80 research outputs found
Development and evaluation of a real-time PCR assay for detection and quantification of blastocystis parasites in human stool samples: prospective study of patients with hematological malignancies.
International audienceBlastocystis anaerobic parasites are widespread worldwide in the digestive tract of many animal species, including humans. Epidemiological Blastocystis studies are often limited by the poor sensitivity of standard parasitological assays for its detection. This report presents a highly sensitive real-time quantitative PCR (qPCR) assay developed to detect Blastocystis parasites in stool samples. The assay targets a partial sequence of the Blastocystis small ribosomal subunit (SSU) rRNA gene, allowing subtyping (ST) of Blastocystis isolates by direct sequencing of qPCR products. This qPCR method was assessed in a prospective study of 186 patients belonging to two cohorts--a group of 94 immunocompromised patients presenting hematological malignancies and a control group of 92 nonimmunocompromised patients. Direct-light microscopy and xenic in vitro stool culture analysis showed only 29% and 52% sensitivity, respectively, compared to our qPCR assay. Of the 27 (14.5%) Blastocystis-positive patients, 8 (4%) experienced digestive symptoms. No correlation was found between symptomatic patients and immune status, parasite load, or parasite subtypes, although subtyping of all isolates revealed a high (63.0%) prevalence of ST4. Two unexpected avian subtypes were found, i.e., ST6 and ST7, which are frequently isolated in Asia but rarely present in Western countries. In conclusion, this qPCR proved by far the most sensitive of the tested methods and allowed subtype determination by direct sequencing of qPCR products. New diagnostic tools such as the qPCR are essential for evaluating the clinical relevance of Blastocystis subtypes and their role in acute or chronic digestive disorders
Genome sequence of the stramenopile Blastocystis, a human anaerobic parasite
International audienceABSTRACT: BACKGROUND: Blastocystis is a highly prevalent anaerobic eukaryotic parasite of humans and animals that is associated with various gastrointestinal and extraintestinal disorders. Epidemiological studies have identified different subtypes but no one subtype has been definitively correlated with disease. RESULTS: Here we report the 18.8 Mb genome sequence of a Blastocystis subtype 7 isolate, which is the smallest stramenopile genome sequenced to date. The genome is highly compact and contains intriguing rearrangements. Comparisons with other available stramenopile genomes (plant pathogenic oomycete and diatom genomes) revealed effector proteins potentially involved in the adaptation to the intestinal environment, which were likely acquired via horizontal gene transfer. Moreover, Blastocystis living in anaerobic conditions harbors mitochondria-like organelles. An incomplete oxidative phosphorylation chain, a partial Krebs cycle, amino acid and fatty acid metabolisms and an iron-sulfur cluster assembly are all predicted to occur in these organelles. Predicted secretory proteins possess putative activities that may alter host physiology, such as proteases, protease-inhibitors, immunophilins and glycosyltransferases. This parasite also possesses the enzymatic machinery to tolerate oxidative bursts resulting from its own metabolism or induced by the host immune system. CONCLUSIONS: This study provides insights into the genome architecture of this unusual stramenopile. It also proposes candidate genes with which to study the physiopathology of this parasite and thus may lead to further investigations into Blastocystis-host interactions
New Insights into Blastocystis spp.: A Potential Link with Irritable Bowel Syndrome
