Biological sex, sex steroids and sex chromosomes contribute to mouse cardiac aging.

After menopause, the incidence of cardiovascular disease rapidly rises in women. The disappearing protection provided by sex steroids is a consequence of the development of many risk factors. Preclinical studies are necessary to understand better the effects of ovarian hormones loss cardiac aging. To mimic menopause in mice and study its consequences, we delayed ovariectomy at 12 months and followed animals for 12 months. Using RNA sequencing, we investigated changes in the myocardial exome with aging. In addition, with four-core genotypes (FCG) transgenic mice, we studied sex chromosome effects on cardiac aging. Heart weight increased from 3 to 24 months (males + 35%, females + 29%). In males, 75% of this increase had occurred at 12 months; in females, only 30%. Gonadectomy of mice at 12 months blocked cardiac hypertrophy in both sexes during the second year of life. The dosage of the X chromosomes did not influence cardiac growth in young and older mice. We performed an RNA sequencing study in young and old mice. We identified new highly expressed genes modulated during aging (Bdh, Myot, Cpxm2, and Slc38a1). The myocardial exome in older animals displayed few differences related to the animal's sex or the presence or absence of sex steroids for a year. We show that the morphological evolution of the heart depends on the biological sex via gonadal sex hormone actions. The myocardial exome of old male and female mice is relatively similar. Our study emphasizes the need to consider sex steroid effects in studying cardiac aging.

Integrative genomic analyses identify candidate causal genes for calcific aortic valve stenosis involving tissue-specific regulation.

There is currently no medical therapy to prevent calcific aortic valve stenosis (CAVS). Multi-omics approaches could lead to the identification of novel molecular targets. Here, we perform a genome-wide association study (GWAS) meta-analysis including 14,819 cases among 941,863 participants of European ancestry. We report 32 genomic loci, among which 20 are novel. RNA sequencing of 500 human aortic valves highlights an enrichment in expression regulation at these loci and prioritizes candidate causal genes. Homozygous genotype for a risk variant near TWIST1, a gene involved in endothelial-mesenchymal transition, has a profound impact on aortic valve transcriptomics. We identify five genes outside of GWAS loci by combining a transcriptome-wide association study, colocalization, and Mendelian randomization analyses. Using cross-phenotype and phenome-wide approaches, we highlight the role of circulating lipoproteins, blood pressure and inflammation in the disease process. Our findings pave the way for the development of novel therapies for CAVS.

Cardiac reverse remodeling in a mouse model with many phenotypical features of heart failure with preserved ejection fraction: effects of modifying lifestyle.

Multiple factors cause heart failure with preserved ejection fraction (HFpEF) and involve various systems. HFpEF prevalence is rapidly rising, and its prognosis remains poor after the first hospitalization. Adopting a more active lifestyle has been shown to provide beneficial clinical outcomes for patients with HFpEF. Using a two-hit HfpEF murine model, we studied cardiac reverse remodeling (RR) after stopping the causing stress and introducing voluntary exercise (VE). We checked in 2-mo-old male and female C57Bl6/J mice the heart's response to angiotensin II (ANG II; 1.5 mg/kg/day for 28 days) fed or not with a high-fat diet (HFD). Then, ANG II and/or the HFD were stopped, and VE was started for an additional 4 wk. ANG II and ANG II + HFD (metabolic-hypertensive stress, MHS) caused cardiac hypertrophy (CH) and myocardial fibrosis, left ventricular (LV) concentric remodeling, atrial enlargement, and reduced exercise capacity. HFD alone induced CH and LV concentric remodeling in female mice only. CH and LV concentric remodeling were reversed 4 wk after stopping ANG II, starting VE, and a low-fat diet. Left atrial enlargement and exercise capacity were improved but differed from controls. We performed bulk LV RNA sequencing and observed that MHS upregulated 58% of the differentially expressed genes (DEGs) compared with controls. In the RR group, compared with MHS animals, 60% of the DEGs were downregulated. In an HfpEF mouse model, we show that correcting hypertension, diet, and introducing exercise can lead to extensive cardiac reverse remodeling.NEW & NOTEWORTHY Using a two-hit murine model of heart failure with preserved ejection fraction (HfpEF), combining elevated blood pressure, obesity, and exercise intolerance in male and female animals, we showed that correction of hypertension, normalization of the diet, and introduction of voluntary exercise could help reverse the remodeling of the left ventricle and double exercise capacity. We also identify genes that escape normalization after myocardial recovery and differences between males' and females' responses to stress and recovery.

