Regulatory sequence-based discovery of anti-defense genes in archaeal viruses.

In silico identification of viral anti-CRISPR proteins (Acrs) has relied largely on the guilt-by-association method using known Acrs or anti-CRISPR associated proteins (Acas) as the bait. However, the low number and limited spread of the characterized archaeal Acrs and Aca hinders our ability to identify Acrs using guilt-by-association. Here, based on the observation that the few characterized archaeal Acrs and Aca are transcribed immediately post viral infection, we hypothesize that these genes, and many other unidentified anti-defense genes (ADG), are under the control of conserved regulatory sequences including a strong promoter, which can be used to predict anti-defense genes in archaeal viruses. Using this consensus sequence based method, we identify 354 potential ADGs in 57 archaeal viruses and 6 metagenome-assembled genomes. Experimental validation identified a CRISPR subtype I-A inhibitor and the first virally encoded inhibitor of an archaeal toxin-antitoxin based immune system. We also identify regulatory proteins potentially akin to Acas that can facilitate further identification of ADGs combined with the guilt-by-association approach. These results demonstrate the potential of regulatory sequence analysis for extensive identification of ADGs in viruses of archaea and bacteria.

Sex-specific association of serum uric acid trajectories with risk of incident retinal arteriosclerosis in Chinese population: A population-based longitudinal study.

Background: The impact of serum uric acid (SUA) trajectories on the development of retinal arteriosclerosis is uncertain. The purpose of this study was to identify adult SUA trajectories by sex and determine their association with risk of retinal arteriosclerosis. Methods: In this longitudinal study, 4,324 participants who were aged between 18 and 60 years without retinal arteriosclerosis at or before baseline (from January 1, 2010, through December 31, 2010) were included. Group-based trajectory modeling was used to identify SUA trajectories during the exposure period (from January 1, 2006, through December 31, 2010). Cox proportional-hazards models were applied to evaluate the associations between SUA trajectories and the risk of incident retinal arteriosclerosis during the outcome period (from January 1, 2011, through December 31, 2019). Results: 4 distinct SUA trajectories were identified in both women and men: low, moderate, moderate-high, and high. During a median follow-up of 9.54 years (IQR 9.53-9.56), 97 women and 295 men had developed retinal arteriosclerosis. In the fully adjusted model, a significant association between the moderate-high SUA trajectory group and incidence of retinal arteriosclerosis was observed only in men (HR: 1.76, 95% CI: 1.17-2.65) compared with the low trajectory group, but not in women (HR: 0.77, 95% CI: 0.39-1.52). Also, the high SUA trajectory group had the highest risk with an adjusted HR of 1.81 (95% CI, 1.04-3.17) in men. However, they did not exhibit a substantially increased risk in women. Conclusion: Higher SUA trajectory groups were significantly associated with an increased risk of incident retinal arteriosclerosis in men but not in women.

Folic acid promotes proliferation and differentiation of porcine pancreatic stem cells into insulin-secreting cells through canonical Wnt and ERK signaling pathway.

Porcine pancreatic stem cells (pPSCs) can be induced to differentiate into insulin-producing cells in vitro and thus serve as a major cells source for ß-cell regeneration. However, this application is limited by the weak cell proliferation ability and low insulin induction efficiency. In this study, we explored the role of folic acid in the proliferation of pPSCs and the formation of insulin-secreting cells. We found that FA-treated pPSCs cells had a high EDU positive rate, and the proliferation marker molecules PCNA, CyclinD1 and c-Myc were up-regulated, while the expression of folate receptor α (FOLRα) was up-regulated. In further research, interference FOLRα or adding canonical Wnt signaling pathway or ERK signaling pathway inhibitors could significantly inhibit the effect of FA on pPSCs proliferation. Meanwhile, during the differentiation of pPSCs into insulin-secreting cells, we found that the maturation marker genes Insulin, NKX6.1, MafA, and NeuroD1 was upregulated in insulin-secreting cell masses differentiationed from pPSCs after FA treatment, and the functional molecules Insulin and C-peptide were increased, the ability to secrete insulin in response to high glucose was also increased. With the addition of Wnt and ERK signaling pathway inhibitors, the pro-differentiation effect of FA was weakened. In conclusion, FA promotes the proliferation of pPSCs by binding to folate receptor α (FOLRα) and increase the efficiency of directed differentiation of pPSCs into insulin-producing cells by regulating canonical Wnt and ERK signaling pathway. This study lays theoretical foundation for solving the bottleneck in the treatment of diabetes with stem cell transplantation in future.