International audienceBlastocystis spp. belong to the phylum Stramenopila, a complex and heterogeneous evolutionary assemblage of heterotrophic and photosynthetic protozoa [1]. Interestingly, this is the only stramenopile living in the lower digestive tract of humans, and it also lives in other mammals, birds, reptiles, amphibians, and insects [1]. Even though isolates were reported to be morphologically indistinguishable, an extensive genetic variation among isolates from both humans and animals has been observed. Thirteen subtypes (ST1-ST13), with the first nine being found in humans, have been identified based on genes coding for the small-subunit ribosomal RNA [2]. Preferential repartition of STs exists among animals that appear to constitute the main reservoir for environmental dissemination and human contamination
Robust estimation of bacterial cell count from optical density
Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data
Children of Senegal River Basin show the highest prevalence of Blastocystissp. ever observed worldwide
Genomics and post-genomics of the intestinal parasite Blastocystis ST7. Evaluation of its pathogenic potential
Blastocystis spp. est un StramĂ©nopile parasite anaĂ©robie frĂ©quemment rencontrĂ© dans le tractus gastro-intestinal de lâhomme et de divers animaux. Ce parasite est associĂ© Ă des troubles gastroâintestinaux aspĂ©cifiques, et semble impliquĂ© dans des dĂ©sordres fonctionnels tels que le syndrome de lâintestin irritable (IBS). Ce travail de thĂšse sâappuie sur le sĂ©quençage du gĂ©nome de Blastocystis sp. ST7 rĂ©alisĂ© en collaboration avec le GĂ©noscope dâEvry, lâUniversitĂ© Nationale de Singapour, lâInstitut Pasteur de Lille et lâUniversitĂ© de Provence. Ce gĂ©nome est constituĂ© dâun gĂ©nome nuclĂ©aire de 18,8 Mpb pour 6020 gĂšnes, et dâun gĂ©nome mitochondrial de 29 kpb localisĂ© dans des organites apparentĂ©s aux mitochondries. Lâanalyse de ce gĂ©nome apporte des informations au niveau de lâĂ©volution de ce microorganisme, de son adaptation Ă lâenvironnement intestinal et de ces facteurs de virulence potentiels. En effet, les analyses in silico de ce gĂ©nome ont montrĂ© que Blastocystis sp. ST7 possĂšde plusieurs gĂšnes codant des protĂ©ines pouvant agir Ă lâinterface entre lâhĂŽte et le parasite et connues chez dâautres protozoaires pour ĂȘtre impliquĂ©es dans des phĂ©nomĂšnes de pathogĂ©nie. Ce sont en particulier des PKS, des NRPS, et des hydrolases dont des protĂ©ases. Dâautre part, des activitĂ©s protĂ©olytiques ont Ă©tĂ© mises en Ă©vidence expĂ©rimentalement dans les surnageants de culture du parasite. Deux protĂ©ases Ă cystĂ©ines (une cathepsine B et une lĂ©gumaĂŻne) pouvant ĂȘtre impliquĂ©es dans la physiopathologie du parasite, ont Ă©tĂ© identifiĂ©es et caractĂ©risĂ©es dans les surnageants, confirmant ainsi nos analyses in silico. Ce travail ouvre de nombreuses pistes intĂ©ressantes Ă explorer pour Ă©valuer lâimpact de ce parasite en santĂ© humaine.Blastocystis spp. is a highly prevalent anaerobic Stramenopile parasite found in the intestinal tract of humans and various animals. This parasite is associated with non specific intestinal disorders, and could be involved in functional disorders such as the irritable bowel syndrome (IBS). In this work, the Blastocystis sp. ST7 genome sequencing project was carried out in collaboration with the GĂ©noscope of Evry, the National University of Singapore, the Pasteur Institute of Lille and the University of Provence. This genome consists in a nuclear genome of 18,8 Mpb encoding 6020 genes, and a mitochondriaâlike genome of 29 kpb localised in the mitochondrionâlike organelles. The analysis of this genome brings information about the evolution of this microâorganism, its adaptation to the intestinal environment and its potential virulence factors. Blastocystis sp. ST7 was predicted to harbor several genes coding proteins that could act at the parasiteâhost interface, and that are known to be involved in the pathogeny of many protozoa. They are PKS, NRPS, and hydrolases among them proteases. In addition, proteolytic activities were highlighted in the parasite culture supernatants. Two cysteine proteases (a cathepsin B and a legumain) were identified and characterized from the supernatants and could play a role in the physiopathology of the parasite, that confirm our in silico analyses. This work opens new ways to evaluate the impact of this parasite in human health
Génomique et post-génomique du parasite intestinal Blastocystis sp. sous-type 7. Evaluation de son pouvoir pathogÚne
