Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

A novel bi-allelic loss-of-function mutation in STIM1 expands the phenotype of STIM1-related diseases.

STIM1, the stromal interaction molecule 1, is the key protein for maintaining calcium concentration in the endoplasmic reticulum by triggering the Store Operated Calcium Entry (SOCE). Bi-allelic mutations in STIM1 gene are responsible for a loss-of-function in patients affected with a CRAC channelopathy syndrome in which severe combined immunodeficiency syndrome (SCID-like), autoimmunity, ectodermal dysplasia and muscle hypotonia are combined. Here, we studied two siblings from a consanguineous Syrian family, presenting with muscle weakness, hyperlaxity, elastic skin, tooth abnormalities, dysmorphic facies, hypoplastic patellae and history of respiratory infections. Using exome sequencing, we have identified a new homozygous frameshift mutation in STIM1: c.685delT [p.(Phe229Leufs*12)], leading to a complete loss of STIM1 protein. In this study, we describe an unusual phenotype linked to STIM1 mutations, combining clinical signs usually observed in different STIM1-related diseases. In particular, we confirmed that the complete loss of STIM1 function is not always associated with severe immune disorders. Altogether, our results broaden the spectrum of phenotypes associated with mutations in STIM1 and opens new perspectives on the pathological mechanisms associated with a defect in the proteins constituting the SOCE complex.

Impact of Endometriosis on Fatigue and Productivity Impairment in a Cross-Sectional Survey of Canadian Women.

OBJECTIVE: To evaluate fatigue burden and productivity impairments in Canadian women with a self-reported diagnosis of endometriosis (DxE). METHODS: From December 2018 to January 2019, Canadian women aged 18-49 years completed an online survey assessing fatigue via the Patient-Reported Outcomes Measurement Information System (PROMIS) Fatigue Short Form 6a questionnaire. Fatigue T-scores were compared between women with and without a DxE, by age and endometriosis symptom severity, using t tests. Women with a DxE completed the Work Productivity and Activity Impairment - Specific Health Problem (WPAI-SHP) questionnaire. The effects of age and hallmark endometriosis symptoms on productivity impairments were assessed via analysis of variance. RESULTS: Survey data included 2004 women with and 26 528 women without a DxE. Mean fatigue T-scores were 58.5 ± 10.1 in women with a DxE and 59.2 ± 10.1 in women with hallmark endometriosis symptoms (i.e., menstrual or non-menstrual pelvic pain/cramping, dyspareunia) versus 55.2 ± 9.4 in women without a DxE (both P < 0.001). Women with moderate or severe endometriosis symptoms had a mean T-score of 61.2 ± 9.4 versus 55.9 ± 10.1 for women with mild symptoms (P < 0.001). Women with moderate or severe hallmark endometriosis symptoms had mean T-scores of 59.6-62.9 versus 57.0-58.2 for women with mild or no symptoms (all comparisons P < 0.01). Women with a DxE reported 17.1% of work time missed, 41.8% impaired work ability, 46.5% overall work impairment, and 41.4% activity impairment per the WPAI-SHP. Women with a DxE aged 30-34 and 35-39 years consistently experienced the greatest effects of fatigue and productivity impairments. CONCLUSIONS: Canadian women with a DxE experience a substantial fatigue burden and significant productivity impairments.

Cross-Sectional Survey of the Impact of Endometriosis Symptoms on Health-Related Quality of Life in Canadian Women.