A Test to Comprehensively Capture the Known Genetic Component of Familial Pulmonary Fibrosis.

The recent European Respiratory Society statement on familial pulmonary fibrosis (FPF) supports the need of genetic testing in the care of patients and their relatives. However, no solution (i.e., a concrete test) was provided to implemented genetic testing in daily practice. Herein, we tabulated and standardized the nomenclature of 128 genetic variants in 20 genes implicated in adult-onset pulmonary fibrosis. The objective was to develop a laboratory developed test (LDT) based on standard Sanger sequencing in order to capture all known FPF-associated variants. Targeted DNA fragments were amplified with harmonized PCR conditions to perform the LDT in a single 96-well plate. The new genetic test was evaluated in 62 sporadic cases of idiopathic pulmonary fibrosis (IPF). As expected in this population, we observed a low yield of disease-causing mutations. More importantly, 100% of targeted variants by the LDT were successfully evaluated. Furthermore, four variants of uncertain significance with in silico-predicted deleterious scores were identified in three patients, suggesting novel pathogenic variants in genes known to cause IPF. Finally, the MUC5B promoter variant rs35705950 was strongly enriched in these patients with a minor allele frequency of 41.1% compared to 10.6% in a matched population-based cohort (n=29,060), leading to an estimation that this variant may explain up to 35% of the population-attributable risk. This LDT provides a solution for rapid clinical translation. Technical laboratory details are provided so that specialised pulmonary centers can implement the LDT in-house in order to expedite the clinical recommendations of experts' panel. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Characterization of vancomycin-resistance vanD gene clusters in the human intestinal microbiota by metagenomics and culture-enriched metagenomics.

Objectives: To characterize vancomycin-resistance vanD gene clusters and potential vanD-carrying bacteria in the intestinal microbiota of healthy volunteers exposed or not to ß-lactam antibiotics. Methods: Stool samples were collected before and after 7 days of cefprozil ß-lactam antibiotic exposure of 18 participants and six control participants who were not exposed to the antibiotic at the same time points. Metagenomic sequencing and culture-enriched metagenomic sequencing (with and without ß-lactam selection) were used to characterize vanD gene clusters and determine potential vanD-carrying bacteria. Alteration by antimicrobials was also examined. Results: Culture enrichment allowed detection of vanD genes in a large number of participants (11/24; 46%) compared to direct metagenomics (2/24; 8%). vanD genes were detected in stool cultures only following ß-lactam exposure, either after ß-lactam treatment of participants or after culture of stools with ß-lactam selection. Six types of vanD gene clusters were identified. Two types of vanD cluster highly similar to those of enterococci were found in two participants. Other vanD genes or vanD clusters were nearly identical to those identified in commensal anaerobic bacteria of the families Lachnospiraceae and Oscillospiraceae and/or bordered by genomic sequences similar or related to these anaerobes, suggesting that they are the origin or carriers of vanD. Conclusions: This study showed that culture-enriched metagenomics allowed detection of vanD genes not detected by direct metagenomics and revealed collateral enrichment of bacteria containing vancomycin-resistance vanD genes following exposure to ß-lactams, with a higher prevalence of the most likely gut commensal anaerobes carrying vanD. These commensal anaerobes could be the reservoir of vanD genes carried by enterococci.

Tumor Mutational Burden by Whole-Genome Sequencing in Resected NSCLC of Never Smokers.

BACKGROUND: Data are scarce about tumor mutational burden (TMB) as a biomarker in never smokers with non-small cell lung cancer (NSCLC). METHODS: TMB was assessed by whole-genome sequencing (WGS) and compared with in silico reduced whole-exome sequencing (WES) and targeted commercial next-generation sequencing (NGS) gene panels in 92 paired tumor-normal samples from never smokers who underwent NSCLC resection with curative intent. Analyses were performed to test for association with survival after surgery and to identify the optimal prognostic TMB cutoff. RESULTS: Tumors of never smokers with NSCLC had low TMB scores (median 1.57 mutations/Mb; range, 0.13-17.94). A TMB cutoff of 1.70 mutations/Mb was associated with a 5-year overall survival of 58% in the high-TMB (42% of cases) compared with 86% in low-TMB patients (Wald P = 0.0029). TMB scores from WGS and WES were highly correlated (Spearman ρ = 0.93, P < 2.2e-16). TMB scores from NGS panels demonstrated high intraindividual fluctuations and identified high-TMB patients with 65% concordance in average compared with WGS. CONCLUSIONS: In resected NSCLC of never smokers, high TMB was associated with worse prognosis. WES provided a good estimate of TMB while targeted NGS panels seem to lack adequate depth and resolution in the setting of low mutation burden. IMPACT: TMB is a prognostic indicator of survival in resected NSCLC from individuals who never smoked. In this setting of low mutation counts, TMB can be accurately measured by WGS or WES, but not NGS panels.

