Inhibition of Syk promotes chemical reprogramming of fibroblasts via metabolic rewiring and H2 S production.

Chemical compounds have recently been introduced as alternative and non-integrating inducers of pluripotent stem cell fate. However, chemical reprogramming is hampered by low efficiency and the molecular mechanisms remain poorly characterized. Here, we show that inhibition of spleen tyrosine kinase (Syk) by R406 significantly promotes mouse chemical reprogramming. Mechanistically, R406 alleviates Syk / calcineurin (Cn) / nuclear factor of activated T cells (NFAT) signaling-mediated suppression of glycine, serine, and threonine metabolic genes and dependent metabolites. Syk inhibition upregulates glycine level and downstream transsulfuration cysteine biosynthesis, promoting cysteine metabolism and cellular hydrogen sulfide (H2 S) production. This metabolic rewiring decreased oxidative phosphorylation and ROS levels, enhancing chemical reprogramming. In sum, our study identifies Syk-Cn-NFAT signaling axis as a new barrier of chemical reprogramming and suggests metabolic rewiring and redox homeostasis as important opportunities for controlling cell fates.

Identification of a small molecule 1,4-bis-[4-(3-phenoxy-propoxy)-but-2-ynyl]-piperazine as a novel inhibitor of the transcription factor p53.

AIM: To identify novel small compound inhibitor of p53 protein. METHODS: Mouse embryonic fibroblasts (MEF) and mouse embryonic stem (ES) cells were tested. Cell proliferation rate was determined using a Cell Proliferation Kit. The mRNA and protein levels of p53-related genes were measured using real-time PCR and Western blotting, respectively. Global response in the p53 signaling network was analyzed using Illumina whole-genome expression BeadChips. RESULTS: Treatment of MEF cells with a small molecule 1,4-bis-[4-(3-phenoxy-propoxy)-but-2-ynyl]-piperazine (G5) at 10 µmol/L for 24 h markedly reduced the mRNA and protein levels of the p53 downstream genes MDM2 and p21. In G5-treated ES cells, a total of 372 differentially expressed genes were identified, and 18 among them were direct downstream genes of p53; 6 out of 9 p53-repressed genes were upregulated, and 5 out of 9 p53-activated genes were downregulated. In both MEF cells and ES cells, treatment of with G5 (10 µmol/L) up to 48 h neither affected the proliferation rate nor caused morphological alterations. CONCLUSION: G5 inhibits p53 activity and simultaneously preserves the normal growth and proliferation of cells, therefore is a new compound for studies of p53-mediated cell manipulation.

Exploration of novel clusters and prognostic value of immune­related signatures and identify HAMP as hub gene in colorectal cancer.

Immune checkpoint inhibitors currently serve an important role in prolonging patients' overall survival. However, the prognostic signatures of immune checkpoint inhibitors in colorectal cancer (CRC) remain uncertain and more knowledge on the genetic characteristics of colorectal cancer is needed. Patients with CRC from The Cancer Genome Atlas were classified into high-immunity group and low-immunity group based on median scores from single-sample gene set enrichment analysis using the GSVA package. We explored immune status by immune scores, stromal scores and tumor purity scores in ESTIMATE package and surveyed the difference of immune cells distribution with CIBERSORT package. Eighteen genes were selected using the LASSO Cox regression method and a prognostic risk model was constructed. Compared with patients in the low-risk group, those in the high-risk group had a significantly shorter survival time. For assessment of the prognostic validity of the risk model, receiver operating characteristic curves with areas under the curve of 0.769, 0.774 and 0.771 for 1, 3 and 5 years respectively. Differences in molecular mechanisms between high- and low-risk groups were analyzed using the clusterProfiler package. Tumor Immune Dysfunction and Exclusion data were downloaded and analyzed. The top 5 enriched pathways in the high-risk group involved 'calcium signaling', 'dilated cardiomyopathy', 'extracellular matrix receptor interaction', 'hypertrophic cardiomyopathy' and 'neuroactive ligand receptor interaction'. HAMP was identified as a hub gene, which was highly expressed in tumor samples. The results of the present study indicate that the prognostic model based on both immune-related genes and HAMP has the potential to support personalized treatment.

Lovastatin promotes the self-renewal of murine and primate spermatogonial stem cells.

