endothelial growth factor (VEGF) in estrogen receptor (ER)-alpha-positive primary breast tumors predict a poor disease outcome for patients treated with first-line tamoxifen for advanced disease. Mutant TP53 may up-regulate VEGF, whereas, on the other hand, wild-type TP53 may decrease VEGF production. EXPERIMENTAL DESIGN: In the present study, we aimed to assess the combined predictive value of TP53 gene mutation and VEGF status of 160 advanced breast cancer patients with ER-positive tumors who were treated with tamoxifen (median follow-up from start of tamoxifen treatment, 64 months). To assess TP53 gene mutation status, the entire open reading frame was sequenced; for VEGF status, an ELISA was used. RESULTS: In univariate analysis, both TP53 gene mutation (28% of the tumors) and a VEGF level above the median value were significantly associated with a short progression-free survival, post-relapse overall survival, and a poor rate of response to tamoxifen. In Cox multivariate regression analysis including the traditional predictive factors, the addition of TP53 gene mutation and VEGF status, alone or in combination, significantly predicted a poor efficacy of tamoxifen treatment. When the two factors were combined, a significantly decreased odds ratio was seen for the rate of response (odds ratio, 0.27). Similarly, an increased hazard ratio (HR) was seen for progression-free survival (HR, 2.32) and post-relapse overall survival (HR, 1.68) in the group with mutant TP53 and high VEGF compared with the group with both risk factors absent. CONCLUSIONS: Combined TP53 gene mutation status and high VEGF levels of ER-positive primary breast tumors independently predict a poor course of the disease of patients with advanced breast cancer treated with tamoxifen. These patients, having unfavorable tumor characteristics, might benefit more from other types of (individualized) treatment protocols

Complete sequencing of TP53 predicts poor response to systemic therapy of advanced breast cancer

TP53 has been implicated in regulation of the cell cycle, DNA repair, and apoptosis. We studied, in primary breast tumors through direct cDNA sequencing of exons 2-11, whether TP53 gene mutations can predict response in patients with advanced disease to either first-line tamoxifen therapy (202 patients, of whom 55% responded) or up-front (poly)chemotherapy (41 patients, of whom 46% responded). TP53 mutations were detected in 90 of 243 (37%) tumors, and one-fourth of these mutations resulted in a premature termination of the protein. The mutations were observed in 32% (65 of 202) of the primary tumors of tamoxifen-treated patients and in 61% (25 of 41) of the primary tumors of the chemotherapy patients. TP53 mutation was significantly associated with a poor response to tamoxifen [31% versu

High-resolution melting (HRM) re-analysis of a polyposis patients cohort reveals previously undetected heterozygous and mosaic APC gene mutations

Cellular mechanisms in basic and clinical gastroenterology and hepatolog

High-throughput Genotyping of Mannose-binding Lectin Variants Using High-resolution DNA-melting Analysis

High Resolution Melting Analysis (HRMA) is a rapid and sensitive method for single nucleotide polymorphism (SNP) analysis. In the present study we present a novel HRMA assay to detect three SNPs in close proximity of each other in the first exon of the gene encoding mannosebinding lectin (MBL), a key molecule of innate immunity. These SNPs have been selected for their known biological and clinical relevance. The three SNPs in MBL2 were simultaneously determined in sixty- nine human DNA samples using HRMA and a single non- fluorescent melting probe, without any post- PCR processing of samples. Combining analyses from amplicon melting and probe melting, we have been able to discriminate ten exon 1 MBL2 genotypes with HRMA, making it a suitable tool for MBL genotyping. A second HRMA assay is presented to detect a relevant polymorphism (Y/X SNP) in the MBL2 promoter region. In conclusion, HRMA is a closed tube assay that is easy to setup and lends itself perfectly for high throughput genotyping of MBL2 variants. The present study thereby facilitates further clinical studies into the role of MBL in inflammatory and infectious disease. (C) 2010 Wiley-Liss, Inc.Genetics of disease, diagnosis and treatmen

Molecular diagnostics of the HBB gene in an Omani cohort using bench-top DNA Ion Torrent PGM technology

Genetics of disease, diagnosis and treatmen

Accurate quantification of dystrophin mRNA and exon skipping levels in Duchenne Muscular Dystrophy

Antisense oligonucleotide (AON)-mediated exon skipping aimed at restoring the reading frame is a promising therapeutic approach for Duchenne muscular dystrophy that is currently tested in clinical trials. Numerous AONs have been tested in (patient-derived) cultured muscle cells and the mdx mouse model. The main outcome to measure AON efficiency is usually the exon-skipping percentage, though different groups use different methods to assess these percentages. Here, we compare a series of techniques to quantify exon skipping levels in AON-treated mdx mouse muscle. We compared densitometry of RT-PCR products on ethidium bromide-stained agarose gels, primary and nested RT-PCR followed by bioanalyzer analysis and melting curve analysis. The digital array system (Fluidigm) allows absolute quantification of skipped vs non-skipped transcripts and was used as a reference. Digital array results show that 1 ng of mdx gastrocnemius muscle-derived mRNA contains B1100 dystrophin transcripts and that 665 transcripts are sufficient to determine exon-skipping levels. Quantification using bioanalyzer or densitometric analysis of primary PCR products resulted in values close to those obtained with digital array. The use of the same technique allows comparison between different groups working on exon skipping in the mdx mouse model. Laboratory Investigation (2010) 90, 1396-1402; doi:10.1038/labinvest.2010.98; published online 10 May 2010Mechanisms of disease, diagnostics and therap

The Complete Genome Sequence of the Murine Pathobiont Helicobacter typhlonius

Molecular Technology and Informatics for Personalised Medicine and Healt