HIF-2α-dependent TGFBI promotes ovarian cancer chemoresistance by activating PI3K/Akt pathway to inhibit apoptosis and facilitate DNA repair process.

Hypoxia-mediated chemoresistance plays a crucial role in the development of ovarian cancer (OC). However, the roles of hypoxia-related genes (HRGs) in chemoresistance and prognosis prediction and theirs underlying mechanisms remain to be further elucidated. We intended to identify and validate classifiers of hub HRGs for chemoresistance, diagnosis, prognosis as well as immune microenvironment of OC, and to explore the function of the most crucial HRG in the development of the malignant phenotypes. The RNA expression and clinical data of HRGs were systematically evaluated in OC training group. Univariate and multivariate Cox regression analysis were applied to construct hub HRGs classifiers for prognosis and diagnosis assessment. The relationship between classifiers and chemotherapy response and underlying pathways were detected by GSEA, CellMiner and CIBERSORT algorithm, respectively. OC cells were cultured under hypoxia or transfected with HIF-1α or HIF-2α plasmids, and the transcription levels of TGFBI were assessed by quantitative PCR. TGFBI was knocked down by siRNAs in OC cells, CCK8 and in vitro migration and invasion assays were performed to examine the changes in cell proliferation, motility and metastasis. The difference in TGFBI expression was examined between cisplatin-sensitive and -resistant cells, and the effects of TGFBI interference on cell apoptosis, DNA repair and key signaling molecules of cisplatin-resistant OC cells were explored. A total of 179 candidate HRGs were extracted and enrolled into univariate and multivariate Cox regression analysis. Six hub genes (TGFBI, CDKN1B, AKAP12, GPC1, TGM2 and ANGPTL4) were selected to create a HRGs prognosis classifier and four genes (TGFBI, AKAP12, GPC1 and TGM2) were selected to construct diagnosis classifiers. The HRGs prognosis classifier could precisely distinguish OC patients into high-risk and low-risk groups and estimate their clinical outcomes. Furthermore, the high-risk group had higher percentage of Macrophages M2 and exhibited higher expression of immunecheckpoints such as PD-L2. Additionally, the diagnosis classifiers could accurately distinguish OC from normal samples. TGFBI was further verified as a specific key target and demonstrated that its high expression was closely correlated with poor prognosis and chemoresistance of OC. Hypoxia upregulated the expression level of TGFBI. The hypoxia-induced factor HIF-2α but not HIF-1α could directly bind to the promoter region of TGFBI, and facilitate its transcription level. TGFBI was upregulated in cisplatin-sensitive and resistant ovarian cancer cells in a cisplatin time-dependent manner. TGFBI interference downregulated DNA repair-related markers (p-p95/NBS1, RAD51, p-DNA-PKcs, DNA Ligase IV and Artemis), apoptosis-related marker (BCL2) and PI3K/Akt pathway-related markers (PI3K-p110 and p-Akt) in cisplatin-resistant OC cells. In summary, the HRGs prognosis risk classifier could be served as a predictor for OC prognosis and efficacy evaluation. TGFBI, upregulated by HIF-2α as an HRG, promoted OC chemoresistance through activating PI3K/Akt pathway to reduce apoptosis and enhance DNA damage repair pathway.

A Novel Germline Mutation of BRCA1 and Integrated Analysis With Somatic Mutation in a Chinese Multi-Cancer Family.

The presence of mutations in the BRCA1 gene (MIM: 113705) is widely recognized as a significant genetic predisposition for ovarian cancer. This study investigated the genomic mutations in a Chinese family with a history of ovarian, breast, and rectal adenocarcinoma. A novel germline mutation (Phe1695Val) in BRCA1 was identified through whole-exome sequencing. Subsequently, we performed whole-genome sequencing to identify somatic mutations and analyze mutational signatures in individuals carrying the novel germline mutation. Our findings revealed a correlation between somatic mutational signatures and the BRCA1 germline mutation in the proband with ovarian cancer, while no such association was observed in the tumor tissue from the patient with breast cancer. Furthermore, distinct somatic driver mutations were identified, a truncated mutation in the TP53 gene in the ovarian tumor tissue, and a hotspot mutation in the PIK3CA gene in the breast cancer. According to our findings, the BRCA1 F1695V mutation is linked to ovarian cancer susceptibility in the family and causes specific somatic mutational profiles.

Building in biologically appropriate multifunctionality in aqueous copper indium selenide-based quantum dots.

