Project description:The aim of this study was to develop a suitable method to preserve fecal samples for metaproteomics analyses when flash-freezing is not an option. Fecal samples were collected from conventional adult C57BL/6 mice and combined into a fecal master mix. The fecal master mix was then split into 48 subsamples that were subjected to different preservation treatments. The following six preservation methods were tested: flash-freezing in liquid nitrogen followed by storage at -80°C, immersion in RNAlater® and storage at room temperature, immersion in RNAlater® and immediate storage at -80°C, immersion in 95% ethanol and storage at room temperature, immersion in a RNAlater-like buffer “NAP buffer” and storage at room temperature, and immersion in an autoclaved RNAlater-like buffer “Autoclaved NAP buffer” and storage at room temperature. Proteins were extracted from the samples after being stored for 1 and 4 weeks. There were 4 replicates per treatment and time-point. Samples were analyzed by LC-MS/MS and the data were analyzed with Proteome Discoverer against a large database of mouse microbiota protein sequences.
Project description:Analysis of breast cancer survivors' gut microbiota after lifestyle intervention, during the COVID-19 lockdown, by 16S sequencing of fecal samples.
Project description:Microbial RNAseq analysis of cecal and fecal samples collected from mice colonized with the microbiota of human twins discordant for obesity. Samples were colleted at the time of sacrifice, or 15 days after colonization from mice gavaged with uncultured or cultured fecal microbiota from the lean twins or their obese co-twins. Samples were sequenced using Illumina HiSeq technology, with 101 paired end chemistry.
Project description:Habitual exercise modulates the composition of the intestinal microbiota. We examined whether transplanting fecal microbiota from trained mice improved skeletal muscle metabolism in high-fat diet-fed mice. The recipient mice that received fecal samples from trained donor mice for 1 week showed elevated levels of metabolic signalings in skeletal muscle. Glucose tolerance was improved by fecal microbiota transplantation after 8 weeks of HFD administration. Intestinal microbiota may mediate exercise-induced metabolic improvement in mice. We performed a microarray analysis to compare the metabolic gene expression profiles in the skeletal muscle from each mouse.
Project description:We found that low protein diet consumption resulted in decrease in the percentage of normal Paneth cell population in wild type mice, indicating that low protein diet could negatively affect Paneth cell function. We performed fecal microbiota composition profiling. Male mice were used at 4-5 weeks of age. Fecal samples were collected for microbiome analysis.
Project description:Microbial RNAseq analysis of cecal and fecal samples collected from mice colonized with the microbiota of human twins discordant for obesity. Samples were colleted at the time of sacrifice, or 15 days after colonization from mice gavaged with uncultured or cultured fecal microbiota from the lean twins or their obese co-twins. Samples were sequenced using Illumina HiSeq technology, with 101 paired end chemistry. Comparisson of microbial gene expression between the microbiota of lean and obese twins fed a Low fat, rich in plant polysaccharide diet.
Project description:We found that western diet consumption resulted in decrease in the percentage of normal Paneth cell population in wild type mice, indicating that western diet could negatively affect Paneth cell function. Subsequent generations of western diet consumption further reduced percentages of normal Paneth cell population. We performed fecal microbiota composition profiling. Male mice were used at 4-5 weeks of age. Fecal samples were collected for microbiome analysis.
Project description:<p><strong>BACKGROUND:</strong> Stool metabolites provide essential insights into the function of the gut microbiome. The current gold standard for storage of stool samples for metabolomics is flash-freezing at - 80 °C which can be inconvenient and expensive. Ambient temperature storage of stool is more practical, however no available methodologies adequately preserve the metabolomic profile of stool. A novel sampling kit (OMNImet.GUT; DNA Genotek, Inc.) was introduced for ambient temperature storage and stabilization of feces for metabolomics; we aimed to test the performance of this kit vs flash-freezing. To do this stool was collected from an infant's diaper was divided into 2 aliquots: 1) flash-frozen and 2) stored in an OMNImet.GUT tube at ambient temperature for 3-4 days. Samples from the same infant were collected at 2 different timepoints to assess metabolite changes over time. Subsequently, all samples underwent metabolomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).</p><p><strong>RESULTS:</strong> Paired fecal samples (flash-frozen and ambient temperature) from 16 infants were collected at 2 timepoints (32 individual samples, 64 aliquots). Similar numbers of metabolites were detected in both the frozen and ambient temperature samples (1126 in frozen, 1107 in ambient temperature, 1064 shared between sample types). Metabolite abundances were strongly correlated between storage methods (median Spearman correlation Rs = 0.785 across metabolites). Hierarchical clustering analysis and principal component analysis showed that samples from the same individuals at a given timepoint clustered closely, regardless of the storage method. Repeat samples from the same individual were compared by paired t-test, separately for the frozen and OMNImet.GUT. The number of metabolites in each biochemical class that significantly changed (p < 0.05) at timepoint 2 relative to timepoint 1 was similar in flash-frozen vs ambient temperature storage. Changes in microbiota modified metabolites over time were also consistent across both methodologies.</p><p><strong>CONCLUSION:</strong> Ambient temperature storage and stabilization of stool in the OMNImet.GUT device yielded comparable metabolomic results to flash freezing in terms of 1) the identity and abundance of detected biochemicals 2) the distinct metabolomic profiles of subjects and 3) changes in metabolites over time that are plausibly microbiota-induced. This method potentially provides a more convenient, less expensive home collection and storage option for stool metabolomic analysis.</p>
Project description:Alterations in intestinal microbiota and intestinal short chain fatty acids profiles have been associated with the pathophysiology of obesity and insulin resistance. Whether intestinal microbiota dysbiosis is a causative factor in humans remains to be clarified We examined the effect of fecal microbial infusion from lean donors on the intestinal microbiota composition, glucose metabolism and small intestinal gene expression. Male subjects with metabolic syndrome underwent bowel lavage and were randomised to allogenic (from male lean donors with BMI<23 kg/m2, n=9) or autologous (reinfusion of own feces, n=9) fecal microbial transplant. Insulin sensitivity and fecal short chain fatty acid harvest were measured at baseline and 6 weeks after infusion. Intestinal microbiota composition was determined in fecal samples and jejunal mucosal biopsies were also analyzed for the host transcriptional response. Insulin sensitivity significantly improved six weeks after allogenic fecal microbial infusion (median Rd: from 26.2 to 45.3 μmol/kg.min, p<0.05). Allogenic fecal microbial infusion increased the overall amount of intestinal butyrate producing microbiota and enhanced fecal harvest of butyrate. Moreover, the transcriptome analysis of jejunal mucosal samples revealed an increased expression of genes involved in a G-protein receptor signalling cascade and subsequently in glucose homeostasis. Lean donor microbial infusion improves insulin sensitivity and levels of butyrate-producing and other intestinal microbiota in subjects with the metabolic syndrome. We propose a model wherein these bacteria provide an attractive therapeutic target for insulin resistance in humans. (Netherlands Trial Register NTR1776).