Performance of an RNA-Based Next-Generation Sequencing Assay for Combined Detection of Clinically Actionable Fusions and Hotspot Mutations in NSCLC.

INTRODUCTION: With its expanding list of approved and emerging therapeutic indications, NSCLC is the exemplar tumor type requiring upfront assessment of several biomarkers to guide clinical management. Next-generation sequencing allows identification of different types of molecular alterations, each with specific analytical challenges. Library preparation using parallel DNA and RNA workflows can overcome most of them, but it increases complexity of laboratory operations, turnaround time, and costs. We describe the performance characteristics of a 15-gene RNA panel on the basis of anchored multiplex polymerase chain reaction for combined detection of clinically relevant oncogenic fusion transcripts and hotspot small variants. METHODS: Formalin-fixed, paraffin-embedded NSCLC clinical samples (N = 58) were used along cell lines and commercial controls to validate the assay's analytical performance, followed by an exploratory prospective cohort (N = 87). RESULTS: The raw assay sensitivity for hotspot mutations and fusions was 83% and 93%, respectively, reaching 100% after filtering for key assay metrics. Those include quantity and quality of input of nucleic acid and sequencing metric from primers on housekeeping genes included in the assay. In the prospective cohort, driver alterations were identified in most cases (≥58%). CONCLUSIONS: This ultrafocused RNA-next-generation sequencing assay offers an advantageous option with single unified workflow for simultaneous detection of clinically relevant hotspot mutations and fusions in NSCLC, focusing on actionable gene targets.

Criibacterium bergeronii gen. nov., sp. nov., a new member of the family Peptostreptococcaceae, isolated from human clinical samples.

A rod-shaped, motile anaerobic bacterium, designated CCRI-22567T, was isolated from a vaginal sample of a woman diagnosed with bacterial vaginosis and subjected to a polyphasic taxonomic study. The novel strain was capable of growth at 30-42 °C (optimum, 42 °C), at pH 5.5-8.5 (optimum, pH 7.0-7.5) and in the presence of 0-1.5 % (w/v) NaCl (optimally at 0.5 % NaCl). The phylogenetic trees based on 16S rRNA gene sequences showed that strain CCRI-22567T forms a distinct evolutionary lineage independent of other taxa in the family Peptostreptococcaceae. Strain CCRI-22567T exhibited 90.1 % 16S rRNA gene sequence similarity to Peptoanaerobacter stomatis ACC19aT and 89.7 % to Eubacterium yurii subsp. schtitka ATCC 43716. The three closest organisms with an available whole genome were compared to strain CCRI-22567T for genomic relatedness assessment. The genomic average nucleotide identities (OrthoANIu) obtained with Peptoanaerobacter stomatis ACC19aT, Eubacterium yurii subsp. margaretiae ATCC 43715 and Filifactor alocis ATCC 35896T were 71.8, 70.3 and 69.6 %, respectively. Strain CCRI-22567T contained C18 : 1 ω9c and C18 : 1 ω9c DMA as the major fatty acids. The DNA G+C content of strain CCRI-22567T based on its genome sequence was 33.8 mol%. On the basis of the phylogenetic, chemotaxonomic and other phenotypic properties, strain CCRI-22567T is considered to represent a new genus and species within the family Peptostreptococcaceae, for which the name Criibacterium bergeronii gen. nov., sp. nov., is proposed. The type strain of Criibacterium bergeronii is CCRI-22567T (=LMG 31278T=DSM 107614T=CCUG 72594T).

Method for isolation of both lactose-fermenting and - non-fermenting Escherichia albertii strains from stool samples.