Advanced nanoplatforms equipped with different functional moieties for theranostics hold appealing promise for reshaping precision medicine. The reliable construction of an individual nanomaterial with intrinsic near-infrared (NIR) photofunction and magnetic domains is much desired but largely unexplored in a direct aqueous synthesis system. Herein, we develop an aqueous phase synthetic strategy for Mn2+ doping of ZnS shell grown on Zn-Cu-In-Se core quantum dots (ZCISe@ZnS:Mn QDs), providing the optimal NIR fluorescence quantum efficiency of up to 18.9% and meanwhile efficiently introducing paramagnetic domains. The relaxometric properties of the water-soluble Mn-doped QDs make them desirable for both the longitudinal and transverse (T1 and T2) magnetic resonance (MR) contrast enhancement due to the shell lattice-doped Mn2+ ions with slow tumbling rates and favoured spin-proton dipolar interactions with surrounding water molecules. Surprisingly, the incorporation of Mn2+ ions into the shell is found to significantly enhance the production of reactive oxygen species (ROS) by combining both the chemodynamic and photodynamic processes upon NIR light irradiation, showing great potential for efficient photo-assisted ablation of cancer cells. Furthermore, a broad-spectrum excitation range beneficial for bright NIR fluorescence imaging of breast cancer has been proven and offers high flexibility in the choice of incident light sources. Multiparametric MR imaging of the brain has also been successfully demonstrated in vivo.

The prevalence of post-traumatic stress disorder in college students by continents and national income during the COVID-19 pandemic: a meta-analysis.

Introduction: The present study aimed to provide a more accurate representation of post-traumatic stress disorder (PTSD) in college students during COVID-19 by performing meta-analyses by continents, national income, and study majors, and comparing the results with estimated pooled prevalence. Methods: Based on the guideline of PRISMA, literature was searched in PubMed, Web of Science, and Embase. The prevalence of PTSD was estimated through a random model based on the different continents and levels of national income, as well as study majors, and compared with the pooled prevalence of PTSD among college students. Results: Totally 381 articles were retrieved from electronic databases and 38 articles were included in the present meta-analysis. The results showed that the pooled prevalence of college students' PTSD was 25% (95% CI: 21-28%). Prevalence estimates of PTSD among college students were statistically significant (p < 0.00001) when stratified with geographical regions, income levels, and study majors. In comparison with the pooled prevalence of PTSD (25%), subgroups of Africa and Europe, lower-middle-income countries, and medical college students possessed higher prevalence estimates. Discussion: The findings of the study showed that the prevalence of PTSD in college students worldwide during COVID-19 was relatively high and varied in different continents and countries with different income levels. Therefore, healthcare providers should pay attention to the psychologically healthy condition of college students during COVID-19.

Intravaginal delivery for CRISPR-Cas9 technology: For example, the treatment of HPV infection.

The increasing incidence of sexually transmitted diseases in women, including human papillomavirus (HPV) infection, has led to the need to develop user-friendly potential prevention methods. At present, although there are several therapeutic parts, none of them has a preventive effect, but they are only limited to providing patients with symptom relief. Researchers have now recognized the need to find effective local preventive agents. One of the potential undiscovered local fungicides is the vaginal delivery of CRISPR/Cas9. CRISPR/Cas9 delivery involves silencing gene expression in a sequence-specific manner in the pathogenic agent, thus showing microbicidal activity. However, vaginal mucosal barrier and physiological changes (such as pH value and variable epithelial thickness in the menstrual cycle) are the main obstacles to effective delivery and cell uptake of CRISPR/Cas9. To enhance the vaginal delivery of CRISPR/Cas9, so far, nano-carrier systems such as lipid delivery systems, macromolecular systems, polymer nanoparticles, aptamers, and cell-penetrating peptides have been extensively studied. In this paper, various nano-carriers and their prospects in the preclinical stage are described, as well as the future significance of CRISPR/Cas9 vaginal delivery based on nano-carriers.

CRISPR/Cas9-HPV-liposome enhances antitumor immunity and treatment of HPV infection-associated cervical cancer.

Increasing evidence shows that human papillomavirus (HPV) E6/E7 deletion in cervical cancer cells may be related to the immunosuppressive tumor microenvironment and adverse reactions or resistance to immune checkpoint blockade. Here, we demonstrate that liposome delivery of CRISPR/cas9 can effectively knock out HPV, which, in turn, induces autophagy and triggers cell death-related immune activation by releasing damage-related molecular patterns. The results of in vivo experiments showed that HPV-targeting guide RNA-liposomes could promote CD8+ T cell infiltration in tumor tissues; enhance the expression of proinflammatory cytokines, such as interleukin-12, tumor necrosis factor-α, and interferon-γ, and reduce regulatory T cells and myeloid suppressor cells. The combination of HPV-targeting guide RNA-liposomes with immune checkpoint inhibitors and antiprogrammed death-1 antibodies produced highly effective antitumor effects. In addition, combination therapy induced immune memory in the cervical cancer model.

miR-532-3p suppresses proliferation and invasion of ovarian cancer cells via GPNMB/HIF-1α/HK2 axis.