The spermatogonial stem cell (SSC) niche is critical for SSC maintenance and subsequent spermatogenesis. Numerous reproductive hazards impair the SSC niche, thereby resulting in aberrant SSC self-renewal and male infertility. However, promising agents targeting the impaired SSC niche to promote SSC self-renewal are still limited. Here, we screen out and assess the effects of Lovastatin on the self-renewal of mouse SSCs (mSSCs). Mechanistically, Lovastatin promotes the self-renewal of mSSCs and inhibits its inflammation and apoptosis through the regulation of isoprenoid intermediates. Remarkably, treatment by Lovastatin could promote the proliferation of undifferentiated spermatogonia in the male gonadotoxicity model generated by busulfan injection. Of note, we demonstrate that Lovastatin could enhance the proliferation of primate undifferentiated spermatogonia. Collectively, our findings uncover that lovastatin could promote the self-renewal of both murine and primate SSCs and have implications for the treatment of certain types of male infertility using small compounds.

The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting.

Although somatic cells can be reprogrammed to pluripotent stem cells (PSCs) with pure chemicals, authentic pluripotency of chemically induced pluripotent stem cells (CiPSCs) has never been achieved through tetraploid complementation assay. Spontaneous reprogramming of spermatogonial stem cells (SSCs) was another non-transgenic way to obtain PSCs, but this process lacks mechanistic explanation. Here, we reconstructed the trajectory of mouse SSC reprogramming and developed a five-chemical combination, boosting the reprogramming efficiency by nearly 80- to 100-folds. More importantly, chemical induced germline-derived PSCs (5C-gPSCs), but not gPSCs and chemical induced pluripotent stem cells, had authentic pluripotency, as determined by tetraploid complementation. Mechanistically, SSCs traversed through an inverted pathway of in vivo germ cell development, exhibiting the expression signatures and DNA methylation dynamics from spermatogonia to primordial germ cells and further to epiblasts. Besides, SSC-specific imprinting control regions switched from biallelic methylated states to monoallelic methylated states by imprinting demethylation and then re-methylation on one of the two alleles in 5C-gPSCs, which was apparently distinct with the imprinting reprogramming in vivo as DNA methylation simultaneously occurred on both alleles. Our work sheds light on the unique regulatory network underpinning SSC reprogramming, providing insights to understand generic mechanisms for cell-fate decision and epigenetic-related disorders in regenerative medicine.

Single-cell multi-omics sequencing of human spermatogenesis reveals a DNA demethylation event associated with male meiotic recombination.

Human spermatogenesis is a highly ordered process; however, the roles of DNA methylation and chromatin accessibility in this process remain largely unknown. Here by simultaneously investigating the chromatin accessibility, DNA methylome and transcriptome landscapes using the modified single-cell chromatin overall omic-scale landscape sequencing approach, we revealed that the transcriptional changes throughout human spermatogenesis were correlated with chromatin accessibility changes. In particular, we identified a set of transcription factors and cis elements with potential functions. A round of DNA demethylation was uncovered upon meiosis initiation in human spermatogenesis, which was associated with male meiotic recombination and conserved between human and mouse. Aberrant DNA hypermethylation could be detected in leptotene spermatocytes of certain nonobstructive azoospermia patients. Functionally, the intervention of DNA demethylation affected male meiotic recombination and fertility. Our work provides multi-omics landscapes of human spermatogenesis at single-cell resolution and offers insights into the association between DNA demethylation and male meiotic recombination.

Single-cell multiomics sequencing reveals the reprogramming defects in embryos generated by round spermatid injection.

Round spermatid injection (ROSI) technique holds great promise for clinical treatment of a proportion of infertile men. However, the compromised developmental potential of ROSI embryos largely limits the clinical application, and the mechanisms are not fully understood. Here, we describe the transcriptome, chromatin accessibility, and DNA methylation landscapes of mouse ROSI embryos derived from early-stage round spermatids using a single-cell multiomics sequencing approach. By interrogating these data, we identify the reprogramming defects in ROSI embryos at the pronuclear stages, which are mainly associated with the misexpression of a cohort of minor zygotic genome activation genes. We screen a small compound, A366, that can significantly increase the developmental potential of ROSI embryos, in which A366 can partially overcome the reprogramming defects by amending the epigenetic and transcriptomic states. Collectively, our study uncovers the reprogramming defects in ROSI embryos for understanding the mechanisms underlying compromised developmental potential and offers an avenue for ROSI technique optimization.

