Project description:Background: Macrophage-based immune dysregulation plays a critical role in development of delayed gastric emptying in animal models of diabetes. Human studies have also revealed loss of anti-inflammatory macrophages and increased expression of genes associated with pro-inflammatory macrophages in full thickness gastric biopsies from gastroparesis patients. Aim: We aimed to determine broader protein expression (proteomics) and protein-based signaling pathways in full thickness gastric biopsies of diabetic (DG) and idiopathic gastroparesis (IG) patients. Additionally, we determined correlations between protein expressions, gastric emptying and symptoms. Methods: Full-thickness gastric antrum biopsies were obtained from nine DG, seven IG patients and five non-diabetic controls. Aptamer-based SomaLogic tissue scan that quantitatively identifies 1300 human proteins was used. Protein fold changes were computed, and differential expressions were calculated using Limma. Ingenuity Pathway Analysis and correlations were carried out. Multiple-testing corrected p-values <0.05 were considered statistically significant. Results: 73 proteins were differentially expressed in DG, 132 proteins in IG and 40 proteins were common to DG and IG. In both DG and IG, “Role of Macrophages, Fibroblasts and Endothelial Cells” was the most statistically significant altered pathway (DG FDR: 7.9x10-9; IG FDR: 6.3x10-12). In DG, properdin expression correlated with GCSI-bloating (r: -0.99, FDR: 0.02) and expressions of prostaglandin G/H synthase 2, protein kinase C zeta type and complement C2 correlated with 4 hr gastric retention (r: -0.97, FDR: 0.03 for all). No correlations were found between proteins and symptoms or gastric emptying in IG. Conclusions: Protein expression changes suggest a central role of macrophage-driven immune dysregulation and complement activation in gastroparesis.
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:Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and main indication for heart transplantation in children. Therapies specific to pediatric DCM remains limited due to lack of a disease model. Our previous study showed that treatment of neonatal rat ventricular myocytes (NRVMs) with non-failing or DCM pediatric patient serum activates the fetal gene program (FGP). Here we show that serum treatment with Proteinase K prevents activation of the FGP, whereas RNase treatment exacerbates it, suggesting that circulating proteins, but not circulating microRNAs, promote these pathological changes. Evaluation of the protein secretome showed that midkine (MDK) is up-regulated in DCM serum, and NRVM treatment with MDK activates the FGP. Changes in gene expression in serum-treated NRVMs, evaluated by next-generation RNA sequencing (RNA-Seq), indicates extracellular matrix remodeling and focal adhesion pathways are upregulated in pediatric DCM serum and serum-treated NRVMs, suggesting alterations in cellular stiffness. Cellular stiffness was evaluated by Atomic Force Microscopy, which showed an increase in stiffness in DCM serum-treated NRVMs. Of the proteins increased in DCM sera, secreted frizzled related protein 1 (sFRP1) was a potential candidate for the increase in cellular stiffness, and sFRP1 treatment of NRVMs recapitulated the increase in cellular stiffness observed in response to DCM-serum treatment. Our results show that serum circulating proteins promote pathological changes in gene expression and cellular stiffness, and circulating miRNAs are protective against pathological changes.
Project description:In this study, we evaluated the utility of proteomics to identify plasma proteins in healthy participants from a phase I clinical trial with IFNβ-1a and pegIFNβ-1a biologics to identify potential pharmacodynamic (PD) biomarkers. Using a linear mixed-effects model with repeated measurement for product-time interaction, we found that 248 and 528 analytes detected by the SOMAscan® assay were differentially expressed (p-value < 6.86E-06) between therapeutic doses of IFNβ-1a or pegIFNβ-1a, and placebo, respectively. We further prioritized signals based on peak change, area under the effect curve over the study duration, and overlap in signals from the two products. Analysis of prioritized datasets indicated activation of IFNB1 signaling and an IFNB signaling node with IL-6 as upstream regulators of the plasma protein patterns from both products. Increased TNF, IL-1B, IFNG, and IFNA signaling also occurred early in response to each product suggesting a direct link between each product and these upstream regulators. In summary, we identified longitudinal global PD changes in a large array of new and previously reported circulating proteins in healthy participants treated with IFNβ-1a and pegIFNβ-1a that may help identify novel single proteomic PD biomarkers and/or composite PD biomarker signatures as well as provide insight into the mechanism of action of these products. Independent replication is needed to confirm present proteomic results and to support further investigation of the identified candidate PD biomarkers for biosimilar product development.
Project description:Maternal plasma samples collected longitudinally from pregnant women were profiled using SomaLogic aptamer-based assays in women with normal pregnancy and those who delivered preterm. DiagnosisGA is the gestational age at diagnosis with any disease indicated by the Group variable, and it is set to NA for normal pregnancies. In the Group variable, sPTD stands for spontaneous preterm delivery, and PPROM for preterm premature rupture of membranes. Additional longitudinal samples of the controls, including the two samples included herein, are also available and described in PMID: 28738067.
