ABSTRACT: Patients treated with benznidazole. Serum samples collected over time following treatment. Candidate biomarkers of cure assessed by PRM. Polar C18 column, positive mode MS.
Project description:A comprehensive screening of glycopeptides as candidate biomarkers from human serum for early diagnosis of NASH hepatocellular carcinoma using a stepped HCD method and PRM evaluation. Glycopeptides from vitronectin(VTNC) has been reported as biomarkers for NASH-related HCCs.
Project description:Serum samples from Chagas disease patients, pre-treatment and 2, 6, 12 and 24 months post-treatment with benznidazole and extracted with methanol. Polar C18 chromatography. PRM analysis of acylcarnitine metabolites.
Project description:Pulmonary sequelae (PS) in patients with chronic paracoccidioidomycosis (PCM) typically include pulmonary fibrosis and emphysema. Knowledge of the molecular pathways involved in PS of PCM is required for treatment and biomarker identification. This non-concurrent cohort study included 29 patients with pulmonary PCM that were followed before and after treatment. From this group, 17 patients evolved to mild/ moderate PS and 12 evolved severe PS. Sera from patients were evaluated before treatment and at clinical cure, serological cure, and apparent cure. A nanoACQUITY UPLC-Xevo QT MS system and PLGS software were used to identify serum differentially expressed proteins, which were then categorized using Cytoscape software and the Reactome pathway database. Seventy-two differentially expressed serum proteins were identified in patients with severe PS compared with patients with mild/moderate PS. Most proteins altered in severe PS were involved in wound healing, inflammatory response, and oxygen transport pathways. Before treatment and at clinical cure, signaling proteins participating in wound healing, complement cascade, cholesterol transport and retinoid metabolism pathways were downregulated in patients with severe PS, whereas signaling proteins in gluconeogenesis and gas exchange pathways were upregulated. At serological cure, the pattern of protein expression reversed. At apparent cure pathways related with tissue repair (fibrosis) became downregulated, and pathway related oxygen transport became upregulated. Additionally, we identified 11 proteins as candidate biomarkers for severe PS. Development of severe PS is related to increased expression of proteins involved in anabolism (gluconeogenesis and oxygen exchange), indicative of the greater cellular activity and replication associated with early dysregulation of wound healing and aberrant tissue repair. Our findings provide new targets to study mechanisms of PS in PCM, as well as potential biomarkers.
Project description:Background: Bone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with 20% fetal bovine serum (FBS). As an alternative to FBS, pooled solvent detergent apheresis platelets, HPGF-C18, was evaluated for BMSC culture. Methods: A comparison of passage 2 BMSC growth revealed that 10% HPGF-C18 produced similar cell numbers as 20% FBS. Marrow aspirates from 5 healthy subjects were cultured for 4 passages in 10% HPGF-C18 or 20% FBS and were analyzed for proliferation, colony formation efficiency (CFE), surface marker expression, suppression of mixed lymphocyte reactions (MLRs), global gene and microRNA expression analysis. BMSC supernatant cytokine and growth factor concentrations were also compared. Results: Primary cultures of marrow aspirates in 10% HPGF-C18 and 20% FBS yielded similar numbers and CFE. After 4 passages, 10% HPGF-C18 and 20% FBS yielded similar numbers of BMSCs, surface marker expression patterns and immunosuppression effects. Gene and microRNA expression analysis revealed that BMSCs cultured under the two conditions had distinct expression profiles. Gene Set Enrichment Analysis (GSEA) revealed HPGF-C18-cultured BMSCs were enriched in metabolic processing and biosynthetic pathways; cell proliferation and cell cycle pathways; and immune response pathways. FBS-cultured BMSCs were enriched in MAPK signaling, TGF-beta signaling, cell adhesion and extracellular matrix pathways. Differently expressed microRNAs were related to the osteogenesis of BMSCs. Supernatant analysis found that HPGF-C18 BMSCs displayed higher levels of PEDF and TGFB1 and lower levels of IL6, VEGF, SDF1 and PLGF. Conclusions: Traditional measures; expansion, surface marker expression and inhibition of MLRs suggest that BMSC cultured in HPGF-C18 and FBS were similar, but analysis at the molecular level revealed many differences. BMSCs cultured in HPGF-C18 should be assessed in specific functional assays that reflect application-specific potency before substituting FBS with HPGF-C18.
Project description:Radiotherapy is a commonly used treatment modality for the local control of breast cancer. However, the clinical signs of radiotherapy response are often not apparent for several weeks post-treatment. We currently lack tools to predict and/or monitor tumor responses during treatment. The aim of this study was to identify tumor secreted biomarkers of radiotherapy response. Estrogen receptor positive (ER+) MCF-7 cells were serum-starved for 2 h, and were then exposed to various doses of radiation (0, 2, 4, 6, 8 or 10 Gy). Conditioned media (CM) was harvested at 1, 2, 4, 8 and 24 h post-radiation for each radiation dose. Samples underwent processing for liquid chromatography-mass spectrometry (LC-MS) following collection. 33 proteins at the 24 h time point were found to have significantly increased secretion levels (up to 12-fold) at all radiation doses compared to untreated cells. To validate the secretomic results and to further investigate the potential use of these proteins as biomarkers of radiosensitivity, the secreted protein levels of candidate biomarkers were assessed through western blot (WB). WB analysis was performed using CM samples to assess the secretion levels from parental and radioresistant (RR) cell lines (developed within our lab) 24 h after the cells had received a single radiation dose of 2 Gy. Secretion levels of our candidate biomarkers were found to be significantly increased from the radiosensitive MCF-7 cells treated with 2 Gy of radiation at 24 h compared to 24 h untreated controls. In comparison, candidate biomarker levels in the CM samples from untreated and radiation-treated MCF-7 RR cells remained low. Currently there are no validated predictive biomarkers to monitor radiotherapy responses during treatment. These biomarkers could have a clinical role in personalising radiotherapy dosing schedules and durations for solid tumours in the neoadjuvant and palliative setting.