OBJECTIVE: Identifying a new target of miR-532-3p and studying its functional mechanism to explore the detailed anti-tumor mechanism of miR-532-3p in ovarian cancer. METHODS: Biological and molecular methods including real-time quantitative PCR (RT-qPCR), Western blotting, colony formation, in vitro migration and invasion assays, glucose consumption and lactate production assays, RNA interference and tumor xenograft mouse models were used to study the role of miR-532-3p and its target in ovarian cancer. mRNA sequencing, dual-luciferase reporter assay and immunohistochemistry (IHC) were used to identify miR-532-3p target. STRING dataset analysis, qPCR and Western blotting were used to investigate the downstream pathway of the target of miR-532-3p. RESULTS: Forced expression of miR-532-3p inhibited the proliferation, migration and invasion of ovarian cancer cells in vitro and the tumor growth in nude mice. RNA sequencing found 299 mRNAs were downregulated in miR-532-3p-overexpressed ovarian cancer cells, and bioinformatic analysis indicated Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB), a type I membrane glycoprotein, was the potential target of miR-532-3p. GPNMB was reduced at both RNA and protein levels in miR-532-3p-overexpressed ovarian cancer cells. Dual-luciferase reporter assay determined GPNMB as the target of miR-532-3p. Interference of GPNMB inhibited the proliferation, migration, invasion, glucose consumption and lactate production of ovarian cancer cells. Knocking down of GPNMB reduced the protein level of HIF-1α without affecting HIF-1α mRNA level. Overexpression of GPNMB reversed the antitumor effect of miR-532-3p. CONCLUSION: miR-532-3p exerted the anti-cancer effect by targeting GPNMB/ HIF-1α/ HK2 pathway to inhibit aerobic glycolysis in ovarian cancer.

Downregulation of DNMT3A Attenuates the Warburg Effect, Proliferation, and Invasion via Promoting the Inhibition of miR-603 on HK2 in Ovarian Cancer.

Background: Ovarian cancer is a highly malignant gynecological cancer. Aerobic glycolysis is one of the features of cancer cell metabolism. Studying the molecular modulation of the Warburg effect in ovarian cancer is significantly valuable for understanding the progression mechanism of ovarian cancer. Materials and Methods: The expression level and prognostic significance of DNMT3A were analyzed using public databases. DNMT3A was overexpressed by plasmid transfection, and DNMT3A was interfered with specific siRNAs transfection. miR-603 was overexpressed by mimic transfection or inhibited by inhibitor transfection. The expression of the molecules was detected by qPCR or western blotting. CCK-8 and transwell assays were used to determine the cell proliferation, migration, and invasion abilities of ovarian cancer. Results: We found that the DNMT3A protein level was higher in ovarian cancer tissues than in normal ovary tissues, but the mRNA level had no significant difference in ovarian cancer tissues and normal ovary tissues. The higher the RNA level of DNMT3A, the poorer prognosis of patients. DNMT3A knocking down impeded the Warburg effect, cell proliferation, migration, and invasion of ovarian cancer cells. Further investigations discovered that DNMT3A promoted ovarian cancer cell malignancy via silencing miR-603. Conclusion: We found that patients who overexpressed DNMT3A showed a poor prognosis. DNMT3A was found to promote the Warburg effect, cell proliferation, migration, and invasion of ovarian cancer by inhibiting the expression of miR-603. As a result, the research revealed that DNMT3A/miR-603/HK2 axis contributed to the Warburg effect of ovarian cancer and DNMT3A may be a potential therapeutic target for ovarian cancer.

Role of adjuvant chemotherapy after concurrent chemoradiotherapy in patients with locally advanced cervical cancer.

