Mutational analysis of the PLCE1 gene in steroid-resistant nephrotic syndrome

International audienceBackground: Mutations in the PLCE1 gene encoding phospholipase C epsilon 1 (PLCε1) have been recently described in patients with early-onset nephrotic syndrome (NS) and diffuse mesangial sclerosis (DMS). In addition, two cases of PLCE1 mutations associated with focal segmental glomerulosclerosis (FSGS) and later NS onset have been reported. Methods: In order to better assess the spectrum of phenotypes associated with PLCE1 mutations, we performed mutational analysis in a worldwide cohort of 139 patients (95 familial cases belonging to 68 families and 44 sporadic cases) with steroid-resistant NS presenting at a median age of 23.0 months (range 0-373). Results: We identified homozygous or compound heterozygous mutations in 33% (8/24) of DMS cases. PLCE1 mutations were found in 8% (6/78) of FSGS cases without NPHS2 mutations. Nine were novel mutations. No clear genotype-phenotype correlation was observed, with either truncating or missense mutations detected in both DMS and FSGS, and leading to a similar renal evolution. Surprisingly, 3 unaffected and unrelated individuals were also found to carry the homozygous mutations identified in their respective families. Conclusion: PLCE1 is a major gene of DMS and is mutated in a non-negligible proportion of FSGS cases without NPHS2 mutations. Although we did not identify additional variants in 19 candidate genes (16 other PLC genes, BRAF, IQGAP1 and NPHS1), we speculate that other modifier genes or environmental factors may play a role in the renal phenotype variability observed in individuals bearing PLCE1 mutations. This observation needs to be considered in the genetic counselling offered to patients

Microbial Ecology of French Dry Fermented Sausages and Mycotoxin Risk Evaluation During Storage

Dry fermented sausages are produced worldwide by well-controlled fermentationprocesses involving complex microbiota including many bacterial and fungal species with key technological roles. However, to date, fungal diversity on sausage casings during storage has not been fully described. In this context, we studied the microbial communities from dry fermented sausages naturally colonized or voluntarily surface inoculated with molds during storage using both culture-dependent and metabarcoding methods. Staphylococci and lactic acid bacteria largely dominated in samples, although some halotolerant genera (e.g., Halomonas, Tetragenococcus, and Celerinatantimonas spp.) were also frequently observed. Fungal populations varied from 7.2 to 9.8 log TFU/cm2 sausage casing during storage, suggesting relatively low count variability among products. Fungal diversity identified on voluntarily inoculated casings was lower (dominated by Penicillium nalgiovense and Debaryomyces hansenii) than naturally environment-inoculated fermented sausages (colonized by P. nalgiovense, Penicillium nordicum, and other Penicillium spp. and sporadically by Scopulariopsis sp., D. hansenii, and Candida zeylanoïdes). P. nalgiovense and D. hansenii were systematically identified, highlighting their key technological role. The mycotoxin risk was then evaluated, and in situ mycotoxin production of selected mold isolates was determined during pilot-scale sausage productions. Among the identified fungal species, P. nalgiovense was confirmed not to produce mycotoxins. However, some P. nordicum, Penicillium chrysogenum, Penicillium bialowienzense, Penicillium brevicompactum, and Penicillium citreonigrum isolates produced one or more mycotoxins in vitro. P. nordicum also produced ochratoxin A during pilotscale sausage productions using “worst-case” conditions in the absence of biotic competition. These data provide new knowledge on fermented sausage microbiota and the potential mycotoxin risk during storage

