SIRT3-dependent mitochondrial redox homeostasis mitigates CHK1 inhibition combined with gemcitabine treatment induced cardiotoxicity in hiPSC-CMs and mice.

Administration of CHK1-targeted anticancer therapies is associated with an increased cumulative risk of cardiac complications, which is further amplified when combined with gemcitabine. However, the underlying mechanisms remain elusive. In this study, we generated hiPSC-CMs and murine models to elucidate the mechanisms underlying CHK1 inhibition combined with gemcitabine-induced cardiotoxicity and identify potential targets for cardioprotection. Mice were intraperitoneally injected with 25 mg/kg CHK1 inhibitor AZD7762 and 20 mg/kg gemcitabine for 3 weeks. hiPSC-CMs and NMCMs were incubated with 0.5 uM AZD7762 and 0.1 uM gemcitabine for 24 h. Both pharmacological inhibition or genetic deletion of CHK1 and administration of gemcitabine induced mtROS overproduction and pyroptosis in cardiomyocytes by disrupting mitochondrial respiration, ultimately causing heart atrophy and cardiac dysfunction in mice. These toxic effects were further exacerbated with combination administration. Using mitochondria-targeting sequence-directed vectors to overexpress CHK1 in cardiomyocyte (CM) mitochondria, we identified the localization of CHK1 in CM mitochondria and its crucial role in maintaining mitochondrial redox homeostasis for the first time. Mitochondrial CHK1 function loss mediated the cardiotoxicity induced by AZD7762 and CHK1-knockout. Mechanistically, mitochondrial CHK1 directly phosphorylates SIRT3 and promotes its expression within mitochondria. On the contrary, both AZD7762 or CHK1-knockout and gemcitabine decreased mitochondrial SIRT3 abundance, thus resulting in respiration dysfunction. Further hiPSC-CMs and mice experiments demonstrated that SIRT3 overexpression maintained mitochondrial function while alleviating CM pyroptosis, and thereby improving mice cardiac function. In summary, our results suggest that targeting SIRT3 could represent a novel therapeutic approach for clinical prevention and treatment of cardiotoxicity induced by CHK1 inhibition and gemcitabine.

Activating transcription factor 3 coordinates differentiation of cardiac and hematopoietic progenitors by regulating glucose metabolism.

The cardiac and hematopoietic progenitors (CPs and HPs, respectively) in the mesoderm ultimately form a well-organized circulation system, but mechanisms that reconcile their development remain elusive. We found that activating transcription factor 3 (atf3) was highly expressed in the CPs, HPs, and mesoderm, in zebrafish. The atf3 -/- mutants exhibited atrial dilated cardiomyopathy and a high ratio of immature myeloid cells. These manifestations were primarily caused by the blockade of differentiation of both CPs and HPs within the anterior lateral plate mesoderm. Mechanistically, Atf3 targets cebpγ to repress slc2a1a-mediated glucose utilization. The high rate of glucose metabolism in atf3 -/- mutants inhibited the differentiation of progenitors by changing the redox state. Therefore, atf3 could provide CPs and HPs with metabolic adaptive capacity to changes in glucose levels. Our study provides new insights into the role of atf3 in the coordination of differentiation of CPs and HPs by regulating glucose metabolism.

Perfluorooctane sulfonate-induced testicular toxicity and differential testicular expression of estrogen receptor in male mice.

Perfluorooctane sulfonate (PFOS, CAS#1763-23-1) causes male reproductive toxicities, but the underlying mechanisms are still unclear. In this study, 0, 0.5 and 10mg/kg/day PFOS were given by oral gavage to adult mice for 5 weeks. In the 10mg/kg group, serum testosterone levels decreased significantly. Sperm counts declined which might be associated with the decreased proliferation and increased apoptosis of germ cells. In relation to increased apoptosis, bax, cleaved caspase-9 and cleaved caspase-3 levels elevated significantly, indicating that PFOS induced germ cell apoptosis by activating the mitochondrial pathway. In addition, the increase in levels of testicular estrogen receptor (ER) ß was observed in both 0.5 and 10mg/kg group, whereas a decrease in ERα expression was only observed in 10mg/kg group. These results suggested that the alterations in testicular ERs expression, together with decreased proliferation and increased apoptosis of germ cells, might be involved in PFOS-induced testicular toxicity.

Association between the XRCC1 Arg194Trp polymorphism and glioma risk: an updated meta-analysis.

Gliomas are the most common type of primary brain tumors. The XRCC1 Arg194Trp variant affects the proliferating cell nuclear antigen( PCNA) binding region, which suggests that this mutation may contribute to gliomagenesis and a number of articles have examine the association between XRCC1 Arg194Trp and the susceptibility to glioma. However, the results were conflicting. Test of heterogeneity, sensitivity analysis, meta- analysis, and assessment of publication bias were all performed in our present meta-analysis, covering a total of 5,407 patients and 7,715 healthy persons. In the overall analysis the XRCC1 Arg194Trp polymorphism showed a significant association with glioma susceptibility in a recessive mode l(for TrpTrp vs ArgArg+ArgTrp: OR=1.918, 95%CI=1.575-2.336, I2=2.3%). In addition, analysis of subgroups presented an increased risk in Asians and populations-based on hospitals. The results suggested that the XRCC1 Arg194Trp polymorphism is a genetic risk factor for glioma, especially in Asian population. To further evaluate gene-gene and gene-environment interactions on XRCC1 polymorphisms and glioma risk, thousands of subjects and tissue-specific biochemical characterizations are required.

miR-142-3p acts as an essential modulator of neutrophil development in zebrafish.