OBJECTIVE: To characterize the health-related quality of life (HRQOL) of Canadian women with a self-reported diagnosis of endometriosis (DxE). METHODS: Canadian women aged 18-49 years completed a survey from December 2018 through January 2019 in which HRQOL was assessed via the 12-item Short Form Health Survey (SF-12) and Endometriosis Health Profile-30 (EHP-30) questionnaire. We used t tests to compare SF-12 scores between women with and without a self-reported DxE, as well as the severity of hallmark endometriosis symptoms, including menstrual pelvic pain/cramping, non-menstrual pelvic pain/cramping, and dyspareunia, for women with a DxE (moderate/severe vs. mild/none). The effects of overall endometriosis symptom severity on HRQOL were assessed via analysis of variance. RESULTS: In total, 26 528 women without a DxE and 2004 women with a self-reported DxE were included. SF-12 scores were significantly lower for women with versus without a DxE (Mental Component Summary: 38.6 vs. 41.2; Physical Component Summary: 47.3 vs. 52.1; both P < 0.001), indicating reduced HRQOL. Moderate/severe hallmark endometriosis symptoms were associated with low SF-12 scores (i.e., worse HRQOL), with the greatest impact from non-menstrual pelvic pain/cramping. EHP-30 scores for women with a DxE ranged from 40.6 to 46.8, with the greatest impairment in self-image. Women with severe endometriosis symptoms had EHP-30 scores 1.3- and >2-fold higher (i.e., worse) than those with moderate and mild symptoms, at 67.5-74.6 versus 51.3-56.9 and 25.6-32.9, respectively. CONCLUSION: Canadian women with a self-reported diagnosis of endometriosis had significantly impaired health-related quality of life, which was inversely correlated with endometriosis symptom severity. This finding highlights a substantial unmet need among women with endometriosis.

Prevalence, Symptomatic Burden, and Diagnosis of Endometriosis in Canada: Cross-Sectional Survey of 30 000 Women.

OBJECTIVE: This study sought to estimate the prevalence of diagnosis of endometriosis (DxE) in Canada and to assess the symptomatic and diagnostic experience of Canadian women with DxE. METHOD: A cross-sectional, online survey of women in Canada aged 18 to 49 was conducted from December 7, 2018 through January 24, 2019. Survey data were weighted by Canadian population statistics to estimate the prevalence, symptomatic burden, and diagnostic experience of DxE. Logistic regressions were used to assess differences in symptom burden between women with and without DxE. RESULTS: The estimated prevalence of DxE was 7.0% (2004 women of 28 532 women surveyed). Almost half (47.5%) of women with DxE were aged 18 to 29 when they received an endometriosis diagnosis, and 84.1% experienced symptoms before diagnosis. More women with versus without DxE experienced menstrual pelvic pain or cramping (70.3% vs. 50.7%), non-menstrual pelvic pain or cramping (49.5% vs. 18.7%), dyspareunia (52.5% vs. 28.0%), and infertility (22.3% vs. 6.3%). Women with DxE were more likely to report severe menstrual pelvic pain or cramping (odds ratio [OR] 2.9; 95% confidence interval [CI] 2.5-3.3), non-menstrual pelvic pain or cramping (OR 3.4; 95% CI 2.8-4.2), general abdominal pain (OR 3.0; 95% CI 2.5-3.6), and pelvic pressure (OR 3.0; 95% CI 2.3-3.8). Women with DxE reported an average 5.4-year diagnostic delay, with a 3.1-year delay from onset of symptoms to physician consultation and a 2.3-year delay between physician consultation and diagnosis. CONCLUSION: Self-reported DxE is prevalent among Canadian women and is associated with a substantial symptomatic burden. The 5.4-year diagnostic delay reported here indicates an important unmet need for more timely diagnosis of endometriosis in Canada.

Educator Sexual Misconduct and Texas Educator Discipline Database Construction.

The purpose of this research is to describe Texas educator sexual misconduct (ESM) by examining 8 years of sanctions issued to educators (N = 1415) for either sexual misconduct or inappropriate relationships with students or minors. We first examine Texas ESM from the perspective of quality database construction and then describe the demographic characteristics of educators sanctioned for ESM between 2008 and 2016. Differences in the demographic characteristics of educators sanctioned for ESM vary according to the definition of ESM employed by the state education agency. Younger and early career educators are more likely to engage in inappropriate relationships with students or minors, whereas older and later-career teachers are more likely to engage in sexual misconduct as that term is defined by the state education agency. Over one-third of educators sanctioned for ESM were either new to the profession or new to their school district when sanctioned. Recommendations are offered for database construction, policy, and practice.

