Project description:Fatal COVID-19 is often complicated by hypoxemic respiratory failure and acute respiratory distress syndrome (ARDS). Mechanisms governing lung injury and repair in ARDS remain poorly understood because there are no biomarker-targeted therapeutics for patients with ARDS. We hypothesized that plasma proteomics may uncover unique biomarkers that correlate with disease severity in COVID-19 ARDS. We analyzed the circulating plasma proteome from 32 patients with ARDS and COVID-19 using an aptamer-based platform, which measures 7289 proteins, and correlated protein measurements with sequential organ failure assessment (SOFA) scores at 2 time points (Days 1 and 7 following ICU admission). We compared differential protein abundance and SOFA scores at each individual time point and identified 119 proteins at Day 1 and 46 proteins at Day 7 that correlated with patient SOFA scores. We modeled the relationship between dynamic protein abundance and changes in SOFA score between Days 1 and 7 and identified 39 proteins that significantly correlated with changes in SOFA score. Using Ingenuity Pathway Analysis, we identified increased ephrin signaling and acute phase response signaling correlated with increased SOFA scores over time, while pathways related to pulmonary fibrosis signaling and wound healing had an inverse relationship with SOFA scores between Days 1 and 7. These findings suggest that persistent inflammation may drive worsened disease severity, while repair processes correlate with improvements in organ dysfunction over time. This approach is generalizable to more diverse ARDS cohorts for identification of protein biomarkers and disease mechanisms as we strive towards targeted therapies in ARDS.
Project description:Current proteomic methods are not well suited to detect protein isoforms. On the one hand, standard shotgun (that is, bottom-up) proteomics involves digestion of proteins into peptides. While this approach identifies many proteins, it results in a loss of isoform information. On the other hand, mass spectrometric analysis of intact proteins (that is, top-down proteomics) distinguishes protein isoforms but only covers a small subset of the proteome. We developed peptide correlation profiling (PepCP) as a method to obtain protein-level information from peptide-centric (that is, bottom-up proteomic) data: First, proteins are fractionated by SDS-PAGE to polypeptides of different length. Second, individual protein fractions are digested into peptides. Third, peptides are identified and quantified in all fractions using quantitative mass spectrometry-based proteomics. Finally, peptide abundance profiles across fractions are analysed to obtain protein-level information.
Project description:Current proteomic methods are not well suited to detect protein isoforms. On the one hand, standard shotgun (that is, bottom-up) proteomics involves digestion of proteins into peptides. While this approach identifies many proteins, it results in a loss of isoform information. On the other hand, mass spectrometric analysis of intact proteins (that is, top-down proteomics) distinguishes protein isoforms but only covers a small subset of the proteome. We developed peptide correlation profiling (PepCP) as a method to obtain protein-level information from peptide-centric (that is, bottom-up proteomic) data: First, proteins are fractionated by SDS-PAGE to polypeptides of different length. Second, individual protein fractions are digested into peptides. Third, peptides are identified and quantified in all fractions using quantitative mass spectrometry-based proteomics. Finally, peptide abundance profiles across fractions are analysed to obtain protein-level information.
Project description:Radiotherapy remains a standard treatment for colorectal cancer. The clinical significance of the conventional treatment is based on accumulated dose and usually consists of single daily irradiations of 1.8 - 2 Gy fractions. However the repopulation of cancer cells during and/or after the treatment suggests that the surviving fraction of cells acquire the radioresistance to cancer therapy. It has been determined a number of radioresistance markers, but only limited data exist on the acquisition of the resistance to fractionated radiation therapy. This research provides the genomic data of gene expression changes in colorectal cancer cell HCT116 line following single and multiple fractions of irradiation, aiming to identify particular genes associated with the effect of fractionated radiotherapy.
Project description:Gradient fractions of RNAi of XAC1 (Tb927.7.2780) in Trypanosoma brucei bloodstream forms. RNAi was induced using tetracycline and cell extracts were fractionated into polysomal and monosome-non-ribosome-associated fractions.
Project description:Prokaryotes are, due to their moderate complexity, particularly amenable to the comprehensive identification of the protein repertoire expressed under different conditions. We applied a generic strategy to identify a complete expressed prokaryotic proteome, which is based on the analysis of RNA and proteins extracted from matched samples. Saturated transcriptome profiling by RNA-seq provided an endpoint estimate of the protein-coding genes expressed under two conditions which mimic the interaction of Bartonella henselae with its mammalian host. Directed shotgun proteomics experiments were carried out on four subcellular fractions. By specifically targeting proteins which are short, basic, low abundant and membrane localized, we could eliminate their initial under-representation compared to the estimated endpoint. A total of 1,250 proteins were identified with an estimated false discovery rate below 1%. This represents 85% of all distinct annotated proteins and around 90% of the expressed protein-coding genes. Genes, whose transcripts were detected, but not their corresponding protein products, were found highly enriched in several genomic islands. Additionally, genes that lacked an ortholog and a functional annotation were not detected at the protein level, and possibly include over-predicted genes in genome annotations. Furthermore, a dramatic membrane proteome re-organization was observed including differential regulation of autotransporters, adhesins and hemin binding proteins. Particularly noteworthy was the complete membrane proteome coverage which included expression of all members of the VirB/D4 type IV secretion system, a key virulence factor. Transcriptome and proteome analysis of B.henselae in two conditions and duplicates: uninduced and induced for host invasion.
Project description:Prokaryotes are, due to their moderate complexity, particularly amenable to the comprehensive identification of the protein repertoire expressed under different conditions. We applied a generic strategy to identify a complete expressed prokaryotic proteome, which is based on the analysis of RNA and proteins extracted from matched samples. Saturated transcriptome profiling by RNA-seq provided an endpoint estimate of the protein-coding genes expressed under two conditions which mimic the interaction of Bartonella henselae with its mammalian host. Directed shotgun proteomics experiments were carried out on four subcellular fractions. By specifically targeting proteins which are short, basic, low abundant and membrane localized, we could eliminate their initial under-representation compared to the estimated endpoint. A total of 1,250 proteins were identified with an estimated false discovery rate below 1%. This represents 85% of all distinct annotated proteins and around 90% of the expressed protein-coding genes. Genes, whose transcripts were detected, but not their corresponding protein products, were found highly enriched in several genomic islands. Additionally, genes that lacked an ortholog and a functional annotation were not detected at the protein level, and possibly include over-predicted genes in genome annotations. Furthermore, a dramatic membrane proteome re-organization was observed including differential regulation of autotransporters, adhesins and hemin binding proteins. Particularly noteworthy was the complete membrane proteome coverage which included expression of all members of the VirB/D4 type IV secretion system, a key virulence factor.
Project description:CAGE sequencing of iPSC and Human dermal fibroblasts, total RNA and fractionated into nuclear, cytoplasmic and chromatin fractions.
Project description:(1) Gene expression profiles during radiation-induced premature, terminal differentiation of exponentially growing progenitor fibroblasts to postmitotic functional cells after single dose (4 Gy) or two fractions (2×4 Gy). (2) Comparison with gene expression profile of confluent cells after fractionated irradiation with 3×4 Gy