Project description:Helicobacter pylori, which is known as pathogens of various gastric diseases, have many types of genome sequence variants. That is part of the reason why pathogenesis and infection mechanisms of the H. pylori-driven gastric diseases have not been well clarified yet. Here we performed a large-scale proteome analysis to profile the heterogeneity of the proteome expression of 7 H. pylori strains by using LC/MS/MS-based proteomics approach combined with a customized database consisting of non-redundant tryptic peptide sequences derived from full genome sequences of 52 H. pylori strains. The non-redundant peptide database enabled us to identify more peptides in the database search of MS/MS data, compared with a simply merged protein database. Using the approach we performed proteome analysis of genome-unknown strains of H. pylori in as large-scale as genome-known ones. Clustering of the H. pylori strains using the proteome profiling slightly differed from the genome profiling and more clearly divided the strains into two groups based on the isolated area. Furthermore, we also identified phosphorylated proteins and sites of the H. pylori strains and obtained phosphorylation motif located in the N-terminus, which are commonly observed in bacteria.
Project description:Saccharomyces cerevisiae is unique among yeasts for its ability to grow rapidly in the complete absence of oxygen. S. cerevisiae is therefore an ideal eukaryotic model to study physiological adaptation to anaerobiosis. Recent transcriptome analyses have identified hundreds of genes that are transcriptionally regulated by oxygen availability but the relevance of this cellular response has not been systematically investigated at the key control level of the proteome. Therefore, the proteomic response of the S. cerevisiae to anaerobiosis was investigated using metabolic stable isotope labeling in aerobic and anaerobic glucose-limited chemostat cultures, followed by proteome analysis to relatively quantify protein expression. Using independent replicate cultures and stringent statistical filtering, a robust dataset of 474 quantified proteins was generated, of which 249 showed differential expression levels. While some of these changes were consistent with previous transcriptome studies, many responses of S. cerevisiae to oxygen availability were hitherto unreported. Comparison of transcriptome and proteome from identical cultivations yielded strong evidence for post-transcriptional regulation of key cellular processes, including glycolysis, amino-acyl tRNA synthesis, purine-nucleotide synthesis and amino-acid biosynthesis. The use of chemostat cultures provided well-controlled and reproducible culture conditions, which are essential for generating robust datasets at different cellular information levels. Integration of transcriptome and proteome data led to new insights in the physiology of anaerobically growing yeast that would not have been apparent from differential analyses at either the messenger RNA or protein level alone, thus illustrating the power of multi-level studies in yeast systems biology. Protein levels versus transcript level: Systematic analysis of the control levels at which the yeast response to anaerobiosis takes place was performed using previously published transcript data obtained from yeast cultures grown under strictly identical conditions as described for the current proteome analysis. Affymetrix microarrays from five aerobic and four anaerobic independent culture replicates were used for this analysis. These comparison data are summarized in the table below. These array data are publicly available at the gene expression repository Gene Expression Omnibus under accession number GSE4804. Keywords: proteomic, nanoflow-LC-MS/MS
Project description:Sarcopenia analysis of the vastus lateralis from rhesus monkeys including metabolomics, proteomics, and contractile protein proteoforms.
Project description:The intestine is an organ responsible for absorption and metabolism of orally administered drugs. It is necessary to examine the human intestinal expression profiles of the genes related to drug absorption, distribution, metabolism, and excretion (ADME), for accurate prediction of pharmacokinetics in the intestine. However, previous studies had issues such as the evaluation limited to specific molecules and regions, and relatively small sample sizes. In this study, to obtain more accurate expression profiles of mRNA and protein in various regions of human intestine, biopsy samples were collected from 38 patients with various regions, duodenum, jejunum, ileum, colon and rectum, and RNA-seq analysis and quantitative proteomics analysis were performed. We performed the expression analysis of drug-metabolizing enzymes (cytochromes P450 (CYP ) and non–CYP enzymes), apical and basolateral drug transporters, and nuclear receptors. Overall, mRNA expression levels of these ADME-related molecules were highly correlated with the protein expression levels. The characteristics of the expression of ADME-related genes in human small and large intestines differed significantly, such as higher or lower expression levels of CYP enzymes in the small or large intestines, respectively. Most CYPs were expressed dominantly in the small intestine. Non-CYPs were expressed in the large intestine, but their expression levels were lower than those in the small intestine. The expression levels of ADME-related genes differed even between the proximal and distal small intestine. The expression of transporters showed their highest abundance in the ileum. The data in the present study contributes to an improved understanding of intestinal ADME of drug candidates, and would be useful for drug discovery research.