摘要: Objectives Fibroblast growth factor 9 (FGF9) is expressed by somatic cells in the seminiferous tubules, yet little information exists about its role in regulating spermatogonial stem cells (SSCs). Materials and Methods Fgf9 overexpression lentivirus was injected into mouse testes, and PLZF immunostaining was performed to investigate the effect of FGF9 on spermatogonia in vivo. Effect of FGF9 on SSCs was detected by transplanting cultured germ cells into tubules of testes. RNA-seq of bulk RNA and single cell was performed to explore FGF9 working mechanisms. SB203580 was used to disrupt p38 MAPK pathway. p38 MAPK protein expression was detected by Western blot and qPCR was performed to determine different gene expression. Small interfering RNA (siRNA) was used to knock down Etv5 gene expression in germ cells. Results Overexpression of Fgf9 in vivo resulted in arrested spermatogenesis and accumulation of undifferentiated spermatogonia. Exposure of germ cell cultures to FGF9 resulted in larger numbers of SSCs over time. Inhibition of p38 MAPK phosphorylation negated the SSC growth advantage provided by FGF9. Etv5 and Bcl6b gene expressions were enhanced by FGF9 treatment. Gene knockdown of Etv5 disrupted the growth effect of FGF9 in cultured SSCs along with downstream expression of Bcl6b. Conclusions Taken together, these data indicate that FGF9 is an important regulator of SSC proliferation, operating through p38 MAPK phosphorylation and upregulating Etv5 and Bcl6b in turn.

摘要: Background Human dermal papilla (DP) cells and melanocytes (hMel) are central players in hair growth and pigmentation, respectively. In hair follicles (HFs), oxygen (O-2) levels average 5%, being coupled with the production of reactive oxygen species (ROS), necessary to promote hair growth. Materials and Methods DP cell and hMel proliferation and phenotype were studied under physiological (5%O-2, physoxia) or atmospheric (21%O-2, normoxia) oxygen levels. hMel-DP cells interactions were studied in indirect co-culture or by directly co-culturing hMel with DP spheroids, to test whether their interaction affected the response to physoxia. Results Physoxia decreased DP cell senescence and improved their secretome and phenotype, as well as hMel proliferation, migration, and tyrosinase activity. In indirect co-cultures, physoxia affected DP cells' alkaline phosphatase (ALP) activity but their signalling did not influence hMel proliferation or tyrosinase activity. Additionally, ROS production was higher than in monocultures but a direct correlation between ROS generation and ALP activity in DP cells was not observed. In the 3D aggregates, where hMel are organized around the DP, both hMel tyrosinase and DP cells ALP activities, their main functional indicators, plus ROS production were higher in physoxia than normoxia. Conclusions Overall, we showed that the response to physoxia differs according to hMel-DP cells interactions and that the microenvironment recreated when in direct contact favours their functions, which can be relevant for hair regeneration purposes.

摘要: Background & Aims Hepatocellular carcinoma (HCC) is a common malignant tumour with high morbidity and mortality. Metabolic regulation by oncogenes is necessary for tumour growth. Testes-specific protease 50 (TSP50) has been found to promote cell proliferation in multiple tumour types. However, the mechanism that TSP50 promotes HCC progression are not known. Methods Hepatocyte proliferation was analysed by MTT and BrdU incorporation after TSP50 transfection. Furthermore, LC-MS/MS, co-immunoprecipitation and GST pull-down assays were performed to analyse protein(s) binding to TSP50. Moreover, the site-specific mutation of G6PD was used to reveal the key site critical for G6PD acetylation mediated by TSP50. Finally, the role of G6PD K171 acetylation regulated by TSP50 in cell proliferation and tumour formation was investigated. Results Our data suggest that the overexpression of TSP50 accelerates hepatocyte proliferation. In addition, G6PD is an important protein that binds to TSP50 in the cytoplasm. TSP50 activates G6PD activity by inhibiting the acetylation of G6PD at the K171 site. In addition, TSP50 promotes the binding of G6PD to SIRT2. Furthermore, the K171ac of G6PD regulated by TSP50 is required for TSP50-induced cell proliferation in vitro and tumour formation in vivo. Additionally, according to The Cancer Genome Atlas (TCGA) programme, TSP50 and G6PD are negatively correlated with the survival of HCC patients. Conclusions Collectively, our findings demonstrate that TSP50-induced cell proliferation and tumour formation are mediated by G6PD K171 acetylation.

摘要: Objectives Using strategy of drug repurposing, antiviral agents against influenza A virus (IAV) and newly emerging SARS-coronavirus 2 (SARS-CoV-2, also as 2019-nCoV) could be quickly screened out. Materials and Methods A previously reported engineered replication-competent PR8 strain carrying luciferase reporter gene (IAV-luc) and multiple pseudotyped IAV and SARS-CoV-2 virus was used. To specifically evaluate the pH change of vesicles containing IAV, we constructed an A549 cell line with endosomal and lysosomal expression of pHluorin2. Results Here, we identified azithromycin (AZ) as an effective inhibitor against multiple IAV and SARS-CoV-2 strains. We found that AZ treatment could potently inhibit IAV infection in vitro. Moreover, using pseudotyped virus model, AZ could also markedly block the entry of SARS-CoV-2 in HEK293T-ACE2 and Caco2 cells. Mechanistic studies further revealed that such effect was independent of interferon signalling. AZ treatment neither impaired the binding and internalization of IAV virions, nor the viral replication, but rather inhibited the fusion between viral and vacuolar membranes. Using a NPC1-pHluorin2 reporter cell line, we confirmed that AZ treatment could alkalize the vesicles containing IAV virions, thereby preventing pH-dependent membrane fusion. Conclusions Overall, our findings demonstrate that AZ can exert broad-spectrum antiviral effects against IAV and SARS-CoV-2, and could be served as a potential clinical anti-SARS-CoV-2 drug in emergency as well as a promising lead compound for the development of next-generation anti-IAV drugs.

摘要: Objectives Gene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell mass (ICM), during pre-implantation embryo development. The extraordinarily longer pre-implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre-implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre-implantation embryos between species. Materials and methods To analyse the functions of SOX2 in lineage segregation and cell proliferation, loss- and gain-of-function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real-time PCR. Results Our results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2-disrupted blastocysts, the expression of the ICM-related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real-time PCR analysis, pluripotency-related genes, excluding OCT4, and proliferation-related genes were decreased in SOX2-targeted blastocysts. In SOX2-overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased. Conclusions Taken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early-stage embryogenesis.