Aims: With the use of concurrent chemoradiotherapy (CCRT) for locally advanced cervical cancer (LACC), survival outcomes are still not optimal. This study was designed to evaluate the efficacy and safety of adjuvant chemotherapy (ACT) for patients with LACC after treatment with CCRT. Methods: Patients diagnosed with stage IIA-IIIB LACC, were retrospectively analyzed. All patients received cisplatin-based CCRT and were divided into two groups: ACT after CCRT (CCRT + ACT group) and observation after CCRT (CCRT group). Overall survival (OS), progression-free survival (PFS) and adverse effects were recorded and analyzed. Results: In total, 375 patients were included; 262 patients accepted ACT after CCRT while the remaining 113 patients chose observation. With a median follow-up of 40 months, no significant differences were found in the OS rates for patients in the CCRT + ACT and CCRT groups at 1 year, 3 years and the end of follow-up. There was also no significant discrepancy in PFS between groups. Subgroup analysis showed the International Federation of Gynecology and Obstetrics (FIGO) stage and age had negligible influence on both OS and PFS. Acute adverse events (grades 3-4) happened more frequently in CCRT + ACT group than in the CCRT group, with significant differences in neutropenia, anemia and creatinine. Conclusion: ACT after CCRT did not show benefit in survival but did induce some adverse effects. Therefore, this regimen is not recommended unless further large-scale randomized controlled trials are executed.

TGF-ß1-based CRISPR/Cas9 Gene Therapy Attenuates Radiation-induced Lung Injury.

BACKGROUND: Radiation-induced lung injury (RILI) is lacking effective therapeutic strategies. In this study, we conducted TGF-ß1-based CRISPR/Cas9 gene therapy for RILI. OBJECTIVE: Mouse lungs were irradiated with a single-dose of 20-Gy gamma rays followed by intravenous administration of Ad-CRISPR-TGF-ß1 or Ad- CRISPR-Null. METHODS: Haematoxylin and eosin staining, as well as Masson staining, were performed to observe lung morphology. Albumin and IgM concentrations in bronchoalveolar lavage fluid were measured by ELISA. Cytokine levels were measured using ELISA and/or real-time PCR with terminal deoxynucleotidyl transferase-mediated nick-end labelling. RESULTS: Ad-CRISPR-TTGF-ß1 improved histopathological and biochemical markers of lung injury, reduced secretion and expression of inflammatory cytokines, and inhibited progression of fibrosis. Importantly, the SK1/S1P axis, which is known to play a key role via S1P1 in TGF-ß1-dependent S1PR pattern remodelling, is responsible for promoting fibrosis. CONCLUSION: Our results indicate novel insights for RILI therapy.

Cloning and sequence analysis of a serine protease gene from Rhizoctonia solani Kühn AG5.

The present study aimed to identify the subtilisin-like proteases (SLPs) of Rhizoctonia solani Kühn potentially involved in the virulence of this phytopathogenic fungus, which has 14 anastomosis groups (AGs) responsible for many crop diseases. Through mycelial microscope observation and strain identification of pathogenic fungus MS-3, it was determined to be R. solani AG-5. Both 5' and 3' rapid amplification of cDNA ends were used to clone the serine protease gene RsSLP from R. solani AG-5. The full-length obtained for RsSLP was 1714 bp with an open reading frame of 1587 bp, encoding a protein of 528 amino acids with a molecular mass of 55.8 kDa. This protein contained a predicted signal peptide for secretion but lacked a transmembrane domain or membrane anchor site. Bioinformatics analysis identified this protein as a serine protease with the Peptidase_S8 and Inhibitor_I9 characteristic domains of SLPs. Phylogenetic analysis suggested that frequent gene duplications of the SLPs occurred in R. solani (RsSLP), and RsSLP shares characteristic sequence features with virulence factors of other phytopathogenic fungi. Because the secretory serine protease RsSLP from R. solani AG5 is similar to the virulence factors of other phytopathogenic fungi, its identification will be helpful in studies considering the roles of these proteases in pathogen virulence.

Enhanced antiviral benefit of combination therapy with anti-HBV and anti-PD1 gRNA/cas9 produces a synergistic antiviral effect in HBV infection.

Targeted therapy for patients with hepatitis B virus (HBV) infection can lead to objective responses, although response times may be short. At the same time, the response rate to programmed cell death-1 (PD-1) treatment was more durable. It is speculated that HBV targeted therapy can synergistically enhance the antitumor activity with PD-1 blockade. To test this hypothesis, we evaluated the effect of crispr-cas9 on HBV and PD-1 in vitro and in vivo. We found that HBV targeting gRNA/cas9 induced a decrease in the expression of HBsAg, while the PD-1 gene could be knocked out by electroporation targeting gRNA / cas9 by polymerase chain reaction. In HBV transgenic mice, the immunophenotype and cytokine expression of human dendritic cells (DCS) were detected by crispr-cas9 system stimulation, flow cytometry and polymerase chain reaction. These results indicate that gRNA/cas9 treatment upregulates the expression of CD80, CD83 and CD86, and significantly increases the mRNA levels of IL-6, IL-12, IL-23 and tumor necrosis factor alpha. The combination of anti HBV and anti PD-1 therapy can inhibit HBV expression and significantly improve the survival of HBV transgenic mice. In addition, the combination therapy increased the production of interferon by T cells, and then enhanced the expression of Th1 related immunostimulatory genes, thereby reducing the transcription of regulatory / inhibitory immune genes. In general, this response can reshape the tumor microenvironment from immunosuppression to immune stimulation. Finally, anti HBV therapy can induce the expression of interferon dependent programmed cell death ligand-1 in HBV transgenic mice in vivo. To sum up, these results demonstrate that the combination of HBV targeted therapy and PD-1 immune checkpoint block has a strong synergistic effect, thus supporting the transformation potential of this combined therapy strategy in clinical treatment of HBV infection.

