Combined bacterial and fungal intestinal microbiota analyses: Impact of storage conditions and DNA extraction protocols

Background The human intestinal microbiota contains a vast community of microorganisms increasingly studied using high-throughput DNA sequencing. Standardized protocols for storage and DNA extraction from fecal samples have been established mostly for bacterial microbiota analysis. Here, we investigated the impact of storage and DNA extraction on bacterial and fungal community structures detected concomitantly. Methods Fecal samples from healthy adults were stored at -80'C as such or diluted in RNAlater0 and subjected to 2 extraction protocols with mechanical lysis: the Powersoil (R) MoBio kit or the International Human Microbiota Standard (IHMS) Protocol Q. Libraries of the 12 samples targeting the V3-V4 16S and the ITS1 regions were prepared using Metabiote (R) (Genoscreen) and sequenced on GS-FLX-454. Sequencing data were analysed using SHAMAN (http://shaman.pasteur.fr/). The bacterial and fungal microbiota were compared in terms of diversity and relative abundance. Results We obtained 171869 and 199089 quality-controlled reads for 16S and ITS, respectively. All 16S reads were assigned to 41 bacterial genera; only 52% of ITS reads were assigned to 40 fungal genera/section. Rarefaction curves were satisfactory in 3/3 and 2/3 subjects for 16S and ITS, respectively. PCoA showed important inter-individual variability of intestinal microbiota largely overweighing the effect of storage or extraction. Storage in RNAlater (R) impacted (downward trend) the relative abundances of 7/41 bacterial and 6/40 fungal taxa, while extraction impacted randomly 18/41 bacterial taxa and 1/40 fungal taxon. Conclusion Our results showed that RNAlater (R) moderately impacts bacterial or fungal community structures, while extraction significantly influences the bacterial composition. For combined bacterial and fungal intestinal microbiota analysis, immediate sample freezing should be preferred when feasible, but storage in RNAlater (R) remains an option under unfavourable conditions or for concomitant metatranscriptomic analysis; and extraction should rely on protocols validated for bacterial analysis, such as IHMS Protocol Q, and including a powerful mechanical lysis, essential for fungal extraction

ER-mitochondria cross-talk is regulated by the Ca(2+) sensor NCS1 and is impaired in Wolfram syndrome.

Communication between the endoplasmic reticulum (ER) and mitochondria plays a pivotal role in Ca(2+) signaling, energy metabolism, and cell survival. Dysfunction in this cross-talk leads to metabolic and neurodegenerative diseases. Wolfram syndrome is a fatal neurodegenerative disease caused by mutations in the ER-resident protein WFS1. Here, we showed that WFS1 formed a complex with neuronal calcium sensor 1 (NCS1) and inositol 1,4,5-trisphosphate receptor (IP3R) to promote Ca(2+) transfer between the ER and mitochondria. In addition, we found that NCS1 abundance was reduced in WFS1-null patient fibroblasts, which showed reduced ER-mitochondria interactions and Ca(2+) exchange. Moreover, in WFS1-deficient cells, NCS1 overexpression not only restored ER-mitochondria interactions and Ca(2+) transfer but also rescued mitochondrial dysfunction. Our results describe a key role of NCS1 in ER-mitochondria cross-talk, uncover a pathogenic mechanism for Wolfram syndrome, and potentially reveal insights into the pathogenesis of other neurodegenerative diseases

Human cryptosporidiosis in immunodeficient patients in France (2015–2017)

International audienceCryptosporidiosis is a common disease in children and immunodeficient individuals. In 2006, a national network was set up on the surveillance of human cryptosporidiosis in France. Since January 2015, the 41 tertiary care hospitals and the 3 private laboratories of the French National Network on the surveillance of human cryptosporidiosis have been able to declare confirmed cases of cryptosporidiosis online. Between 2015 and 2017, 210 cases of cryptosporidiosis were declared in immunodeficient patients in France; Cryptosporidium parvum and Cryptosporidium hominis represented 66% and 22% of cases, respectively. A peak was observed in autumn. Cryptosporidiosis occurred mainly in a context of solid organ transplantation (SOT) (49%) and of HIV infection (30%). In SOT recipients, cryptosporidiosis appeared more frequently in the first 6 months post transplantation. Regarding cases declared in SOT recipients, mycophenolate mofetil was used in 68%. A mortality rate of 6% was observed. Present results underline the importance of screening for cryptosporidiosis in immunocompromised patients suffering from diarrhea, especially in the course of major cell mediated immunodeficiency or even systematic screening before SOT. Exclusive Cryptosporidium free water feeding could be suggested during major cell mediated immunodeficiency