Initially, Escherichia albertii has been described as a non-lactose fermenting bacterium and methods used to isolate it were first based on this phenotypic property. However, a recent study showed a variable lactose fermentation phenotype for E. albertii suggesting that this microorganism could have been underestimated by previous studies using isolation methods based on lactose fermentation. In this study, we present a method for the isolation and identification of both lactose fermenting and non-fermenting-E. albertii cells in stool samples, said method combining culture and isolation on mEA agar, an indole test, as well as an E. albertii-specific PCR assay for formal species identification. The ability of the procedure to detect E. albertii strains was verified using 19 E. albertii strains and 132 non-E. albertii strains representing 88 species of different origins majoritary belonging to the Enterobacteriaceae family. All indole-positive white colonies grown on mEA agar were subjected to E. albertii-specific PCR amplification; all E. albertii strains tested were detected with this assay and none of the non-E. albertii strains tested was detected. To demonstrate the ability of the procedure to directly detect E. albertii in stool samples, E. albertii-inoculated stools were tested and for all inoculated samples, E. albertii colonies were easily detected and identified. The present study provides a method enable to recover both lactose-fermenting and -non-fermenting E. albertii strains from clinical samples. This method could help to provide a better portrait of the prevalence and pathogenicity of E. albertii in clinical samples.

Draft Genome Sequence of a Sporulating and Motile Strain of Lachnotalea glycerini Isolated from Water in Québec City, Canada.

Lachnotalea glycerini CCRI-19302 belongs to the genus Lachnotalea The strain was isolated from a water sample harvested in Québec City, Canada. The genome assembly comprised 4,694,231 bp, with 34.6% GC content. This is the first documentation to report the genome sequence of a sporulating and motile strain of L. glycerini.

Draft Genome Sequence of Romboutsia maritimum sp. nov. Strain CCRI-22766T, Isolated from Coastal Estuarine Mud.

The Romboutsia maritimum sp. nov. CCRI-22766T strain was isolated from coastal estuarine mud in New Zealand. The genome assembly comprised 2,854,352 bp, with 27.1% G+C content. This is the first documentation that reports the genome sequence of R. maritimum.

Draft Genome Sequence of Romboutsia weinsteinii sp. nov. Strain CCRI-19649T Isolated from Surface Water.

Romboutsia weinsteinii sp. nov. CCRI-19649T belongs to the genus Romboutsia The strain was isolated from a water sample harvested in Québec City, Québec, Canada. The genome assembly comprised 4,134,593 bp with a 29.3% GC content. This is the first documentation that reports the genome sequence of R. weinsteinii.

Use of phylogenetical analysis to predict susceptibility of pathogenic Candida spp. to antifungal drugs.

Successful treatment of a Candida infection relies on 1) an accurate identification of the pathogenic fungus and 2) on its susceptibility to antifungal drugs. In the present study we investigated the level of correlation between phylogenetical evolution and susceptibility of pathogenic Candida spp. to antifungal drugs. For this, we compared a phylogenetic tree, assembled with the concatenated sequences (2475-bp) of the ATP2, TEF1, and TUF1 genes from 20 representative Candida species, with published minimal inhibitory concentrations (MIC) of the four principal antifungal drug classes commonly used in the treatment of candidiasis: polyenes, triazoles, nucleoside analogues, and echinocandins. The phylogenetic tree revealed three distinct phylogenetic clusters among Candida species. Species within a given phylogenetic cluster have generally similar susceptibility profiles to antifungal drugs and species within Clusters II and III were less sensitive to antifungal drugs than Cluster I species. These results showed that phylogenetical relationship between clusters and susceptibility to several antifungal drugs could be used to guide therapy when only species identification is available prior to information pertaining to its resistance profile. An extended study comprising a large panel of clinical samples should be conducted to confirm the efficiency of this approach in the treatment of candidiasis.

Draft Genome Sequence of Criibacterium bergeronii gen. nov., sp. nov., Strain CCRI-22567T, Isolated from a Vaginal Sample from a Woman with Bacterial Vaginosis.

Criibacterium bergeronii gen. nov., sp. nov., CCRI-22567 is the type strain of the new genus Criibacterium The strain was isolated from a woman with bacterial vaginosis. The genome assembly comprised 2,384,460 bp, with 34.4% G+C content. This is the first genome announcement of a strain belonging to the genus Criibacterium.

The initial state of the human gut microbiome determines its reshaping by antibiotics.

