Project description:We compared the microbiota of paired mouse caecal contents and faeces by applying a multi-omic approach, including 16S rDNA sequencing, shotgun metagenomics, and shotgun metaproteomics. The aim of the study was to verify whether faecal samples are a reliable proxy for the mouse colonic luminal microbiota, as well as to identify changes in taxonomy and functional activity between caecal and faecal microbial communities, which have to be carefully considered when using stool as sample for mouse gut microbiota investigations.
Project description:The intestinal microbiota plays a key role in shaping host homeostasis by regulating metabolism, immune responses and behaviour. Its dysregulation has been associated with metabolic, immune and neuropsychiatric disorders and is accompanied by changes in bacterial metabolic regulation. Although proteomic is well suited for analysis of individual microbes, metaproteomic of faecal samples is challenging due to the physical structure of the sample, presence of contaminating host proteins and coexistence of hundreds of species. Furthermore, there is a lack of consensus regarding preparation of faecal samples, as well as downstream bioinformatic analyses following metaproteomic data acquisition. Here we assess sample preparation and data analysis strategies applied to mouse faeces in a typical LC-MS/MS metaproteomic experiment. We show that low speed centrifugation (LSC) of faecal samples leads to high protein identification rates but possibly enriched for a subset of taxa. During database search, two-step search strategies led to dramatic and underestimated accumulation of false positive protein identifications. Regarding taxonomic annotation, the MS-identified peptides of unknown origin were annotated with highest sensitivity and specificity using the Unipept software. Comparison of matching metaproteome and metagenome data revealed a positive correlation between protein and gene abundances. Notably, nearly all functional categories of detected protein groups were differentially abundant in the metaproteome compared to what would be expected from the metagenome, highlighting the need to perform metaproteomic when studying complex microbiome samples.
Project description:Age-dependent changes of the gut-associated microbiome have been linked to increased frailty and systemic inflammation. This study found that age-associated changes of the gut microbiome of BALB/c and C57BL/6 mice could be reverted by co-housing of aged (22 months old) and adult (3 months old) mice for 30-40 days or faecal microbiota transplantation (FMT) from adult into aged mice. This was demonstrated using high-throughput sequencing of the V3-V4 hypervariable region of bacterial 16S rRNA gene isolated from faecal pellets collected from 3-4 months old adult and 22-23 months old aged mice before and after co-housing or FMT.
Project description:To further decipher the alteration of gene expression profile of irradiated mice with or without faecal microbiota transplantation (FMT), we performed FMT for 10 days following total body irradiaton (6.5 Gy gamma ray). Twenty-one days after irradiation, the mice were euthanized and the small intestine tissues excised.
Project description:Behaviour is derived from complex molecular interactions within the brain, in response to specific environmental stimuli. In some rare cases, the direct causes of behaviour have been attributed to the interactions of a single or small group of gene transcripts and proteins. We conducted two experiments with the hope of defining some of the molecular interactors for four separate behaviours: sugar feeding, locomotor activity in a novel field, and acoustic startle reflex, and prepulse inhibition of the acoustic startle reflex, which have been linked to prefrontal cortex dopaminergic function or as predictors of sensorimotor gating in diseases such as schizophrenia. Rats with high and low response phenotypes were selected to determine the differences between these two extremes of behaviour. From our analyses, transcripts of Homer1, a neuronal scaffolding protein which interacts with group1 metabotropic glutamate receptors, were found to be significantly correlated with array data in both experiments, and with behaviour data across three separate tests in the second experiment, indicating that this gene's transcripts and probably downstream protein interactions have a significant correlation with behaviour phenotype in the inbred Lewis rat. Future areas of pursuit for this data should involve modification of the expression of Homer1 in an isolated fashion to determine a pharmacological threshold for differences in behaviour. This SuperSeries is composed of the SubSeries listed below.
Project description:The human gut microbiota is crucial for degrading dietary fibres from the diet. However, some of these bacteria can also degrade host glycans, such as mucins, the main component of the protective gut mucus layer. Specific microbiota species and mucin degradation patterns are associated with inflammatory processes in the colon. Yet, it remains unclear how the utilization of mucin glycans affects the degradation of dietary fibres by the human microbiota. Here, we used three dietary fibres (apple pectin, β-glucan and xylan) to study in vitro the dynamics of colon mucin and dietary fibre degradation by the human faecal microbiota. The dietary fibres showed clearly distinguishing modulatory effects on faecal microbiota composition. The utilization of colon mucin in cultures led to alterations in microbiota composition and metabolites. Metaproteome analysis showed the central role of the Bacteroides in degradation of complex fibres while Akkermansia muciniphila was the main degrader of colonic mucin. This work demonstrates the intricacy of complex glycan metabolism by the gut microbiota and how the utilization of host glycans leads to alterations in the metabolism of dietary fibres. Metaproteomics analysis of this data reveals the functional activities of the bacteria in consortia, by this contributing to a better understanding of the complex metabolic pathways within the human microbiota that can be manipulated to maximise beneficial microbiota-host interactions.
Project description:A phylogenetic microarray targeting 66 families described in the human gut microbiota has been developped aud used to monitor the gut microbiota's structure and diversity. The microarray format provided by Agilent and used in this study is 8x15K. A study with a total of 4 chips was realized. Arrays 1 and 2: Hybridization with 100ng of labelled 16S rRNA gene amplicons from a mock community sample and 250ng of labelled 16S rRNA gene amplicons from 1 faecal sample. Each Agilent-030618 array probe (4441) was synthetized in three replicates. Arrays 3 and 4: Hybridization with 250ng of labelled 16S rRNA gene amplicons from 2 faecal samples. Each Agilent-40558 array probe (4441) was synthetized in three replicates.