Project description:Protein-based targeting reagents, such as antibodies and non-antibody scaffold proteins, are rapidly inactivated in the upper gastrointestinal (GI) tract. Hydrochloric acid in gastric juice denatures proteins and activates pepsin, concentrations of which reach 1 mg/mL in the mammalian stomach. Here we present gastrobodies, a protein scaffold derived from Kunitz soybean trypsin inhibitor (SBTI). SBTI is highly resistant to the challenges of the upper GI tract, including digestive proteases, pH 2 and bile acids. Computational prediction of SBTI’s evolvability identified two nearby loops for randomization, to create a potential recognition surface which was experimentally validated by alanine scanning. We established display of SBTI on full-length pIII of M13 phage. Phage selection of gastrobody libraries against the glucosyltransferase domain of Clostridium difficile toxin B (GTD) identified hits with nanomolar affinity and enzyme inhibitory activity. Anti-GTD binders retained high stability to acid, digestive proteases and heat. We use mass spectrometry to identify cut sites outside of the gastrobody.
Project description:Cholesterol gallstone is a common disease, but the pathogenesis of gallstone remains a mystery. The pathogenetic factors of gallstone disease include a genetic background, cholesterol condensation and bile supersaturation, while the risk factors may include fatty liver, diabetes, obesity and microbiome dysbiosis. Dysbiosis refers to altered bacterial composition and abundance, which is found to be associated with the pathogenesis of variety of diseases. Mass spectrometry-based proteomics emerges as a powerful tool to identify protein expressions of human samples. A number of proteomic studies have been performed to analyze human protein contents of bile samples. All these studies focus on the analysis of human proteins, and no study to date has been performed to analyze the protein expression of microbiome in bile samples associated with gallstone disease. In current study, we performed a label-free metaproteomic analysis of gallbladder bile samples from cholesterol gallstone disease patients and normal individuals. Maxquant was used in the comprehensive database searching to infer protein groups with quantitation. We attempt to investigate the dysbiosis changes, potential virulence factor of microbiome and host immune responses which contribute to gallstone formation and cholelithiasis.
Project description:Cholesterol gallstone is a common disease, but the pathogenesis of gallstone remains a mystery. The pathogenetic factors of gallstone disease include a genetic background, cholesterol condensation and bile supersaturation, while the risk factors may include fatty liver, diabetes, obesity and microbiome dysbiosis. Dysbiosis refers to altered bacterial composition and abundance, which is found to be associated with the pathogenesis of variety of diseases. Mass spectrometry-based proteomics emerges as a powerful tool to identify protein expressions of human samples. A number of proteomic studies have been performed to analyze human protein contents of bile samples. All these studies focus on the analysis of human proteins, and no study to date has been performed to analyze the protein expression of microbiome in bile samples associated with gallstone disease. In current study, we performed a label-free metaproteomic analysis of gallbladder bile samples from cholesterol gallstone disease patients and normal individuals. ProteoStorm was used for the first-step comprehensive database searching to identify microbial and host peptides, and Maxquant was used in the second-step refined database searching to infer protein groups with quantitation. We attempt to investigate the dysbiosis changes, potential virulence factor of microbiome and host immune responses which contribute to gallstone formation and cholelithiasis.
Project description:Bile and its individual components, mainly bile acids, are important for digestion and drive bacteria community dynamics in the upper gastrointestinal tract of chickens. However, specific responses to bile acids have been characterized in only a few commensal bacteria, and it is unclear how other members of the microbiota respond to biliary stress. Here, we used label-free LC-MS/MS to assess the proteomic response of a common inhabitant of the chicken upper intestinal tract, Turicibacter spp. MMM721, to 24 hours of growth in anaerobic growth media supplemented with 0.1% whole chicken bile, 0.1% taurochenodeoxycholic acid, or 0.1% taurocholic acid. 70, 46, and 8 differentially expressed proteins were identified in Turicibacter spp. MMM721 cultured with supplements of whole chicken bile, TCDCA, and TCA, respectively, when compared to unsupplemented controls. Many of the differentially expressed proteins were involved in ribosomal processes, post-translational modifications and chaperones, and modifications to the cell surface. To our knowledge, this work represents the first description of the Turicibacter spp. MMM721 proteomic response to bile and bile acid exposure. Ultimately, the T. bilis MMM721 response to whole bile and bile acids is highly complex, with numerous proteins from a variety of functional categories being induced.