Human pegivirus isolates characterized by deep sequencing from hepatitis C virus-RNA and human immunodeficiency virus-RNA-positive blood donations, France.

Human pegivirus (HPgV, formerly GBV-C) is a member of the genus Pegivirus, family Flaviviridae. Despite its identification more than 20 years ago, both natural history and distribution of this viral group in human hosts remain under exploration. Analysis of HPgV genomes characterized up to now points out the scarcity of French pegivirus sequences in databases. To bring new data regarding HPgV genomic diversity, we investigated 16 French isolates obtained from hepatitis C virus-RNA and human immunodeficiency virus-RNA-positive blood donations following deep sequencing and coupled molecular protocols. Initial phylogenetic analysis of 5'-untranslated region (5'-UTR)/E2 partial sequences permitted to assign HPgV isolates to genotypes 2 (n = 15) and 1 (n = 1), with up to 16% genetic diversity observed for both regions considered. Seven nearly full-length representative genomes were characterized subsequently, with complete polyprotein coding sequences exhibiting up to 13% genetic diversity; closest nucleotide (nt) divergence with available HPgV sequences was in the range 7% to 11%. A 36 nts deletion located on the NS4B coding region (N-terminal part, 12 amino acids) of the genotype 1 HPgV genome characterized was identified, along with single nucleotide deletions in two genotype 2, 5'-UTR sequences.

Oceanobacillus timonensis sp. nov. and Oceanobacillus senegalensis sp. nov., two new moderately halophilic, Gram-stain positive bacteria isolated from stools sample of healthy young Senegalese.

Oceanobacillus timonensis Marseille-P3532T (CSUR P3532, CCUG 70981) and Oceanobacillus senegalensis Marseille-P3587T (CSUR P3587, CCUG 70613), are the type strains of O. timonensis sp. nov. and O. senegalensis sp. nov., respectively. They are moderately halophilic, aerobic, motile and Gram-stain positive bacteria. The strains P3532T and P3587T were isolated from stools with 3.8% and 2.1% sodium chloride (NaCl) of healthy 10 year old female and male 7-year-old children, respectively and living respectively at Dielmo and N'diop two villages in Senegal (West Africa). This study aimed to describe the genome and phenotypic characteristics of O. timonensis Marseille-P3532T and O. senegalensis Marseille-P3587T. The genomes are 4,485,335 bp long for O. timonensis and 4,300,331 bp for O. senegalensis with 38.78% and 36.92% G+C content, respectively. They contain 4306 and 3979 protein-coding and 87 and 273 RNAs genes, respectively.

Repertoire of the gut microbiota from stomach to colon using culturomics and next-generation sequencing.

BACKGROUND: Most studies on the human microbiota have analyzed stool samples, although a large proportion of the absorption of nutrients takes place in upper gut tract. We collected samples from different locations along the entire gastrointestinal tract from six patients who had simultaneously undergone upper endoscopy and colonoscopy, to perform a comprehensive analysis using culturomics with matrix assisted laser desorption ionisation - time of flight (MALDI-TOF) identification and by metagenomics targeting the 16S ribosomal ribonucleic acid (rRNA) gene. RESULTS: Using culturomics, we isolated 368 different bacterial species, including 37 new species. Fewer species were isolated in the upper gut: 110 in the stomach and 106 in the duodenum, while 235 were isolated from the left colon (p < 0.02). We isolated fewer aero-intolerant species in the upper gut: 37 from the stomach and 150 from the left colon (p < 0.004). Using metagenomics, 1,021 species were identified. The upper gut microbiota was revealed to be less rich than the lower gut microbiota, with 37,622 reads from the stomach, 28,390 from the duodenum, and 79,047 from the left colon (p < 0.009). There were fewer reads for aero-intolerant species in the upper gut (8,656 in the stomach, 5,188 in the duodenum and 72,262 in the left colon, p < 0.02). Patients taking proton pump inhibitors (PPI) were then revealed to have a higher stomach pH and a greater diversity of species in the upper digestive tract than patients not receiving treatment (p < 0.001). CONCLUSION: Significant modifications in bacterial composition and diversity exist throughout the gastrointestinal tract. We suggest that the upper gut may be key to understanding the relationship between the gut microbiota and health.