Human Papillomavirus Oncogene Manipulation Using Clustered Regularly Interspersed Short Palindromic Repeats/Cas9 Delivered by pH-Sensitive Cationic Liposomes.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology enables targeted gene editing, but cancer gene therapy with this approach requires improvements to enable safe and efficient delivery of CRISPR/Cas9 to tumors. We developed and evaluated a self-assembled liposome to selectively deliver CRISPR/Cas9 to cancer tissues. Our CRISPR/Cas9 system effectively inhibited proliferation of human papillomavirus (HPV) 16-positive cervical cancer cells and induced apoptosis by inactivating the HR-HPV16E6/E7 oncogene. Based on this system, we prepared a long-circulating pH-sensitive cationic nano-liposome complex with a high cell targeting and gene knockout rate. Intratumoral injection of cationic liposomes targeted to splicing HPV16 E6/E7 in nude mice significantly inhibited tumor growth without significant toxicity in vivo. Liposomes that targeted HPV16 E6/E7 splicing were established as a basis for treatment of HPV16-positive cervical cancer drug candidates. Our study demonstrates that this liposome offers an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine-based cancer therapeutics.

TSPY1 suppresses USP7-mediated p53 function and promotes spermatogonial proliferation.

Testis-specific protein Y-linked 1 (TSPY1) is expressed predominantly in adult human spermatogonia and functions in the process of spermatogenesis; however, our understanding of the underlying mechanism is limited. Here we observed that TSPY1, as an interacting partner of TSPY-like 5 (TSPYL5), enhanced the competitive binding of TSPYL5 to ubiquitin-specific peptidase 7 (USP7) in conjunction with p53. This activity, together with its promotion of TSPYL5 expression by acting as a transcription factor, resulted in increased p53 ubiquitylation. Moreover, TSPY1 could decrease the p53 level by inducing the degradation of ubiquitinated USP7. We demonstrated that the promotion of p53 degradation by TSPY1 influenced the activity of p53 target molecules (CDK1, p21, and BAX) to expedite the G2/M phase transition and decrease cell apoptosis, accelerating cell proliferation. Taken together, the observations reveal the significance of TSPY1 as a suppressor of USP7-mediated p53 function in inhibiting p53-dependent cell proliferation arrest. By simulating TSPY1 function in Tspy1-deficient spermatogonia derived from mouse testes, we found that TSPY1 could promote spermatogonial proliferation by decreasing the Usp7-modulated p53 level. The findings suggest an additional mechanism underlying the regulation of spermatogonial p53 function, indicating the significance of TSPY1 in germline homeostasis maintenance and the potential of TSPY1 in regulating human spermatogonial proliferation via the USP7-mediated p53 signaling pathway.

Spermatogenic phenotype of testis-specific protein, Y-encoded, 1 (TSPY1) dosage deficiency is independent of variations in TSPY-like 1 (TSPYL1) and TSPY-like 5 (TSPYL5): a case-control study in a Han Chinese population.