SerpinB1 promotes the proliferation of porcine pancreatic stem cells through the STAT3 signaling pathway.

Porcine pancreatic stem cells (pPSCs) can be induced to insulin-secreting cells and therefore considered the most promising seeding cells for curing human diabetes in future. However, insufficient pPSCs number is one of the bottleneck problems before its clinical application. SerpinB1 is a serine protease inhibitor in neutrophils and can directly promote the proliferation of ß cells. Whether SerpinB1 is involved in pPSC proliferation and differentiation remains unknown. The effects of SerpinB1 on pPSCs proliferation were measured by Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine, qRT-PCR, western blot, and flow cytometry assays. We found that pPSCs did not efficiently reach the S phase when SerpinB1 expression was knocked down with short hairpin RNA (sh-SerpinB1), the expression of Cyclin D1, CDK-2, and PCNA also decreased. Meanwhile, cell viability and proliferation ability were both declined. Further analyses showed that the expression level of phosphorylated STAT3/STAT3was downregulated, along with an upregulation of p53 and p21. We used a two-step induction method to induce pPSCs to insulin-secreting cells and found that SerpinB1 expression in insulin-secreting cells was higher than in pPSCs. Meanwhile, the protein expression level of phosphorylated STAT3/STAT3 was increased while p53 and p21 was decreased in induced insulin-secreting cells in comparison with control cells. The insulin-secreting cells derived from the sh-SerpinB1 cells secreted less insulin and showed poor sensitivity to high glucose than control group. However, the insulin-secreting cells derived from the ov-SerpinB1 cells has a quite contrary tendency. In conclusion, this study demonstrates that SerpinB1 promotes the proliferation of pPSCs through the STAT3 signaling pathway, and SerpinB1 is a key factor for maintaining the viability of pPSCs during the transition to insulin-secreting cells.

Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells.

Diabetes mellitus affects a large number of men of reproductive age and it usually leads to serious reproductive disorders. However, the underlying mechanisms and specific therapies still remain largely unknown. We observed Leydig cell loss in the testes of diabetic mice. Continuous high glycemic status of testes stimulated expression of Caspase12, Grp78, and Chop, the three ERS response factors; this might induce cell cycle arrest and apoptosis of Leydig cells in response to ERS. In these diabetic mouse models, melatonin alleviated apoptosis of testicular stromal cell induced by ERS, and promoted SSCs self-renewal by recovering Leydig cells secretion of CSF1 after 8 weeks of treatment. To explore the relationship between CSF-1 and ERS in Leydig cells, we treated Leydig tumor cell line with an activator Tuniamycin and an inhibitor 4-Phenylbutyrate of ERS. Our data showed that the CSF-1 expression in mouse Leydig cell lines decreased six-fold while reversely increasing five-fold in the 4-Phenylbutyrate-treated group. Thus, melatonin likely alleviates the loss of Leydig cells in diabetic testes and provides a healthier niche for SSCs to self-renew and continually provide healthy sperm for male fertility.

Double sex and mab-3 related transcription factor 1 regulates differentiation and proliferation in dairy goat male germline stem cells.

The protein encoded by double sex and mab-3 related transcription factor 1 (Dmrt1) gene contains a double sex/mab-3 domain, which was considered as one of the most conservative structures in sex determination. However, its effect on spermatogenesis of dairy goat spermatogonial stem cells (SSCs) remains to be clarified. For the first time, the roles of Dmrt1 in spermatogenesis of livestock are highlighted. Here, we investigated the expression pattern of Dmrt1 in the testes of dairy goats. Dmrt1 primarily located in undifferentiated SSCs. Moreover, Dmrt1 enhanced differentiation and proliferation of mGSCs. On the contrary, the level of meiosis was down-regulated, as Dmrt1 determines whether SSCs undergo mitosis and spermatogonial differentiation or meiosis. In the busulfan-treated mice testes, Dmrt1 repair germ cell damage was emphasized as well. Our results exposed that Dmrt1 maintenance mGSCs in two ways: facilitating proliferation and self-renewal of SSCs; and reducing the inflammatory response caused by reproductive injury. These findings identify a central role for Dmrt1 in controlling population stability and injury restoring of SSCs.

Resveratrol controlled the fate of porcine pancreatic stem cells through the Wnt/ß-catenin signaling pathway mediated by Sirt1.

