Project description:Mice were challneged with Mycobacterium abscessus cell wall particles intra- tracheally or saline. Lung was harvested after 3 weeks. RNA extracted from lungs and RNASeq was performed to identify differentially expressed genes.
Project description:We report the first use of ex vivo lung perfusion (EVLP) in the genetic and physiologic modification of lungs from deceased pulmonary arterial hypertension (PAH) patients and propose this as a translational platform to both (1) derive clinically relevant mechanistic insights into pulmonary pathophysiology and (2) to test treatments on human lungs. The EVLP consist in the perfusion of the lungs out of the body during 6 hours. It is a well established protocol in where basically lungs are on a table connected to a close circuit containing a special perfusion solution that is circulated through the pulmonary vein and artery using a pump. The circuit contains also a deoxygenator. The perfusion temperature and flow are adjusted gradually and after 20 mins of perfusion the ventilation is initiated. Every hour lungs are recruited in order to assess pulmonary function and collect perfusate samples. In addition to perfusate, tissue samples from the lower lobe of the left lung and bronchial alveolar lavage (BAL) are collected at times T0, 3 and 6 hr.
Project description:In-vivo Gene Signatures of Mycobacterium tuberculosis In C3HeB/FeJ Mice In this experiment we have compared Mtb transcriptomics in-vivo, using samples derived from chronically infected C3HeB/FeJ mice, which produce human like caseating lesions, unlike other murine species, to Mtb cultured in-vitro. Similarly, the genome-wide expression of MtbdeldosR, MtbdeldosS and MtbdeldosT mutants is also compared between lungs from C3HeB/FeJ mice and in-vitro culturing.
Project description:Neutrophils are accumulated in mouse lungs following Mycobacterium tuberculosis infections. Increased influx of neutrophils is associated with increased susceptibility to disease, but the underlying mechanisms of neutrophil mediated Tuberculosis disease pathogenesis is poorly understood. To understand the transcriptomic differences between resistant (C57 BL/6) and susceptible (C3HeB/FeJ) mouse lung neutrophils, we sorted them at Day 29 post infection and analyzed them by bulk mRNA sequencing.
Project description:Detection of species-specific proteotypic peptides for accurate and easy characterization of infectious non-tuberculous mycobacteria such as Mycobacterium avium subsp. paratuberculosis, Mycobacterium marinum and Mycobacterium vaccae is essential. Therefore, we carried out reanalysis of publicly available M. avium subsp. paratuberculosis, M. marinum and M. vaccae proteomic dataset PXD027444, PXD003766 and PASS00954 by proteome database search and followed by spectral library generation. The raw DDA data were searched against their respective reference proteome databases using Proteome Discoverer and FragPipe. The resulting peptide spectrum matches were converted into a spectral library using BiblioSpec.
Project description:A growing body of evidence suggests that epithelial cells have special roles during pneumonia. The purpose of this study was to elucidate epithelial-specific responses during lung infection. Mice received intratracheal instillations of Streptococcus pneumoniae (10^6 CFU) into the left lung. 15 hours after the challenge, left lungs were collected. Single cell supensions were generated and sorted into 2 separate populations, epithelial cells (CD45-EpCAM+) and others (all non-epithelial cells). Epithelial cells from uninfected mice were also collected.
Project description:Gut microbiome research is rapidly moving towards the functional characterization of the microbiota by means of shotgun meta-omics. Here, we selected a cohort of healthy subjects from an indigenous and monitored Sardinian population to analyze their gut microbiota using both shotgun metagenomics and shotgun metaproteomics. We found a considerable divergence between genetic potential and functional activity of the human healthy gut microbiota, in spite of a quite comparable taxonomic structure revealed by the two approaches. Investigation of inter-individual variability of taxonomic features revealed Bacteroides and Akkermansia as remarkably conserved and variable in abundance within the population, respectively. Firmicutes-driven butyrogenesis (mainly due to Faecalibacterium spp.) was shown to be the functional activity with the higher expression rate and the lower inter-individual variability in the study cohort, highlighting the key importance of the biosynthesis of this microbial by-product for the gut homeostasis. The taxon-specific contribution to functional activities and metabolic tasks was also examined, giving insights into the peculiar role of several gut microbiota members in carbohydrate metabolism (including polysaccharide degradation, glycan transport, glycolysis and short-chain fatty acid production). In conclusion, our results provide useful indications regarding the main functions actively exerted by the gut microbiota members of a healthy human cohort, and support metaproteomics as a valuable approach to investigate the functional role of the gut microbiota in health and disease.
Project description:Cancer cachexia has been linked to gut bacterial alterations, but alterations of gut viruses, mostly bacteriophages, have not yet been explored. We performed shotgun metagenomic sequencing of DNA from stool samples of 78 cachectic and 42 non-cachectic cancer patients. K-mer-based matching to reference databases revealed abundance variations of bacteria and viruses. Beyond bacterial alterations, cachectic patients exhibited significantly lower bacteriophage abundance, predominantly affecting Caudovirales and Siphoviridae species (double-stranded DNA) but also Inoviridae and Microviridae families (single-stranded DNA).