Testis-specific protein, Y-encoded, 1 (TSPY1) is involved in the regulation of spermatogenic efficiency via highly variable copy dosage, with dosage deficiency of the multicopy gene conferring an increased risk of spermatogenic failure. TSPY-like 1 (TSPYL1) and TSPY-like 5 (TSPYL5), two autosomal homologous genes originating from TSPY1, share a core sequence that encodes a functional nucleosome assembly protein (NAP) domain with TSPY1. To explore the potential effects of TSPYL1 and TSPYL5 on the TSPY1-related spermatogenic phenotype, we investigated the expression of these genes in 15 healthy and nonpathological human tissues (brain, kidney, liver, pancreas, thymus, prostate, spleen, muscle, leucocytes, placenta, intestine, ovary, lung, colon and testis) and explored associations between their variations and spermatogenic failure in 1558 Han Chinese men with different spermatogenic conditions, including 304 men with TSPY1 dosage deficiency. TSPYL1 and TSPYL5 were expressed in many different tissues, including the testis. An unreported rare variant that is likely pathogenic (c.1057A>G, p.Thr353Ala) and another of uncertain significance (c.1258C>T, p.Arg420Cys) in the NAP-coding sequence of TSPYL1 were observed in three spermatogenesis-impaired patients with heterozygous status. The distribution differences in the alleles, genotypes and haplotypes of eight TSPYL1- and TSPYL5-linked common variants did not reach statistical significance in comparisons of patients with spermatogenic failure and controls with normozoospermia. No difference in sperm production was observed among men with different genotypes of the variants. Similar results were obtained in men with TSPY1 dosage deficiencies. Although the distribution of missense variants of TSPYL1 found in the present and other studies suggests that patients with spermatogenic failure may have a statistically significant greater burden of rare variations in TSPYL1 relative to normozoospermic controls, the functional evidence suggests that TSPYL1 contributes to impaired spermatogenesis. Moreover, the present study suggests that the effects of TSPYL1 and TSPYL5 on the spermatogenic phenotype of TSPY1 dosage deficiency are limited, which may be due to the stability of their function resulting from high sequence conservation.

Copy number variation of functional RBMY1 is associated with sperm motility: an azoospermia factor-linked candidate for asthenozoospermia.

STUDY QUESTION: What is the influence of copy number variation (CNV) in functional RNA binding motif protein Y-linked family 1 (RBMY1) on spermatogenic phenotypes? SUMMARY ANSWER: The RBMY1 functional copy dosage is positively correlated with sperm motility, and dosage insufficiency is an independent risk factor for asthenozoospermia. WHAT IS KNOWN ALREADY: RBMY1, a multi-copy gene expressed exclusively in the adult testis, is one of the most important candidates for male infertility in the azoospermia factor (AZF) region of the Y-chromosome. RBMY1 encodes an RNA-binding protein that serves as a pre-mRNA splicing regulator during spermatogenesis, and male mice deficient in Rbmy are sterile. STUDY DESIGN, SIZE, DURATION: A total of 3127 adult males were recruited from 2009 to 2016; of this group, the dosage of RBMY1 functional copy were investigated in 486 fertile males. In the remaining 2641 males with known spermatogenesis status, 1070 Y-chromosome haplogroup (Y-hg) O3* or O3e carriers without chromosomal aberration or known AZF structure mutations responsible for spermatogenic impairment, including 506 men with normozoospermia and 564 men with oligozoospermia or/and asthenozoospermia, were screened, and the RBMY1 functional copy dosage and copy conversion were determined to explore their associations with sperm phenotypes. The correlation between RBMY1 dosage and its mRNA level or RBMY1 protein level and the correlation between sperm RBMY1 level and motility were analysed in 15 testis tissue samples and eight semen samples. Ten additional semen samples were used to confirm the subcellular localization of RBMY1 in individual sperm. PARTICIPANTS/MATERIALS, SETTING, METHODS: All the Han volunteers donating whole blood, semen and testis tissue were from southwest China. RBMY1 copy number, copy conversion, mRNA/protein amount and protein location in sperm were detected using the AccuCopy® assay method, paralog ratio test, quantitative PCR, western blotting and immunofluorescence staining methods, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: This study identified Y-hg-independent CNV of functional RBMY1 in the enrolled population. A difference in the distribution of RBMY1 copy number was observed between the group with normal sperm motility and the group with asthenozoospermia. A positive correlation between the RBMY1 copy dosage and sperm motility was identified, and the males with fewer than six copies of RBMY1 showed an elevated risk for asthenozoospermia relative to those with six RBMY1 copies, the most common dosage in the population. The RBMY1 copy dosage was positively correlated with its mRNA and protein level in the testis. Sperm with high motility were found to carry more RBMY1 protein than those with relatively low motility. The RBMY1 protein was confirmed to predominantly localize in the neck and mid-piece region of sperm as well as the principal piece of the sperm tail. Our population study completes a chain of evidence suggesting that RBMY1 influences the susceptibility of males to asthenozoospermia by modulating sperm motility. LIMITATIONS REASONS FOR CAUTION: High sequence similarity between the RBMY1 functional copies and a large number of pseudogenes potentially reduces the accuracy of the copy number detection. The mechanism underlying the CNV in RBMY1 is still unclear, and the effect of the structural variations in the RBMY1 copy cluster on the copy dosage of other protein-coding genes located in the region cannot be excluded, which may potentially bias our observations. WIDER IMPLICATIONS OF THE FINDINGS: Asthenozoospermia is a multi-factor complex disease with a limited number of proven susceptibility genes. This study identified a novel genomic candidate independently contributing to the condition, enriching our understanding of the role of AZF-linked genes in male reproduction. Our finding provides insight into the physiological and pathological characteristics of RBMY1 in terms of sperm motility, supplies persuasive evidence of the significance of RBMY1 copy number analysis in the clinical counselling of male infertility resulting from asthenozoospermia. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the National Natural Science Foundation of China (Nos. 81370748 and 30971598). The authors have no conflicts of interest.