Project description:The performances of label-based SWATH-MS, SRM and PRM to accurately quantify ten candidate biomarkers of beef meat tenderness or marbling, in a cohort of 64 bovine muscle tissues expected to cover a wide biological range of these traits, were evaluated. Limits of quantification, dynamic range and quantification performances were assessed. Moreover, protein amounts for all proteins detected in SWATH-MS were estimated in a label-free manner.
Project description:The development of biomarkers that can predict viral rebound following discontinuation of antiretroviral therapy (ART) in HIV-1-infected humans would be an important advance in HIV-1 cure research. In a prior study, we initiated ART in 20 rhesus macaques on days 0, 1, 2, and 3 following SIVmac251 infection prior to plasma viremia1. Following 6 months of suppressive ART, we discontinued ART and observed viral rebound in 9 of 20 animals. Here we show that transcriptomic and proteomic signatures of inflammation and immune activation in peripheral blood during ART suppression predicted viral rebound following ART discontinuation. Higher levels of proinflammatory and cellular immune activation pathways, including TNF, IL-1, IL-6, monocyte, and T cell activation signaling pathways, correlated with viral rebound following ART discontinuation. Immune modulatory IL-10 and TGF-b signaling also correlated with viral rebound. We then validated these candidate biomarkers of viral rebound in a second cohort of SIV-infected, ART-suppressed macaques. Taken together, these data suggest that persistent upregulation of inflammatory and immune activation pathways despite suppressive ART may represent a peripheral blood biomarker signature of the rebound-competent viral reservoir. The development of interventions that target the viral reservoir and modulate this signature may open new avenues in HIV-1 cure research.
Project description:Project is john hopkins humna serum project, this project we try to search the biomarker candidates by comparing infected progressors and infected non-progressors. This is data backup for the positive mode polar C18 column dataset.
Project description:Colorectal cancer (CRC) is one of the most prevalent and lethal cancer diseases worldwide. Here, we aimed to identify and quantify CRC serum biomarkers by combining a robust label-free quantification procedure followed of two consecutive steps of targeted parallel reaction monitoring (PRM) for biomarker validation in a fully inclusive proteomic strategy. For the discovery phase pooled serum samples were used for shotgun proteomics and label-free quantification. On the identification phase, 116 potential biomarkers were selected based on their statistical significance and their relative expression in disease stages respect to healthy stage and their functional relation with cancer progression. Verification phase was conducted in 2 steps. In the first step, 318 peptides from 116 proteins were used for PRM verification giving place to 23 PRM-quantifiable, potential CRC biomarkers. In a second step, 7 peptides corresponding to CO9, APOC3, CRP, THSB1, ECM1 and IGF2 proteins were reproducibly confirmed by PRM in every CRC stage for these unfractionated samples. Finally, a different cohort composed by individual serum samples was used in the final validation phase. In individual serum samples, 5 peptides belonging to 4 proteins were consistently quantified and validated. ROC analyses indicated that peptides GWVTDGFSSLK and LCNNPTPQFGGK were suitable candidates for predicting the separation between control and CRC patients. Two assays for absolute quantification of significant peptides in serum samples were established using AQUA peptides. In conclusion, a set of serum peptides were validated by PRM as potential biomarkers for differentiating control from CRC patients.
Project description:Colorectal cancer (CRC) is one of the most prevalent and lethal cancer diseases worldwide. Here, we aimed to identify and quantify CRC serum biomarkers by combining a robust label-free quantification procedure followed of two consecutive steps of targeted parallel reaction monitoring (PRM) for biomarker validation in a fully inclusive proteomic strategy. For the discovery phase pooled serum samples were used for shotgun proteomics and label-free quantification. On the identification phase, 116 potential biomarkers were selected based on their statistical significance and their relative expression in disease stages respect to healthy stage and their functional relation with cancer progression. Verification phase was conducted in 2 steps. In the first step, 318 peptides from 116 proteins were used for PRM verification giving place to 23 PRM-quantifiable, potential CRC biomarkers. In a second step, 7 peptides corresponding to CO9, APOC3, CRP, THSB1, ECM1 and IGF2 proteins were reproducibly confirmed by PRM in every CRC stage for these unfractionated samples. Finally, a different cohort composed by individual serum samples was used in the final validation phase. In individual serum samples, 5 peptides belonging to 4 proteins were consistently quantified and validated. ROC analyses indicated that peptides GWVTDGFSSLK and LCNNPTPQFGGK were suitable candidates for predicting the separation between control and CRC patients. Two assays for absolute quantification of significant peptides in serum samples were established using AQUA peptides. In conclusion, a set of serum peptides were validated by PRM as potential biomarkers for differentiating control from CRC patients.