Draft Genome and Description of Eisenbergiella massiliensis Strain AT11T: A New Species Isolated from Human Feces After Bariatric Surgery.

A novel strain of a Gram-stain negative, non-motile, non-spore forming rod-shaped, obligate anaerobic bacterium, designated AT11T, was isolated from a stool sample of a morbidly obese woman living in Marseille, France. This bacterium was characterized using biochemical, chemotaxonomic, and phylogenetic methods. The 16S rRNA gene sequence analysis showed that strain AT11T had a 97.8% nucleotide sequence similarity with Eisenbergiella tayi strain B086562T, the closest species with standing in nomenclature. The major cellular fatty acids of the novel isolate were C16:0 followed by saturated or unsaturated C18 fatty acids (C18:1n9, C18:1n5 and C18:0). The draft genome of strain AT11T is 7,114,554 bp long with 48% G+C content. 6176 genes were predicted, including 6114 protein-coding genes and 62 were RNAs (with 2 5S rRNA genes, two 16S rRNA genes, two 23S rRNA genes, and 56 tRNA genes). The digital DNA-DNA hybridization (dDDH) relatedness between the new isolate and E. tayi strain B086562T was 23.1% ± 2.2. Based on the phenotypic, chemotaxonomic, genomic, and phylogenetic characteristics, Eisenbergiella massiliensis sp. nov., is proposed. The type strain is AT11T (= DSM 100838T = CSUR P2478T).

Characterization of a novel Gram-stain-positive anaerobic coccus isolated from the female genital tract: Genome sequence and description of Murdochiella vaginalis sp. nov.

Strain Marseille-P2341T , a nonmotile, nonspore-forming, Gram-stain-positive anaerobic coccus, was isolated in the vaginal specimen of a patient with bacterial vaginosis using culturomics. Its growth occurred at temperatures ranging from 25 to 42°C, with pH between 6.5 and 8.5, and at NaCl concentrations lower than 5%. The major fatty acids were C18:1n9 (27.7%) and C16:0 (24.4%). Its genome is 1,671,491 bp long with 49.48 mol% of G+C content. It is composed of 1,501 genes: 1,446 were protein-coding genes and 55 were RNAs. Strain Marseille-P2341T shared 97.3% of 16S rRNA gene sequence similarity with Murdochiella asaccharolytica, the phylogenetically closest species. These results enabled the classification of strain Marseille-P2341T as a new species of the genus Murdochiella for which we proposed the name Murdochiella vaginalis sp. nov. The type strain is strain Marseille-P2341T (=DSM 102237, =CSUR P2341).

Mycobacterium ahvazicum sp. nov., the nineteenth species of the Mycobacterium simiae complex.

Four slowly growing mycobacteria isolates were isolated from the respiratory tract and soft tissue biopsies collected in four unrelated patients in Iran. Conventional phenotypic tests indicated that these four isolates were identical to Mycobacterium lentiflavum while 16S rRNA gene sequencing yielded a unique sequence separated from that of M. lentiflavum. One representative strain AFP-003T was characterized as comprising a 6,121,237-bp chromosome (66.24% guanosine-cytosine content) encoding for 5,758 protein-coding genes, 50 tRNA and one complete rRNA operon. A total of 2,876 proteins were found to be associated with the mobilome, including 195 phage proteins. A total of 1,235 proteins were found to be associated with virulence and 96 with toxin/antitoxin systems. The genome of AFP-003T has the genetic potential to produce secondary metabolites, with 39 genes found to be associated with polyketide synthases and non-ribosomal peptide syntases and 11 genes encoding for bacteriocins. Two regions encoding putative prophages and three OriC regions separated by the dnaA gene were predicted. Strain AFP-003T genome exhibits 86% average nucleotide identity with Mycobacterium genavense genome. Genetic and genomic data indicate that strain AFP-003T is representative of a novel Mycobacterium species that we named Mycobacterium ahvazicum, the nineteenth species of the expanding Mycobacterium simiae complex.