Project description:Purpose: To analyze human and bacteria proteomic profiles in bile, exposed to a tumor vs. non-tumor microenvironment, in order to identify differences between these conditions, which may contribute to a better understanding of pancreatic carcinogenesis. Patients and Methods: Using liquid chromatography and mass spectrometry, human and bacteria proteomic profiles of a total of 20 bile samples (7 from gallstone (GS) patients, and 13 from pancreatic head ductal adenocarcinoma (PDAC) patients) that were collected during surgery, and taken directly from the gallbladder were compared. g:Profiler and KEGG (Kyoto Encyclopedia of Genes and Genomes) Mapper Reconstruct Pathway was used as the main comparative platform focusing on over-represented biological pathways among human proteins and interaction pathways among bacterial proteins. Results: Three bacterial infection pathways were over-represented in the human PDAC group of proteins. IL-8 is the only human protein that coincides in the three pathways and this protein is only present in the PDAC group. Quantitative and qualitative differences in bacterial proteins suggest a dysbiotic microenvironment in the PDAC group, supported by significant participation of antibiotic biosynthesis enzymes. Prokaryote interaction signaling pathways highlight the presence of zeatin in the GS group and surfactin in the PDAC group, the former in the metabolism of terpenoids and polyketides, and the latter in both metabolisms of terpenoids, polyketides and quorum sensing. Based on our findings, we propose a bacterial-induced carcinogenesis model for the biliary tract. Conclusion: To the best of our knowledge this is the first study with the aim of comparing human and bacteria bile proteins in a tumor vs. non-tumor microenvironment. We proposed a new carcinogenesis model for the biliary tract based on bile metaproteomic findings. Our results suggest that bacteria may be key players in biliary tract carcinogenesis, in a long-lasting dysbiotic and epithelially harmful microenvironment, in which specific bacterial species biofilm formation is of utmost importance. Our finding should be further explored in future using in vitro and in vivo investigations
2020-07-08 | PXD020151 | Pride
Project description:Upper respiratory tract microbiota sequencing from Covid-19 patients
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:Glomus tumors (GT) are perivascular tumors mostly occurring in the distal extremities. Rare cases occur in the digestive tract and may be misdiagnosed with neuroendocrine or gastrointestinal stromal tumors. We aimed to specify the features of GT of the upper digestive tract. We investigated two cases using whole exome sequencing (WES) and RNA-sequencing, and present clinical, histological, phenotypic and molecular features of 16 cases. WES did not reveal any commonly involved cellular pathway. By contrast, RNA-sequencing disclosed a t(1:5)(p13;q32) translocation between MIR143HG and NOTCH2 in both cases. The deducted fusion protein sequence corresponded to the NOTCH2 intracellular domain known as NICD2, which acts as transcription factor. These data were confirmed by high expression of the transcripts of genes targeted by NOTCH cellular pathway (HES and HEY gene families). In our retrospective multicentric series of 16 GT of upper digestive tract MIR143HG-NOTCH2 translocation was detected in 14 (88%) cases. By contrast, it was present in only 2/6 (33%) GT of the distal extremities. Most digestive GT raised from the stomach (n=13), and the others from duodenal (2) or oesophagous (1). All digestive GT were positive for α-smooth muscle actin and transgelin, and negative for cytokeratin AE1/AE3, chromogranine, DOG1, KIT and S100. Most cases were positive for H-caldesmon (n=14) and/or for synaptophysin (n=10). Desmin, CD34 or CD56 were positive in only one case each. Nuclear expression of NOTCH2 was detected in the 14 cases containing the fusion transcripts. The present study shows that MIR143HG-NOTCH2 translocation is present in most digestive GT. This fusion transcript is associated with activation of the NOTCH2 pathway and may drive tumor development. Detection of nuclear NOTCH2 expression may be helpful for diagnosis.