Blastocystis spp. is a highly prevalent anaerobic Stramenopile parasite found in the intestinal tract of humans and various animals. This parasite is associated with non specific intestinal disorders, and could be involved in functional disorders such as the irritable bowel syndrome (IBS). In this work, the Blastocystis sp. ST7 genome sequencing project was carried out in collaboration with the GĂ©noscope of Evry, the National University of Singapore, the Pasteur Institute of Lille and the University of Provence. This genome consists in a nuclear genome of 18,8 Mpb encoding 6020 genes, and a mitochondriaâlike genome of 29 kpb localised in the mitochondrionâlike organelles. The analysis of this genome brings information about the evolution of this microâorganism, its adaptation to the intestinal environment and its potential virulence factors. Blastocystis sp. ST7 was predicted to harbor several genes coding proteins that could act at the parasiteâhost interface, and that are known to be involved in the pathogeny of many protozoa. They are PKS, NRPS, and hydrolases among them proteases. In addition, proteolytic activities were highlighted in the parasite culture supernatants. Two cysteine proteases (a cathepsin B and a legumain) were identified and characterized from the supernatants and could play a role in the physiopathology of the parasite, that confirm our in silico analyses. This work opens new ways to evaluate the impact of this parasite in human health.Blastocystis spp. est un StramĂ©nopile parasite anaĂ©robie frĂ©quemment rencontrĂ© dans le tractus gastro-intestinal de lâhomme et de divers animaux. Ce parasite est associĂ© Ă des troubles gastroâintestinaux aspĂ©cifiques, et semble impliquĂ© dans des dĂ©sordres fonctionnels tels que le syndrome de lâintestin irritable (IBS). Ce travail de thĂšse sâappuie sur le sĂ©quençage du gĂ©nome de Blastocystis sp. ST7 rĂ©alisĂ© en collaboration avec le GĂ©noscope dâEvry, lâUniversitĂ© Nationale de Singapour, lâInstitut Pasteur de Lille et lâUniversitĂ© de Provence. Ce gĂ©nome est constituĂ© dâun gĂ©nome nuclĂ©aire de 18,8 Mpb pour 6020 gĂšnes, et dâun gĂ©nome mitochondrial de 29 kpb localisĂ© dans des organites apparentĂ©s aux mitochondries. Lâanalyse de ce gĂ©nome apporte des informations au niveau de lâĂ©volution de ce microorganisme, de son adaptation Ă lâenvironnement intestinal et de ces facteurs de virulence potentiels. En effet, les analyses in silico de ce gĂ©nome ont montrĂ© que Blastocystis sp. ST7 possĂšde plusieurs gĂšnes codant des protĂ©ines pouvant agir Ă lâinterface entre lâhĂŽte et le parasite et connues chez dâautres protozoaires pour ĂȘtre impliquĂ©es dans des phĂ©nomĂšnes de pathogĂ©nie. Ce sont en particulier des PKS, des NRPS, et des hydrolases dont des protĂ©ases. Dâautre part, des activitĂ©s protĂ©olytiques ont Ă©tĂ© mises en Ă©vidence expĂ©rimentalement dans les surnageants de culture du parasite. Deux protĂ©ases Ă cystĂ©ines (une cathepsine B et une lĂ©gumaĂŻne) pouvant ĂȘtre impliquĂ©es dans la physiopathologie du parasite, ont Ă©tĂ© identifiĂ©es et caractĂ©risĂ©es dans les surnageants, confirmant ainsi nos analyses in silico. Ce travail ouvre de nombreuses pistes intĂ©ressantes Ă explorer pour Ă©valuer lâimpact de ce parasite en santĂ© humaine
Génomique et post-génomique du parasite intestinal Blastocystis sp. sous-type 7. Evaluation de son pouvoir pathogÚne
Blastocystis spp. est un StramĂ©nopile parasite anaĂ©robie frĂ©quemment rencontrĂ© dans le tractus gastro-intestinal de l homme et de divers animaux. Ce parasite est associĂ© Ă des troubles gastro intestinaux aspĂ©cifiques, et semble impliquĂ© dans des dĂ©sordres fonctionnels tels que le syndrome de l intestin irritable (IBS). Ce travail de thĂšse s appuie sur le sĂ©quençage du gĂ©nome de Blastocystis sp. ST7 rĂ©alisĂ© en collaboration avec le GĂ©noscope d Evry, l UniversitĂ© Nationale de Singapour, l Institut Pasteur de Lille et l UniversitĂ© de Provence. Ce gĂ©nome est constituĂ© d un gĂ©nome nuclĂ©aire de 18,8 Mpb pour 6020 gĂšnes, et d un gĂ©nome mitochondrial de 29 kpb localisĂ© dans des organites apparentĂ©s aux mitochondries. L analyse de ce gĂ©nome apporte des informations au niveau de l Ă©volution de ce microorganisme, de son adaptation Ă l environnement