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RT-qPCR is the accepted technique for the quantification of microRNA (miR) expression: however, stem-loop RT-PCR, the most frequently used method for quantification of miRs, is time- and reagent-consuming as well as inconvenient for scanning. We established a new method called ‘universal stem-loop primer’ (USLP) with 8 random nucleotides instead of a specific sequence at the 3′ end of the traditional stem-loop primer (TSLP), for screening miR profile and to semi-quantify expression of miRs. Peripheral blood samples were cultured with phytohaemagglutinin (PHA), and then 87 candidate miRs were scanned in cultured T cells. By USLP, our study revealed that the expression of miR-150-5p (miR-150) decreased nearly 10-fold, and miR-155-5p (miR-155) increased more than 7-fold after treated with PHA. The results of the dissociation curve and gel electrophoresis showed that the PCR production of the USLP and TSLP were specificity. The USLP method has high precision because of its low ICV (ICV<2.5%). The sensitivity of the USLP is up to 103 copies/µl miR. As compared with the TSLP, USLP saved 75% the cost of primers and 60% of the test time. The USLP method is a simple, rapid, precise, sensitive, and cost-effective approach that is suitable for screening miR profiles. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is the most frequent endocrinopathy in women of reproductive age. It is difficult to treat PCOS because of its complex etiology and pathogenesis. Here, we characterized the roles of gut microbiota on the pathogenesis and treatments in letrozole (a nonsteroidal aromatase inhibitor) induced PCOS rat model. Changes in estrous cycles, hormonal levels, ovarian morphology and gut microbiota by PCR-DGGE and real-time PCR were determined. The results showed that PCOS rats displayed abnormal estrous cycles with increasing androgen biosynthesis and exhibited multiple large cysts with diminished granulosa layers in ovarian tissues. Meanwhile, the composition of gut microbiota in letrozole-treated rats was different from that in the controls. Lactobacillus, Ruminococcus and Clostridium were lower while Prevotella was higher in PCOS rats when compared with control rats. After treating PCOS rats with Lactobacillus and fecal microbiota transplantation (FMT) from healthy rats, it was found that the estrous cycles were improved in all 8 rats in FMT group, and in 6 of the 8 rats in Lactobacillus transplantation group with decreasing androgen biosynthesis. Their ovarian morphologies normalized. The composition of gut microbiota restored in both FMT and Lactobacillus treated groups with increasing of Lactobacillus and Clostridium, and decreasing of Prevotella. These results indicated that dysbiosis of gut microbiota was associated with the pathogenesis of PCOS. Microbiota interventions through FMT and Lactobacillus transplantation were beneficial for the treatments of PCOS rats. [ABSTRACT FROM AUTHOR]

Fatal Ebola virus infection is characterized by a systemic inflammatory response similar to septic shock. Ebola glycoprotein (GP) is involved in this process through activating dendritic cells (DCs) and macrophages. However, the mechanism is unclear. Here, we showed that LSECtin (also known as CLEC4G) plays an important role in GP-mediated inflammatory responses in human DCs. Anti-LSECtin mAb engagement induced TNF-α and IL-6 production in DCs, whereas silencing of LSECtin abrogated this effect. Intriguingly, as a pathogen-derived ligand, Ebola GP could trigger TNF-α and IL-6 release by DCs through LSECtin. Mechanistic investigations revealed that LSECtin initiated signaling via association with a 12-kDa DNAX-activating protein (DAP12) and induced Syk activation. Mutation of key tyrosines in the DAP12 immunoreceptor tyrosine-based activation motif abrogated LSECtin-mediated signaling. Furthermore, Syk inhibitors significantly reduced the GP-triggered cytokine production in DCs. Therefore, our results demonstrate that LSECtin is required for the GP-induced inflammatory response, providing new insights into the EBOV-mediated inflammatory response. [ABSTRACT FROM AUTHOR]