Cytoprotection of baicalein against oxidative stress-induced cardiomyocytes injury through the Nrf2/Keap1 pathway.

Baicalein is one of the major flavonoids found in the root of Scutellaria baicalensis Georgi. Previous studies suggest that baicalein displays protective effect on experimental cardiac models in vitro and in vivo. However, the mode of action remains unclear. Here, we showed that baicalein conferred cardioprotective effect against oxidative stress-induced cell injury in H9c2 cells and human embryonic stem cells-derived cardiomyocytes. Immunoprecipitation with anti-NF-E2-related factor 2 (Nrf2) antibody in baicalein-treated cells demonstrated that baicalein effectively disrupted the association between Nrf2 and Kelch-like epichlorohydrin-associated protein 1 (Keap1). In addition, the unbounded Nrf2 translocated from cytoplasm to nucleus and increased Nrf2/heme oxygenase-1 (HO-1) content in a time-dependent manner. Moreover, antioxidant response element transcriptional activity was enhanced by baicalein treatment, and the Nrf2 siRNA transfection could block the cytoprotective effect of baicalein. Taken together, these results demonstrate that baicalein protected cardiomyocytes against oxidative stress-induced cell injury through the Nrf2/Keap1 pathway.

Quick, sensitive and specific detection and evaluation of quantification of minor variants by high-throughput sequencing.

Minor variants have significant implications in quasispecies evolution, early cancer detection and non-invasive fetal genotyping but their accurate detection by next-generation sequencing (NGS) is hampered by sequencing errors. We generated sequencing data from mixtures at predetermined ratios in order to provide insight into sequencing errors and variations that can arise for which simulation cannot be performed. The information also enables better parameterization in depth of coverage, read quality and heterogeneity, library preparation techniques, technical repeatability for mathematical modeling, theory development and simulation experimental design. We devised minor variant authentication rules that achieved 100% accuracy in both testing and validation experiments. The rules are free from tedious inspection of alignment accuracy, sequencing read quality or errors introduced by homopolymers. The authentication processes only require minor variants to: (1) have minimum depth of coverage larger than 30; (2) be reported by (a) four or more variant callers, or (b) DiBayes or LoFreq, plus SNVer (or BWA when no results are returned by SNVer), and with the interassay coefficient of variation (CV) no larger than 0.1. Quantification accuracy undermined by sequencing errors could neither be overcome by ultra-deep sequencing, nor recruiting more variant callers to reach a consensus, such that consistent underestimation and overestimation (i.e. low CV) were observed. To accommodate stochastic error and adjust the observed ratio within a specified accuracy, we presented a proof of concept for the use of a double calibration curve for quantification, which provides an important reference towards potential industrial-scale fabrication of calibrants for NGS.