intestinal et de ces facteurs de virulence potentiels. En effet, les analyses in silico de ce gĂ©nome ont montrĂ© que Blastocystis sp. ST7 possĂšde plusieurs gĂšnes codant des protĂ©ines pouvant agir Ă l interface entre l hĂŽte et le parasite et connues chez d autres protozoaires pour ĂȘtre impliquĂ©es dans des phĂ©nomĂšnes de pathogĂ©nie. Ce sont en particulier des PKS, des NRPS, et des hydrolases dont des protĂ©ases. D autre part, des activitĂ©s protĂ©olytiques ont Ă©tĂ© mises en Ă©vidence expĂ©rimentalement dans les surnageants de culture du parasite. Deux protĂ©ases Ă cystĂ©ines (une cathepsine B et une lĂ©gumaĂŻne) pouvant ĂȘtre impliquĂ©es dans la physiopathologie du parasite, ont Ă©tĂ© identifiĂ©es et caractĂ©risĂ©es dans les surnageants, confirmant ainsi nos analyses in silico. Ce travail ouvre de nombreuses pistes intĂ©ressantes Ă explorer pour Ă©valuer l impact de ce parasite en santĂ© humaine.Blastocystis spp. is a highly prevalent anaerobic Stramenopile parasite found in the intestinal tract of humans and various animals. This parasite is associated with non specific intestinal disorders, and could be involved in functional disorders such as the irritable bowel syndrome (IBS). In this work, the Blastocystis sp. ST7 genome sequencing project was carried out in collaboration with the GĂ©noscope of Evry, the National University of Singapore, the Pasteur Institute of Lille and the University of Provence. This genome consists in a nuclear genome of 18,8 Mpb encoding 6020 genes, and a mitochondria like genome of 29 kpb localised in the mitochondrion like organelles. The analysis of this genome brings information about the evolution of this micro organism, its adaptation to the intestinal environment and its potential virulence factors. Blastocystis sp. ST7 was predicted to harbor several genes coding proteins that could act at the parasite host interface, and that are known to be involved in the pathogeny of many protozoa. They are PKS, NRPS, and hydrolases among them proteases. In addition, proteolytic activities were highlighted in the parasite culture supernatants. Two cysteine proteases (a cathepsin B and a legumain) were identified and characterized from the supernatants and could play a role in the physiopathology of the parasite, that confirm our in silico analyses. This work opens new ways to evaluate the impact of this parasite in human health.CLERMONT FD-Bib.Ă©lectronique (631139902) / SudocSudocFranceF
Resolvin D1 reduces inflammation in co-cultures of primary human macrophages and adipocytes by triggering macrophages
Obesity leads to chronic inflammation of the adipose tissue which is tightly associated with the metabolic syndrome, type 2 diabetes and cardiovascular disease. Inflammation of the adipose tissue is mainly characterized by the presence of crown-like structures composed of inflammatory macrophages in the neighborhood of adipocytes. Resolvin D1 (RvD1), a potent anti-inflammatory and pro-resolving lipid mediator derived from the omega-3 fatty acid docosahexaenoic acid, has been shown to reduce the inflammatory tone of adipose tissue in animal models but the underlying mechanism is not clear. We investigated the effect of RvD1 on the inflammatory state of a human co-culture system of adipocytes and macrophages. For this, human mesenchymal stem cells were differentiated into mature adipocytes and overlaid with human primary macrophages. In this co-culture, 10-500 nM RvD1 dose-dependently reduced the secretion of the pro-inflammatory cytokine IL-6 (-21%) and its soluble receptor IL-6Rα (-22%), of the chemokine MCP-1 (-13%), and of the adipokine leptin (-22%). Similarly, we observed a reduction in secretion of the soluble receptor IL-6Rα (-20%), and TNF-α (-11%) when macrophages alone were treated with RvD1, while no change of cytokine secretion was observed when adipocytes were treated with RvD1. We conclude that RvD1 polarizes macrophages to an anti-inflammatory phenotype, which in turn modulates inflammation in adipocytes
Genomic analysis of the Bacillus megaterium Mes11: New insights into nitroreductase genes associated with the degradation of mesotrione
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