Neutrophils play critical roles in vertebrate innate immune responses. As an emerging regulator in normal myelopoiesis, the precise roles of microRNA in the development of neutrophils have yet to be clarified. Using zinc-finger nucleases, we have successfully generated heritable mutations in miR-142a and miR-142b and showed that hematopoietic-specific miR-142-3p is completely deleted in miR-142 double mutant zebrafish. The lack of miR-142-3p resulted in aberrant reduction and hypermaturation of neutrophils in definitive myelopoiesis, as well as impaired inflammatory migration of neutrophils in the fetal stage. Furthermore, the adult myelopoiesis in the miR-142-3p-deficient zebrafish was also affected, producing irregular hypermature neutrophils with increased cell size and a decreased nucleocytoplasmic ratio. Additionally, miR-142-3p-deficient zebrafish are expected to develop a chronic failure of myelopoiesis with age. Transcriptome analysis showed an aberrant activation of the interferon γ (IFN-γ) signaling pathway in myelomonocytes after miR-142-3p deletion. We found that the reduced number and hypermaturation of neutrophils caused by loss of miR-142-3p was mainly mediated by the abnormally activated IFN-γ signaling, especially the upregulation of stat1a and irf1b. Taken together, we uncovered a novel role of miR-142-3p in maintaining normal neutrophil development and maturation.

The preparation and application of N-terminal 57 amino acid protein of the follicle-stimulating hormone receptor as a candidate male contraceptive vaccine.

Follicle-stimulating hormone receptor (FSHR), which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and development. This study introduces a method for the preparation and purification of human FSHR 57-amino acid protein (FSHR-57aa) as well as determination of its immunogenicity and antifertility effect. A recombinant pET-28a(+)-FSHR-57aa plasmid was constructed and expressed in Escherichia coli strain BL21 Star TM (DE3) and the FSHR-57aa protein was separated and collected by cutting the gel and recovering activity by efficient refolding dialysis. The protein was identified by Western blot and high-performance liquid chromatography analysis with a band of nearly 7 kDa and a purity of 97.4%. Male monkeys were immunized with rhFSHR-57aa protein and a gradual rising of specific serum IgG antibody was found which reached a plateau on day 112 (16 weeks) after the first immunization. After mating of one male with three female monkeys, the pregnancy rate of those mated with males immunized against FSHR-57aa was significantly decreased while the serum hormone levels of testosterone and estradiol were not disturbed in the control or the FSHR-57aa groups. By evaluating pathological changes in testicular histology, we found that the blood-testis barrier remained intact, in spite of some small damage to Sertoli cells. In conclusion, our study demonstrates that the rhFSHR-57aa protein might be a feasible male contraceptive which could affect sperm production without disturbing hormone levels.

Pten regulates homeostasis and inflammation-induced migration of myelocytes in zebrafish.

BACKGROUND: Loss of the tumor suppressor phosphatase and tensin homolog (PTEN) is frequently observed in hematopoietic malignancies. Although PTEN has been implicated in maintaining the quiescence of hematopoietic stem cells (HSCs), its role in hematopoiesis during ontogeny remains largely unexplored. METHODS: The expression of hematopoietic marker genes was analyzed via whole mount in situ hybridization assay in ptena and ptenb double mutant zebrafish. The embryonic myelopoiesis was characterized by living imaging and whole mount in situ immunofluorescence with confocal microscopy, as well as cell-specific chemical staining for neutrophils and macrophages. Analyses of the involved signaling pathway were carried out by inhibitor treatment and mRNA injection. RESULTS: Pten-deficient zebrafish embryos exhibited a strikingly increased number of myeloid cells, which were further characterized as being immune deficient. In accordance with this finding, the inhibition of phosphoinositide 3-kinase (PI3K) or the mechanistic target of rapamycin (mTOR) corrected the expansive myelopoiesis in the pten-deficient embryos. Further mechanistic studies revealed that the expression of cebpa, a critical transcription factor in myeloid precursor cells, was downregulated in the pten-deficient myeloid cells, whereas the injection of cebpa mRNA markedly ameliorated the dysmyelopoiesis induced by the loss of pten. CONCLUSIONS: Our data provide in vivo evidence that definitive myelopoiesis in zebrafish is critically regulated by pten via the elevation of cebpa expression.

TERT rs2736098 polymorphism and cancer risk: results of a meta-analysis.