Efficacy and Safety of Single- or Double-Drug Antidiabetic Regimens in the Treatment of Type 2 Diabetes Mellitus: A Network Meta-Analysis.

A network meta-analysis was performed in order to compare the efficacy and safety of single- or double-drug antidiabetic regimens in the treatment of type 2 diabetes mellitus (T2DM). PubMed and Cochrane Library searches were conducted since inception to February 2017. Randomized controlled trials (RCTs) of different antidiabetic regimens in the treatment of T2DM were included in this study. Direct and indirect evidences were combined to calculate the odds ratio (OR) or weighted mean difference (WMD) and its 95% confidence interval (95%CI), and in order to draw the surface under the cumulative ranking curves (SUCRA). A total of 19 RCTs meeting our inclusion criteria were finally incorporated into our network meta-analysis, including 19 single- or double-drug antidiabetic regimens. The network meta-analysis showed that the anti-hyperglycemic effects of Sitagliptin + Metformin, Empagliflozin + Metformin, Exenatide + Metformin, Vildagliptin + Metformin, Taspoglutide + Metformin, and Pioglitazone + Metformin were better than individual Metformin regimens. Dulaglutide + Metformin and Taspoglutide + Metformin regimens were comparatively less safe than individual Metformin regimens. The cluster ranking analysis based on SUCRA values suggested that Taspoglutide + Metformin regimens had best efficacy and worst safety among the different therapy regimens. Our data confirmed previous observations and suggested that Taspoglutide + Metformin regimen may have better efficacy for the treatment of T2DM among 19 therapy regimens, while its incidence of adverse events was relatively higher. J. Cell. Biochem. 118: 4536-4547, 2017. © 2017 Wiley Periodicals, Inc.

Functional Variations in the NOS3 Gene Are Associated With Erectile Dysfunction Susceptibility, Age of Onset and Severity in a Han Chinese Population.

BACKGROUND: Impaired function of endothelial nitric oxide synthase (eNOS) is involved in the pathologic processes of erectile dysfunction (ED), and three functional polymorphisms (G894T, T-786C, and a tandem repeat of 27 bp in intron 4) in the NOS3 gene, which encodes eNOS, are associated with the clinical characteristics of ED in several populations. AIM: To investigate the effect of these variations of NOS3 on ED phenotypes and the response to sildenafil in a Han Chinese population. METHODS: This case-control study enrolled 112 patients with ED and 156 age-matched healthy men. Their medical history and laboratory data were collected. ED severity and response to sildenafil were assessed using the five-item International Index of Erectile Function (IIEF-5) score. Routine polymerase chain reaction and Sanger sequencing were used to genotype the three polymorphisms of NOS3. OUTCOMES: The frequencies of alleles, genotypes, and haplotypes of the loci in patients and controls; the IIEF-5 scores of patients carrying the risk and non-risk genotype; and the frequencies of risk and non-risk genotypes in patients with different ages at onset and responses to sildenafil were assessed. RESULTS: The frequencies of drinkers and diabetic and hyperlipidemic patients in the ED group were higher than those in the age-matched control group (P < .05). The distributions of alleles (G894T, P < .005; T-786C, P < .015), genotypes (G894T, P < 0.015; T-786C, P < .010), and haplotypes (G894T/T-786C, P < .015) of the NOS3 polymorphisms were significantly different between patients with ED and controls. An increased risk for earlier onset of ED was observed in the G894T risk genotype carriers (odds ratio = 3.572; P < .020). Patients with the risk genotype of T-786C exhibited lower IIEF-5 scores than patients with the non-risk genotype (8.2 ± 4.5 vs 12.2 ± 5.0; P < .015). The influence of the T-786C or G894T genotype on the response to sildenafil was not observed. CLINICAL TRANSLATION: The detectable effect of NOS3 functional polymorphisms on ED suggests their application potential as a molecular biomarker in predicting ED susceptibility and severity in the Han Chinese population. STRENGTHS & LIMITATIONS: This study provides strong evidence that NOS3 functional variation is an independent risk factor for ED in the Han Chinese population, which should be confirmed in larger cohorts considering the limited number of subjects in this study. CONCLUSION: These results are the first to identify a clear association between NOS3 functional variation and ED susceptibility, age at onset, and severity in the Han Chinese population. Yang B, Liu L, Peng Z, et al. Functional Variations in the NOS3 Gene Are Associated With Erectile Dysfunction Susceptibility, Age of Onset and Severity in a Han Chinese Population. J Sex Med 2017;14:551-557.