Detection of novel RNA viruses from free-living gorillas, Republic of the Congo: genetic diversity of picobirnaviruses.

Most of the emerging infectious diseases reported so far originated in wildlife. Therefore, virological surveillance of animals and particularly great apes is of great interest to establish the repertory of viruses associated with healthy hosts. This will further help to identify the emergence of new viruses and predict the possibility of interspecies transmission. In this study, we performed shotgun viral metagenomics on stool samples collected from seventeen free-living wild gorillas from the Republic of the Congo. The analysis revealed the presence of novel RNA viruses (picobirnaviruses, partitivirus, and Picornavirales (posa-like and dicistrovirus-like viruses)). Among these, picobirnavirus-related sequences were abundantly covered in the stools. Based on genetic variations both in capsid and RdRp proteins of picobirnaviruses, at least 96 variants were identified and most of them were novel. Among the 96, 22 variants had a nearly complete genome or segment. A comprehensive sequence analysis identified a potential new genogroup/genetic cluster and the presence of a short linear amino acid motif (ExxRxNxxxE) in a hypothetical protein. The sequence analysis of posa-like virus and dicistrovirus showed that these two viruses were novel members in the respective viral families. In conclusion, the identification of novel RNA viruses and their genetic diversity increases our knowledge about viruses that are associated with stools of wild gorillas and contributes to the initiatives in the search for potential emerging zoonotic viruses.

Sediminibacillus massiliensis sp. nov., a moderately halophilic, Gram-positive bacterium isolated from a stool sample of a young Senegalese man.

A Gram-positive, moderately halophilic bacterium, referred to as strain Marseille-P3518T, was isolated from a stool sample with 2% NaCl concentration from a healthy 15-year-old male living in Dielmo, a village in Senegal. Cells are aerobic, rod-shaped and motile and display endospore formation. Strain Marseille-P3518T can grow in a medium with 0-20% (w/v) sodium chloride (optimally at 5-7.5% w/v). The major fatty acids were 12-methyl-tetradecanoic acid (45.8%), 13-methyl-tetradecanoic acid (26.9%) and 12-methyl-tridecanoic acid (12.8%). The genome is 4,347,479 bp long with 42.1% G+C content. It contains 4282 protein-coding and 107 RNA genes. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that strain Marseille-P3518T is a member of the Bacillaceae family and is closely related to Sediminibacillus albus (97.4% gene sequence similarity). Strain Marseille-P3518T was clearly differentiated from its phylogenetic neighbors on the basis of phenotypic and genotypic features. Strain Marseille-P3518T is, therefore, considered to be a novel representative of the genus Sediminibacillus, for which the name Sediminibacillus massiliensis sp. nov. is proposed, and the type strain is Marseille-P3518T (CSUR P3518T, DSM69894).

Microbial Culturomics Broadens Human Vaginal Flora Diversity: Genome Sequence and Description of Prevotella lascolaii sp. nov. Isolated from a Patient with Bacterial Vaginosis.

Microbial culturomics is a new subfield of postgenomic medicine and omics biotechnology application that has broadened our awareness on bacterial diversity of the human microbiome, including the human vaginal flora bacterial diversity. Using culturomics, a new obligate anaerobic Gram-stain-negative rod-shaped bacterium designated strain khD1T was isolated in the vagina of a patient with bacterial vaginosis and characterized using taxonogenomics. The most abundant cellular fatty acids were C15:0 anteiso (36%), C16:0 (19%), and C15:0 iso (10%). Based on an analysis of the full-length 16S rRNA gene sequences, phylogenetic analysis showed that the strain khD1T exhibited 90% sequence similarity with Prevotella loescheii, the phylogenetically closest validated Prevotella species. With 3,763,057 bp length, the genome of strain khD1T contained (mol%) 48.7 G + C and 3248 predicted genes, including 3194 protein-coding and 54 RNA genes. Given the phenotypical and biochemical characteristic results as well as genome sequencing, strain khD1T is considered to represent a novel species within the genus Prevotella, for which the name Prevotella lascolaii sp. nov. is proposed. The type strain is khD1T ( = CSUR P0109, = DSM 101754). These results show that microbial culturomics greatly improves the characterization of the human microbiome repertoire by isolating potential putative new species. Further studies will certainly clarify the microbial mechanisms of pathogenesis of these new microbes and their role in health and disease. Microbial culturomics is an important new addition to the diagnostic medicine toolbox and warrants attention in future medical, global health, and integrative biology postgraduate teaching curricula.