Mutations des gènes du SRA et anomalies du développement rénal

La dysgénésie tubulaire rénale (DTR) autosomique récessive est une anomalie sévère du développement rénal. Elle se manifeste par une anurie fœtale persistante responsable d'oligoamnios et, le plus souvent, de mort périnatale. Il s'y associe une hypotension réfractaire observée chez les nouveau-nés survivants et un retard d'ossification de la voûte crânienne. La néphropathie est caractérisée histologiquement par l'absence, ou la présence d'un nombre très réduit de tubes proximaux différenciés, associées constamment à une anomalie majeure de l'expression rénale de rénine. Nous avons récemment montré que cette néphropathie était liée à des mutations des gènes codant pour les différentes protéines du système rénine-angiotensine (SRA). L'étude de 46 familles confirme le rôle majeur des mutations des gènes du SRA à l'origine de la DTR récessive autosomique : des mutations homozygotes ou hétérozygotes composites ont été identifiées dans 41 familles (F). Elles touchaient les gènes REN (9F), AGT (3F), AGTR1 (3F) ou ACE (26F). Le phénotype est le même quel que soit le gène muté. Les parents sont asymptomatiques. Cette étude souligne l'importance du SRA dans le développement du rein humain. L'identification du défaut moléculaire permet de proposer à la famille un conseil génétique précis et à leur demande un diagnostic prénatal précoce

Individual and combined toxicological effects of deoxynivalenol and zearalenone on human hepatocytes in in vitro chronic exposure conditions

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Unraveling microbial ecology of industrial-scale Kombucha fermentations by metabarcoding and culture-based methods.

Kombucha, historically an Asian tea-based fermented drink, has recently become trendy in Western countries. Producers claim it bears health-enhancing properties that may come from the tea or metabolites produced by its microbiome. Despite its long history of production, microbial richness and dynamics have not been fully unraveled, especially at an industrial scale. Moreover, the impact of tea type (green or black) on microbial ecology was not studied. Here, we compared microbial communities from industrial-scale black and green tea fermentations, still traditionally carried out by a microbial biofilm, using culture-dependent and metabarcoding approaches. Dominant bacterial species belonged to Acetobacteraceae and to a lesser extent Lactobacteriaceae, while the main identified yeasts corresponded to Dekkera, Hanseniaspora and Zygosaccharomyces during all fermentations. Species richness decreased over the 8-day fermentation. Among acetic acid bacteria, Gluconacetobacter europaeus, Gluconobacter oxydans, G. saccharivorans and Acetobacter peroxydans emerged as dominant species. The main lactic acid bacteria, Oenococcus oeni, was strongly associated with green tea fermentations. Tea type did not influence yeast community, with Dekkera bruxellensis, D. anomala, Zygosaccharomyces bailii and Hanseniaspora valbyensis as most dominant. This study unraveled a distinctive core microbial community which is essential for fermentation control and could lead to Kombucha quality standardization

Use of metabarcoding and source tracking to identify desirable or spoilage autochthonous microorganism sources during black olive fermentations

International audienceThis study aimed at investigating the influence of the process environment and raw materials as sources of microorganisms during Nyons black table olive fermentations. Fermented olives and/or brine from spoiled fermentation tanks were analyzed and compared to good quality samples from fermentations collected during 3 consecutive harvest years. Fresh olives, salt and different process environment samples were also analyzed. Microbial diversity of all samples was analyzed using 16S and ITS2 amplicon sequencing and SourceTracker tool was used to investigate links between environment, raw materials and fermentation samples. First, comparison of microbial diversity in control and most spoiled fermentations revealed striking differences in bacterial composition with an overall higher diversity in spoiled fermentations especially for lactic acid bacteria with Lentilactobacillus buchneri, Lentilactobacillus parafarraginis dominating in brine and Pediococcus parvulus, Pediococcus ethanolidurans dominating in olive fruits. Fungal communities were similar in composition although higher abundances of Pichia membranifaciens and Penicillium carneum/roqueforti were observed in spoiled samples. Secondly, process environment samples were characterized by high bacterial and fungal diversity, especially compared to fresh olive fruits. Overall, dominant fungal species in control fermentations were also found in most environmental samples revealing a "house mycobiota". SourceTracker analysis further highlighted the contribution of brine and water from the optical sorter as a source of fungi. Most interestingly, spoilage fungi and most bacteria were retrieved in brine and environmental samples while others such as P. ethanolidurans were only found in environmental samples indicating that the studied spoilage originated from a fermentation deviation rather than a punctual contamination. Taken altogether, these results highlighted the positive and negative influence of the process environment and emphasized the relevance of studying it to better understand microbial vectors occurring during food fermentations, especially natural ones