Microbiome studies have demonstrated the high inter-individual diversity of the gut microbiota. However, how the initial composition of the microbiome affects the impact of antibiotics on microbial communities is relatively unexplored. To specifically address this question, we administered a second-generation cephalosporin, cefprozil, to healthy volunteers. Stool samples gathered before antibiotic exposure, at the end of the treatment and 3 months later were analysed using shotgun metagenomic sequencing. On average, 15 billion nucleotides were sequenced for each sample. We show that standard antibiotic treatment can alter the gut microbiome in a specific, reproducible and predictable manner. The most consistent effect of the antibiotic was the increase of Lachnoclostridium bolteae in 16 out of the 18 cefprozil-exposed participants. Strikingly, we identified a subgroup of participants who were enriched in the opportunistic pathogen Enterobacter cloacae after exposure to the antibiotic, an effect linked to lower initial microbiome diversity and to a Bacteroides enterotype. Although the resistance gene content of participants' microbiomes was altered by the antibiotic, the impact of cefprozil remained specific to individual participants. Resistance genes that were not detectable prior to treatment were observed after a 7-day course of antibiotic administration. Specifically, point mutations in beta-lactamase blaCfxA-6 were enriched after antibiotic treatment in several participants. This suggests that monitoring the initial composition of the microbiome before treatment could assist in the prevention of some of the adverse effects associated with antibiotics or other treatments.

Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology.

Recombinase polymerase amplification (RPA) technology relies on three major proteins, recombinase proteins, single-strand binding proteins, and polymerases, to specifically amplify nucleic acid sequences in an isothermal format. The performance of RPA with respect to sequence mismatches of closely-related non-target molecules is not well documented and the influence of the number and distribution of mismatches in DNA sequences on RPA amplification reaction is not well understood. We investigated the specificity of RPA by testing closely-related species bearing naturally occurring mismatches for the tuf gene sequence of Pseudomonas aeruginosa and/or Mycobacterium tuberculosis and for the cfb gene sequence of Streptococcus agalactiae. In addition, the impact of the number and distribution of mismatches on RPA efficiency was assessed by synthetically generating 14 types of mismatched forward primers for detecting five bacterial species of high diagnostic relevance such as Clostridium difficile, Staphylococcus aureus, S. agalactiae, P. aeruginosa, and M. tuberculosis as well as Bacillus atropheus subsp. globigii for which we use the spores as internal control in diagnostic assays. A total of 87 mismatched primers were tested in this study. We observed that target specific RPA primers with mismatches (n > 1) at their 3'extrimity hampered RPA reaction. In addition, 3 mismatches covering both extremities and the center of the primer sequence negatively affected RPA yield. We demonstrated that the specificity of RPA was multifactorial. Therefore its application in clinical settings must be selected and validated a priori. We recommend that the selection of a target gene must consider the presence of closely-related non-target genes. It is advisable to choose target regions with a high number of mismatches (≥36%, relative to the size of amplicon) with respect to closely-related species and the best case scenario would be by choosing a unique target gene.

Molecular method for detection of total coliforms in drinking water samples.

This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality.

Abilities of the mCP Agar method and CRENAME alpha toxin-specific real-time PCR assay to detect Clostridium perfringens spores in drinking water.

We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed of C. perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C. perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP(-)/rtPCR(+) colonies were identified as C. perfringens, whereas 3 mCP(+)/rtPCR(-) colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME + cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water. CRENAME + cpa rtPCR detected as few as one C. perfringens CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME + cpa rtPCR also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection.

Ability of three DNA-based assays to identify presumptive Escherichia coli colonies isolated from water by the culture-based mFC agar method.

We tested the ability of three PCR assays, targeting uidA and tuf genes to correctly identify Escherichia coli colonies isolated from water and we compared them to two ß-glucuronidase-based culture methods (Colilert(®) and Readycult(®)), in terms of specificity and sensitivity. E. coli isolates recovered on mFC agar were first tested for the presence of the uidA positive colonies were presumed to be E. coli. For further characterization, uidA-negative colonies were subsequently identified using the Vitek 2 automated system. Colilert(®) and Readycult(®) detected 436 and 442 of 468 colonies identified as E. coli on mFC corresponding to sensitivities of 93.2 and 94.4%, respectively. None of the 59 non-E. coli isolates was detected by both methods for a specificity of 100%. Two (2) uidA and 1 tuf PCR assays were also tested. The uidA PCR assays yielded positive signals for 447 (95.5%) and 434 (92.7%) of 468 E. coli isolates tested respectively, whereas the tuf PCR assay showed a sensitivity of 100%. None of the 59 non-E. coli isolates was detected by both uidA PCR assays (100% specificity), whereas tuf PCR false-positive signals were obtained with Escherichia fergusonii and Escherichia albertii. However, since these 2 species are principally found in the feces of mammals and birds, their detection indicates a fecal contamination. Consequently, using a 1-h tuf rtPCR assay to confirm the identity of E. coli colonies on mFC agar is as specific, more sensitive, and potentially more cost-efficient than culture methods based on ß-glucuronidase detection.