Draft Genome Sequence of Kingella negevensis SW7208426, the First European Strain of K. negevensis Isolated from a Healthy Child in Switzerland.

We report here the draft genome of Kingella negevensis strain SW7208426, isolated from the oropharynx of a healthy 6-year-old boy in Geneva, Switzerland. To our knowledge, this is the first genome report of the newly described K. negevensis species from Europe.

Isolation and characterization of Kingella negevensis sp. nov., a novel Kingella species detected in a healthy paediatric population.

We herein report the isolation and characterization of 21 Gram-stain-negative strains cultivated from the oropharynx of healthy children in Israel and Switzerland. Initially described as small colony variants of Kingella kingae, phenotypic analysis, biochemical analysis, phylogenetic analysis based on sequencing of the partial 16S rRNA gene and five housekeeping genes (abcZ, adk, G6PD, groEL and recA), and whole genome sequencing and comparison between members of the genera Kingella and Neisseria provided evidence for assigning them to the genus Kingella. Cellular fatty acids included important amounts of C12 : 0, C14 : 0, C16 : 0 and C16 : 1n7. Digital DNA-DNA hybridization between the isolates Sch538T and K. kingae ATCC 23330T revealed relatedness of 19.9 %. Comparative analysis of 16S rRNA gene sequences available in GenBank allowed matches to strains isolated in the USA, suggesting a wider geographical distribution. A novel species named Kingella negevensis sp. nov. is proposed, as most strains have been isolated in the Negev, a desert region of southern Israel. The type strain is Sch538T (=CCUG 69806T=CSUR P957).

Gut Bacteria Missing in Severe Acute Malnutrition, Can We Identify Potential Probiotics by Culturomics?

Severe acute malnutrition is the world-leading cause of children under-five's death. Recent metagenomics studies have established a link between gut microbiota and severe acute malnutrition, describing an immaturity with a striking depletion in oxygen-sensitive prokaryotes. Amoxicillin and therapeutic diet cure most of the children with severe acute malnutrition but an irreversible disruption of the gut microbiota is suspected in the refractory and most severe cases. In these cases, therapeutic diet may be unable to reverse the microbiota alteration leading to persistent impaired development or death. In addition, as enteric sepsis is a major cause of death in this context, identification of missing gut microbes to be tested as probiotics (live bacteria that confer a benefit to the host) to restore rapidly the healthy gut microbiota and prevent the gut pathogenic invasion is of foremost importance. In this study, stool samples of malnourished patients with kwashiorkor and healthy children were collected from Niger and Senegal and analyzed by culturomics and metagenomics. We found a globally decreased diversity, a decrease in the hitherto unknown diversity (new species isolation), a depletion in oxygen-sensitive prokaryotes including Methanobrevibacter smithii and an enrichment in potentially pathogenic Proteobacteria, Fusobacteria and Streptococcus gallolyticus. A complex of 12 species identified only in healthy children using culturomics and metagenomics were identified as probiotics candidates, providing a possible, defined, reproducible, safe, and convenient alternative to fecal transplantation to restore a healthy gut microbiota in malnourished children. Microbiotherapy based on selected strains has the potential to improve the current treatment of severe acute malnutrition and prevent relapse and death by reestablishing a healthy gut microbiota.