Hepatotoxicity of fusariotoxins, alone and in combination, towards the HepaRG human hepatocyte cell line

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The infectious process of Colletotrichum lupini, a major threat for lupin crops

International audienceColletotrichum lupini is a species within the Colletotrichum acutatum species complex (CAsc) causing anthracnose, a serious disease affecting lupin crops worldwide. In addition to its economic impact, C. lupini is a useful model to study host speciation process since it is known to infect only lupins. To date, this pathogenic fungus has been poorly described. In this context, our work aims at getting better insight into the genome, the physiology and the infectious process of the fungus. First, based on the recent annotation of two C. lupini reference genomes, we identified specific regions of C. lupini that are absent in the genome of other species from the CAsc. In order to better understand the infectious process of the fungi, transcriptomic and proteomic analyses were performed on C. lupini infected lupin seeds during transition from the biotrophic to the necrotrophic stage. The results allowed us to identify specific pathogenicity gene markers of both stages. The expression of these markers has been further evaluated using a set of C. lupini strains. These results together with the description of the physiology of C. lupini during the first steps of the infection will significantly increase knowledge in host speciation process and ultimately contribute (i) to the early detection of the fungus in the field and (ii) to the development of new resistant lupin varieties

Technical note: High-throughput method for antifungal activity screening in a cheese-mimicking model

In this study, we developed a high-throughput antifungalactivity screening method using a cheese-mimickingmatrix distributed in 24-well plates. This method allowed rapid screening of a large variety of antifungalagent candidates: bacterial fermented ingredients, bacterial isolates, and preservatives. Using the proposedmethod, we characterized the antifungal activity of 44 lactic acid bacteria (LAB) fermented milk-based ingredients and 23 LAB isolates used as protective cultures against 4 fungal targets (Mucor racemosus, Penicillium commune, Galactomyces geotrichum, and Yarrowia lipolytica). We also used this method to determine theminimum inhibitory concentration of a preservative, natamycin, against 9 fungal targets. The results underlinedthe strain-dependency of LAB antifungal activity, the strong effect of fermentation substrate on this activity,and the effect of the screening medium on natamycin minimum inhibitory concentration. Our methodcould achieved a screening rate of 1,600 assays per week and can be implemented to evaluate antifungal activityof microorganisms, fermentation products, or purified compounds compatible with dairy technology

Diversity of spoilage fungi associated with various French dairy products

Yeasts and molds are responsible for dairy product spoilage, resulting in significant food waste and economiclosses. Yet, few studies have investigated the diversity of spoilage fungi encountered in dairy products. In thepresent study, 175 isolates corresponding to 105 from various spoiled dairy products and 70 originating fromdairy production environments,were identified using sequencing of the ITS region, the partial β-tubulin, calmodulinand/or EFα genes, and the D1–D2 domain of the 26S rRNA gene for filamentous fungi and yeasts, respectively.Among the 41 species found in spoiled products, Penicillium commune and Penicillium bialowiezense were themost common filamentous fungi, representing around 10% each of total isolateswhile Meyerozyma guilliermondiiand Trichosporon asahii were the most common yeasts (4.8% each of total isolates). Several species (e.g. Penicilliumantarcticum, Penicillium salamii and Cladosporium phyllophilum) were identified for the first time in dairyproducts or their environment. In addition, numerous species were identified in both spoiled products andtheir corresponding dairy production environment suggesting that the latter acts as a primary source of contamination.Secondly, the resistance to chemical preservatives (sodiumbenzoate, calciumpropionate, potassiumsorbateand natamycin) of 10 fungal isolates representative of the observed biodiversity was also evaluated.Independently of the fungal species, natamycin had the lowest minimum inhibitory concentration (expressedin gramof preservative/l), followed by potassiumsorbate, sodiumbenzoate and calciumpropionate. In the testedconditions, Cladosporium halotolerans and Didymella pinodella were the most sensitive fungi while Yarrowialipolytica and Candida parapsilosis were the most resistant towards the tested preservatives. This study providesinteresting information on the occurrence of fungal contaminants in dairy products and environments thatmayhelp developing adequate strategies for fungal spoilage control