Porcine pancreatic stem cells (PSCs) are considered promising transplant materials that may be used to treat diabetes, but some problems, such as insufficient cell number and low differentiation efficiency, should be solved before its clinical application. Resveratrol is a natural polyphenolic compound that can alleviate the complications of diabetes. In this study, we aimed to explore the specific effect of resveratrol on porcine PSCs. We treated porcine PSCs with 10 µM, 25 µM resveratrol to explore the effect of resveratrol on porcine PSCs. We found that 10 µM resveratrol improved the proliferation of porcine PSCs, increased the expression of A-ß-catenin (active ß-catenin), Pcna, C-Myc, Bcl-2 and sirtuin-1 (Sirt1), and decreased the expression of P53, Caspase3. While 25 µM resveratrol had almost opposite effect compared with 10 µM resveratrol group. The utilization of Dickkopf-related protein 1 (DKK1, Wnt signaling pathway inhibitor) and nicotinamide (Sirt1 inhibitor) suggested that resveratrol regulated cell proliferation by controlling Wnt signaling pathway and this effect was mediated by Sirt1. Our results further revealed that 10 µM resveratrol promoted the formation of ß-like cells regulated by Wnt/ß-catenin signal pathway. Relatively low-dose resveratrol could improve porcine PSCs fate. It lays theoretical foundation for diabetes treatment with cell transplantation in future.

Autophagy is essential for the differentiation of porcine PSCs into insulin-producing cells.

Porcine pancreatic stem cells (PSCs) are seed cells with potential use for diabetes treatment. Stem cell differentiation requires strict control of protein turnover and lysosomal digestion of organelles. Autophagy is a highly conserved process that controls the turnover of organelles and proteins within cells and contributes to the balance of cellular components. However, whether autophagy plays roles in PSC differentiation remains unknown. In this study, we successfully induced porcine PSCs into insulin-producing cells and found that autophagy was activated during the second induction stage. Inhibition of autophagy in the second stage resulted in reduced differentiational efficiency and impaired glucose-stimulated insulin secretion. Moreover, the expression of active ß-catenin increased while autophagy was activated but was suppressed when autophagy was inhibited. Therefore, autophagy is essential to the formation of insulin-producing cells, and the effects of autophagy on differentiation may be regulated by canonical Wnt signalling pathway.

Autophagy stimulated proliferation of porcine PSCs might be regulated by the canonical Wnt signaling pathway.

Porcine pancreatic stem cells (PSCs) are one kind of the potential cells for treatment of human diabetes. Autophagy is a highly conserved cellular degradation process in which it helps to maintain the balance between the synthesis, degradation and subsequent recycling of cellular components. However, how autophagy contributes to PSCs has not yet been investigated. Here, we established GFP-LC3 transfected porcine PSC lines in which the accumulation of autophagosomes can be efficiently visualized to evaluate the autophagic activity. Moreover, we observed that starved PSCs which showed increased autophagic activity exhibited an increased tendency to proliferate through the results of BrdU, flow cytometry and western blotting. Furthermore, increased expression of active ß-catenin after inducing autophagy indicated that it might be the canonical Wnt signaling that autophagy activated to exert the function on the stimulation of PSCs proliferation. Collectively, these results demonstrated that autophagy stimulated proliferation of PSCs might be regulated by the canonical Wnt signaling pathway. Our results for the first time shed light on a role of autophagy for stimulating the proliferation of porcine PSCs.

Karrikins delay soybean seed germination by mediating abscisic acid and gibberellin biogenesis under shaded conditions.

Karrikins (KAR) are a class of signal compounds, discovered in wildfire smoke, which affect seed germination. Currently, numerous studies have focused on the model plant Arabidopsis in the KAR research field, rather than on crops. Thus the regulatory mechanisms underlying KAR regulation of crop seed germination are largely unknown. Here, we report that KAR delayed soybean seed germination through enhancing abscisic acid (ABA) biosynthesis, while impairing gibberellin (GA) biogenesis. Interestingly, KAR only retarded soybean seed germination under shaded conditions, rather than under dark and white light conditions, which differs from in Arabidopsis. Phytohormone quantification showed that KAR enhanced ABA biogenesis while impairing GA biosynthesis during the seed imbibition process, and subsequently, the ratio of active GA4 to ABA was significantly reduced. Further qRT-PCR analysis showed that the transcription pattern of genes involved in ABA and GA metabolic pathways are consistent with the hormonal measurements. Finally, fluridone, an ABA biogenesis inhibitor, remarkably rescued the delayed-germination phenotype of KAR-treatment; and paclobutrazol, a GA biosynthesis inhibitor, inhibited soybean seed germination. Taken together, these evidences suggest that KAR inhibit soybean seed germination by mediating the ratio between GA and ABA biogenesis.