Inhibition of miRNA-212/132 improves the reprogramming of fibroblasts into induced pluripotent stem cells by de-repressing important epigenetic remodelling factors

MicroRNAs (miRNAs) repeatedly have been demonstrated to play important roles in the generation of induced pluripotent stem cells (iPSCs). To further elucidate the molecular mechanisms underlying transcription factor-mediated reprogramming we have established a model, which allows for the efficient screening of whole libraries of miRNAs modulating the generation of iPSCs from murine embryonic fibroblasts. Applying this model, we identified 14 miRNAs effectively inhibiting iPSC generation, including miR-132 and miR-212. Intriguingly, repression of these miRNAs during iPSC generation also resulted in significantly increased reprogramming efficacy. MiRNA target evaluation by qRT-PCR, Western blot, and luciferase assays revealed two crucial epigenetic regulators, the histone acetyl transferase p300 as well as the H3K4 demethylase Jarid1a (KDM5a) to be directly targeted by both miRNAs. Moreover, we demonstrated that siRNA-mediated knockdown of either p300 or Jarid1a recapitulated the miRNA effects and led to a significant decrease in reprogramming efficiency. Thus, conducting a full library miRNA screen we here describe a miRNA family, which markedly reduces generation of iPSC and upon inhibition in turn enhances reprogramming. These miRNAs, at least in part, exert their functions through repression of the epigenetic modulators p300 and Jarid1a, highlighting these two molecules as an endogenous epigenetic roadblock during iPSC generation

Hepatocyte-specific suppression of microRNA-221-3p mitigates liver fibrosis.

Fibrosis, a cardinal feature of a dysfunctional liver, significantly contributes to the ever-increasing mortality due to end-stage chronic liver diseases. The crosstalk between hepatocytes and hepatic stellate cells (HSCs) plays a key role in the progression of fibrosis. Although ample efforts have been devoted to elucidate the functions of HSCs during liver fibrosis, the regulatory functions of hepatocytes remain elusive. Using an unbiased functional microRNA (miRNA) screening, we investigated the ability of hepatocytes to regulate fibrosis by fine-tuning gene expression via miRNA modulation. The in vivo functional analyses were performed by inhibiting miRNA in hepatocytes using adeno-associated virus in carbon-tetrachloride- and 3,5-di-diethoxycarbonyl-1,4-dihydrocollidine-induced liver fibrosis. Blocking miRNA-221-3p function in hepatocytes during chronic liver injury facilitated recovery of the liver and faster resolution of the deposited extracellular matrix. Furthermore, we demonstrate that reduced secretion of C-C motif chemokine ligand 2, as a result of post-transcriptional regulation of GNAI2 (G protein alpha inhibiting activity polypeptide 2) by miRNA-221-3p, mitigates liver fibrosis. Collectively, miRNA modulation in hepatocytes, an easy-to-target cell type in the liver, may serve as a potential therapeutic approach for liver fibrosis

Performance of T-Track^® TB, a Novel Dual Marker RT-qPCR-Based Whole-Blood Test for Improved Detection of Active Tuberculosis

Tuberculosis (TB) is one of the leading causes of death by an infectious disease. It remains a major health burden worldwide, in part due to misdiagnosis. Therefore, improved diagnostic tests allowing the faster and more reliable diagnosis of patients with active TB are urgently needed. This prospective study examined the performance of the new molecular whole-blood test T-Track® TB, which relies on the combined evaluation of IFNG and CXCL10 mRNA levels, and compared it to that of the QuantiFERON®-TB Gold Plus (QFT-Plus) enzyme-linked immunosorbent assay (ELISA). Diagnostic accuracy and agreement analyses were conducted on the whole blood of 181 active TB patients and 163 non-TB controls. T-Track® TB presented sensitivity of 94.9% and specificity of 93.8% for the detection of active TB vs. non-TB controls. In comparison, the QFT-Plus ELISA showed sensitivity of 84.3%. The sensitivity of T-Track® TB was significantly higher (p ® TB with QFT-Plus to diagnose active TB was 87.9%. Out of 21 samples with discordant results, 19 were correctly classified by T-Track® TB while misclassified by QFT-Plus (T-Track® TB-positive/QFT-Plus-negative), and two samples were misclassified by T-Track® TB while correctly classified by QFT-Plus (T-Track® TB-negative/QFT-Plus-positive). Our results demonstrate the excellent performance of the T-Track® TB molecular assay and its suitability to accurately detect TB infection and discriminate active TB patients from non-infected controls