Evidence for the involvement of the proximal copy of the MAGEA9 gene in Xq28-linked CNV67 specific to spermatogenic failure.

Spermatogenic failure characterized by impaired sperm production is a common multifactorial disease with molecular and cytogenetic causes for its extreme phenotype that include azoospermia and severe oliogzoospermia. Recently, a high-resolution array-comparative genomic hybridization analysis of the X chromosome and a subsequent cohort study revealed three X-linked microdeletions (CNV64, CNV67, and CNV69) that were associated with decreased sperm production in a mixed group that included Spanish and Italian males. To confirm their spermatogenic effect, we examined the hemizygous deletions and copy dosage of the MAGE family member A9 (MAGEA9) gene, which is a potential X-linked candidate for the CNV67-related spermatogenic phenotype, to investigate their association with spermatogenic failure in 1722 Han males from southwest China. The individuals in this group consisted of 884 patients with idiopathic azoospermia/oliogzoospermia and 838 controls with normozoospermia. Our results showed that both CNV64 and CNV69 were more common in patients than in controls. Similar to that reported previously, the CNV67 was also identified as being specific to spermatogenic failure in our population, although it was rare. More importantly, the paralog ratio tests and sequence family variant analyses provided evidence that the CNV67 might cause a partial deletion of the proximal copy of the MAGEA9 and suggests that CNV67-related spermatogenic failure may be attributed to the functional defect of the Cancer/Testis gene. Our findings highlight the potential of the Xq-linked CNV67 to serve as a novel detection target in the etiological diagnosis of spermatogenic failure and male infertility, although its pathogenic mechanism remains to be elucidated.

Genetic variants of ADAM17 are implicated in the pathological process of Kawasaki disease and secondary coronary artery lesions via the TGF-ß/SMAD3 signaling pathway.

UNLABELLED: Kawasaki disease (KD) is a systemic vasculitis childhood disease frequently complicating coronary artery lesions (CALs). Recently, the gene encoding a disintegrin and metalloprotease 17 (ADAM17) was found to modify vascular pathology in humans by differentially regulating the transforming growth factor-ß (TGF-ß) signaling pathway, which affects KD/CAL susceptibility. To explore the potential role of ADAM17 in KD occurrence and outcomes, we investigated the association of 28 single nucleotide polymorphisms (SNPs) in ADAM17 and three pathway genes of TGF-ß signaling with KD phenotypes in a Han Chinese population, including 392 KD patients and 421 non-KD controls. Three ADAM17 SNPs showed an association with KD risk, which was further confirmed by haplotype analysis. The effect of ADAM17 on KD was also shown by multi-variable logistic regression analysis. In two-locus model analyses with SNPs in ADAM17 and TGF-ß signaling pathway genes, stronger compound effects on the risk of KD and secondary CAL formation were observed relative to comparable single SNPs. CONCLUSION: Our results suggest that ADAM17 contributes to the KD risk and is involved in secondary CAL formation via the TGF-ß/SMAD3 signaling pathway. This further enriches our understanding of the importance of the signaling pathway in KD occurrence and outcomes. WHAT IS KNOWN: • The transforming growth factor (TGF)-ß/SMAD3 signaling pathway greatly influences susceptibility to Kawasaki disease (KD) and secondary coronary artery lesions (CALs) and/or the treatment response of intravenous immunoglobulin. • A disintegrin and metalloprotease 17 (ADAM17) effectively reduces TGF-ß signaling by cleaving TGF-ß receptor type-1, while ADAM17 genetic variants modify human vascular pathology by differentially regulating this signaling although it is unknown whether ADAM17 contributes to KD phenotypes. What is New: • ADAM17 genetic variants were shown to be associated with KD risk, even when excluding the influence of TGF-ß signaling pathway genes, suggesting that ADAM17 is an important KD susceptibility-related genetic locus. • The more significant compound effects of two-locus models, combining single nucleotide polymorphisms (SNPs) in ADAM17 and other TGF-ß signaling pathway genes including TGFB2 and SMAD3, on KD phenotypes relative to single SNPs suggest that ADAM17 is also involved in secondary CAL formation and confers the risk of KD/CALs via the TGF-ß/SMAD3 signaling pathway.