Targeting APLN/APJ restores blood-testis barrier and improves spermatogenesis in murine and human diabetic models.

Type 2 diabetes mellitus is one of the most prevalent metabolic diseases presenting with systemic pathologies, including reproductive disorders in male diabetic patients. However, the molecular mechanisms that contributing to spermatogenesis dysfunction in diabetic patients have not yet been fully elucidated. Here, we perform STRT-seq to examine the transcriptome of diabetic patients' testes at single-cell resolution including all major cell types of the testis. Intriguingly, whereas spermatogenesis appears largely preserved, the gene expression profiles of Sertoli cells and the blood-testis barrier (BTB) structure are dramatically impaired. Among these deregulate pathways, the Apelin (APLN) peptide/Apelin-receptor (APJ) axis is hyper-activated in diabetic patients' testes. Mechanistically, APLN is produced locally by Sertoli cells upon high glucose treatment, which subsequently suppress the production of carnitine and repress the expression of cell adhesion genes in Sertoli cells. Together, these effects culminate in BTB structural dysfunction. Finally, using the small molecule APLN receptor antagonist, ML221, we show that blocking APLN/APJ significantly ameliorate the BTB damage and, importantly, improve functional spermatogenesis in diabetic db/db mice. We also translate and validate these findings in cultured human testes. Our findings identify the APLN/APJ axis as a promising therapeutic target to improve reproduction capacity in male diabetic patients.

Cell-fate transition and determination analysis of mouse male germ cells throughout development.

Mammalian male germ cell development is a stepwise cell-fate transition process; however, the full-term developmental profile of male germ cells remains undefined. Here, by interrogating the high-precision transcriptome atlas of 11,598 cells covering 28 critical time-points, we demonstrate that cell-fate transition from mitotic to post-mitotic primordial germ cells is accompanied by transcriptome-scale reconfiguration and a transitional cell state. Notch signaling pathway is essential for initiating mitotic arrest and the maintenance of male germ cells' identities. Ablation of HELQ induces developmental arrest and abnormal transcriptome reprogramming of male germ cells, indicating the importance of cell cycle regulation for proper cell-fate transition. Finally, systematic human-mouse comparison reveals potential regulators whose deficiency contributed to human male infertility via mitotic arrest regulation. Collectively, our study provides an accurate and comprehensive transcriptome atlas of the male germline cycle and allows for an in-depth understanding of the cell-fate transition and determination underlying male germ cell development.

[Geochemical character of precipitation in summer of Shanghai 2008-2009].

The chemical compositions of the rainwater collected in Shanghai in Summer of 2008-2009 were investigated. The chemical character and pollutant source of rainwater were evaluated depended on HYSPLIT model, ions tracer techniques, correlation and principal component analysis. The results showed that: (1) the mean pH in rain was 4.72 and 4.68; (2) the frequency of acid rain was 53.30% and 63.30%, respectively, in 2008 and 2009; (3) ionic concentration was SO4(2-) > NH4+ > NO3- > Cl- > Ca2+ > Na+ > Mg2+ > K+, in which the secondary components like SO4(2-), NO3- and NH4+ contributed significantly to total ions of rainwater and they accounted for 55.01% and 65.97% of total ions in 2008 and 2009, respectively, which indicate the severe secondary pollution in Shanghai; (4) the ratio of SO4(2-) to NO3- in Summer precipitation in 2008 and 2009 was 3.19 and 2.13, respectively, which implies sulfuric-nitrous mixed type of precipitation; (5) the content of DOC varied from 1.36 mg/L to 10.69 mg/L and average value was 2.44 mg/L in rainwater; (6) SO4(2-) and NO3- were mainly in the form of (NH4) 2SO4 and NH4NO3, which showed the dominant neutralization effect of NH4+ over Ca2+ in Summer. Source identification indicated that SO4(2-), NH4+, NO3-, K+ and most Ca2+ derived from anthropogenic sources, while Mg2+ and Cl- derived from both marine and non-marine but non-marine was over marine. The chemistry of precipitation in Shanghai was impacted by local pollutants and the long-and moderate-range transport by Southwest monsoon according to backward trajectory analysis.