Elevated levels of protein phosphatase 1 and phosphatase 2A may contribute to cardiac dysfunction in diabetes

AbstractAlthough protein phosphorylation and dephosphorylation are known to regulate the activities of different enzymes, sufficient information on the role of dephosphorylation in cardiac function is not available. Since protein phosphatases mediate dephosphorylation, it is possible that cardiac dysfunction induced by diabetes may be due to alterations in the activities of these enzymes. We therefore determined cardiac protein phosphatase activity as well as protein contents of phosphatase 1 and phosphatase 2A in diabetic animals. For this purpose, rats were made diabetic by administering a single intravenous injection of streptozotocin (65 mg/kg body weight) and hearts were examined after 1, 2, 3, 4 and 8 weeks. Some of the 4-week diabetic animals received subcutaneous injections of insulin (3 U/day) for a further period of 4 weeks. Cardiac dysfunction was evident after 2 weeks of inducing diabetes and deteriorated further with time. A significant increase in protein phosphatase activity appeared after 1 week and persisted until 8 weeks. Increased protein phosphatase activity in the diabetic heart was associated with a corresponding increase in the protein contents of both phosphatase 1 and phosphatase 2A. Insulin treatment partly prevented the changes observed in diabetic animals. The results suggest that increased protein phosphatase activities and subsequent enhanced protein dephosphorylation may play a role in diabetes-induced cardiac dysfunction

A detailed analysis of next generation sequencing reads of microRNA expression in Barrett’s Esophagus: absolute versus relative quantification

Background Next generation sequencing (NGS) is a state of the art technology for microRNA (miRNA) analysis. The quantitative interpretation of the primary output of NGS i.e. the read counts for a miRNA sequence that can vary by several orders of magnitude (1 to 107) remains incompletely understood. Findings NGS (SOLiD 3 technology) was performed on biopsies from 6 Barrett’s esophagus (BE) and 5 Gastroesophageal Reflux Disease (GERD) patients. Read sequences were aligned to miRBase 18.0. Differential expression analysis was adjusted for false discovery rate of 5%. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for 36 miRNA in a validation cohort of 47 patients (27 BE and 20 GERD). Correlation coefficients, accuracy, precision and recall of NGS compared to qRT-PCR were calculated. Increase in NGS reads was associated with progressively lower Cq values, p 1000 vs. 500 vs. 100 vs. <100). The accuracy, precision and recall of NGS to label a miRNA as differentially expressed were 0.71, 0.88 and 0.74 respectively. Conclusion Absolute NGS reads correlated modestly with qRT-PCR but fold changes correlated highly. NGS is robust at relative but not absolute quantification of miRNA levels and accurate for high-throughput identification of differentially expressed miRNA.The current work was supported by a pilot grant from the American Cancer Society (AB and LKC), the American College of Gastroenterology Junior Faculty Development Award (AB) and Hall Family Foundation (LKC)

MicroRNA Expression can be a Promising Strategy for the Detection of Barrett's Esophagus: A Pilot Study

Clinical and Translational Gastroenterology is an open-access journal published by Nature Publishing Group.Patient outcomes for esophageal adenocarcinoma (EAC) have not improved despite huge advances in endoscopic therapy because cancers are being diagnosed late. Barrett's esophagus (BE) is the primary precursor lesion for EAC, and thus the non-endoscopic molecular diagnosis of BE can be an important approach to improve EAC outcomes if robust biomarkers for timely diagnosis are identified. MicroRNAs (miRNAs) are tissue-specific novel biomarkers that regulate gene expression and may satisfy this requirement.The current work was supported by a pilot grant from the American Cancer Society (A.B. and L.K.C.), the American College of Gastroenterology Junior Faculty Development Award (A.B.) and grants from Hall Family Foundation (L.K.C.) and Kansas IDeA Network of Biomedical Research Excellence (A.B., L.K.C.). None of the funding bodies had any role in design, in the collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication

Discovery and Validation of Barrett's Esophagus MicroRNA Transcriptome by Next Generation Sequencing

Objective: Barrett's esophagus (BE) is transition from squamous to columnar mucosa as a result of gastroesophageal reflux disease (GERD). The role of microRNA during this transition has not been systematically studied. Design: For initial screening, total RNA from 5 GERD and 6 BE patients was size fractionated. RNA <70 nucleotides was subjected to SOLiD 3 library preparation and next generation sequencing (NGS). Bioinformatics analysis was performed using R package “DEseq”. A p value<0.05 adjusted for a false discovery rate of 5% was considered significant. NGS-identified miRNA were validated using qRT-PCR in an independent group of 40 GERD and 27 BE patients. MicroRNA expression of human BE tissues was also compared with three BE cell lines. Results: NGS detected 19.6 million raw reads per sample. 53.1% of filtered reads mapped to miRBase version 18. NGS analysis followed by qRT-PCR validation found 10 differentially expressed miRNA; several are novel (-708-5p, -944, -224-5p and -3065-5p). Up- or down- regulation predicted by NGS was matched by qRT-PCR in every case. Human BE tissues and BE cell lines showed a high degree of concordance (70–80%) in miRNA expression. Prediction analysis identified targets that mapped to developmental signaling pathways such as TGFβ and Notch and inflammatory pathways such as toll-like receptor signaling and TGFβ. Cluster analysis found similarly regulated (up or down) miRNA to share common targets suggesting coordination between miRNA. Conclusion: Using highly sensitive next-generation sequencing, we have performed a comprehensive genome wide analysis of microRNA in BE and GERD patients. Differentially expressed miRNA between BE and GERD have been further validated. Expression of miRNA between BE human tissues and BE cell lines are highly correlated. These miRNA should be studied in biological models to further understand BE development