OBJECTIVE: Several studies have demonstrated associations between the TERT rs2736098 single nucleotide polymorphisms (SNPs) and susceptibility to cancer development. However, there are conflicting results. A systematic meta-analysis was therefore performed to establish the cancer risk associated with the polymorphism. METHODS: In this meta-analysis, a total of 6 case-control studies, including 5,567 cases and 6,191 controls, were included. Crude odds ratios with 95% confidence intervals were used to assess the strength of associations in several genetic models. RESULTS: Our results showed no association reaching the level of statistical significance for overall risk. Interestingly, in the stratified analyses (subdivided by ethnicity), significantly increased risks were found in the Asian subgroup which indicates the TERT rs2736098 polymorphism may have controversial involvement in cancer susceptibility. CONCLUSIONS: Overall, this meta-analysis indicates that the TERT rs2736098 polymorphism may have little involvement in cancer susceptibility.

Genetic variants in TP53 and MDM2 associated with male infertility in Chinese population.

The TP53, a transcriptional regulator and tumor suppressor, is functionally important in spermatogenesis. MDM2 is a key regulator of the p53 pathway and modulates p53 activity. Both proteins have been functionally linked to germ cell apoptosis, which may affect human infertility, but very little is known on how common polymorphisms in these genes may influence germ cell apoptosis and the risk of male infertility. Thus, this study was designed to test whether three previously described polymorphisms 72Arg>Pro (rs1042522) and the Ex2+19C>T (rs2287498) in TP53, and the 5' untranslated region (5' UTR) 309T>G (rs937283) in MDM2, are associated with idiopathic male infertility in a Chinese population. The three polymorphisms were genotyped using OpenArray assay in a hospital-based case-control study, including 580 infertile patients and 580 fertile controls. Our analyses revealed that TP53 Ex2+19C>T and MDM2 309T>G polymorphisms are associated with male infertility. Furthermore, we detected a nearly statistically significant additive interaction between TP53 rs2287498 and MDM2 rs937283 for the development of male infertility (P(interaction)=0.055). In summary, this study found preliminary evidence, demonstrating that genetic variants in genes of the TP53 pathway are risk factors for male infertility.

Involvement of IGF-I signaling pathway in the regulation of steroidogenesis in mouse Leydig cells treated with fenvalerate.

Exposure to fenvalerate has been shown to be associated with decreased steroid hormone production by mouse Leydig tumor cells (MLTC-1) in our previous study and the interference with cAMP-PKA pathway cannot explain this inhibitory effect completely. In this study, the same cell line was used to investigate the potential involvement of insulin-like growth factor I (IGF-I) signaling pathway in the downregulation of steroidogenesis by fenvalerate. Results showed that fenvalerate treatment decreased IGF-I secretion significantly which was consistent with the reduced expression of IGF-I mRNA. Then inhibitors of the two downstream pathways of IGF-I were added to the medium. The addition of LY294002 (inhibitor of phosphatidylinositol (PI)-3-kinase) did not alter the declining trend of progesterone production with increasing dosages of fenvalerate treatment while the addition of UO126 (inhibitor of extracellular signal-regulated kinases 1/2 (ERK1/2)) markedly attenuated this trend, which strongly indicated the possible involvement of pathway ERK1/2. In addition, phosphorylation of ERK1/2 was also suppressed by fenvalerate. The results suggest that the mechanism by which fenvalerate decreased steroid hormone production might involve the impairment of IGF-I signal pathway by attenuating the IGF-I production and ERK1/2 phosphorylation.

BCL2 Ala43Thr is a functional variant associated with protection against azoospermia in a Han-Chinese population.

Apoptosis is very common during various stages of mammalian germ cell development and differentiation, and the BCL2 gene is one of the most important apoptotic regulators. Although its genetic variants are reported to be involved in cancers and autoimmune diseases, little information is available regarding BCL2 polymorphisms in male spermatogenesis. In the present study, single nucleotide polymorphisms (SNPs) in coding regions of the BCL2 gene were examined in a hospital-based, case-control study including 198 infertile patients with idiopathic azoospermia and 183 fertile controls. Subsequently, a functional study was conducted for comparison of paclitaxel-induced cytotoxicity and apoptosis between the BCL2 variant and the wild type in vitro. Three SNPs were found in exon 2--A21G (rs1801018), G127A (rs1800477), and C300T (rs61733416)--with the latter first reported in the Han-Chinese population. The frequency of G127A (GA+AA) genotype was significantly lower in azoospermic, infertile men compared to the age-matched controls (P = 0.01). This genotype may confer a lower risk of azoospermia (adjusted odd ratio [OR] = 0.448, 95% confidence interval = 0.226-0.889). In addition, HeLa cells expressing the BCL2 Ala43Thr (G127A), similar to the control cells, were more sensitive to paclitaxel-induced cytotoxicity and apoptosis than those expressing wild-type BCL2. Consistently, the cleaved PARP and p-BCL2 proteins were subsequently increased after paclitaxel treatment, as also predicted by the bioinformatics analysis. Considering the decreased antiapoptotic function of BCL2, these results suggest that the Ala43Thr variant is associated with protection against azoospermia in the Han-Chinese population.