Investigation on the mechanisms of human sperm DNA damage based on the proteomics analysis by SWATH-MS.

BACKGROUND: Spermatozoa have the task of delivering an intact paternal genome to the oocyte and supporting successful embryo development. The detection of sperm DNA fragmentation (SDF) has been emerging as a complementary test to conventional semen analysis for male infertility evaluation, but the mechanism leading to SDF and its impact on assisted reproduction remain unclear. Therefore, the study identified and analyzed the differentially expressed proteins of sperm with high and low SDF. METHODS: Semen samples from men attended the infertility clinic during June 2020 and August 2020 were analyzed, and sperm DNA fragmentation index (DFI) was detected by the sperm chromatin structure assay. Semen samples with low DFI (< 30%, control group) and high DFI (≥ 30%, experimental group) were optimized by density gradient centrifugation (DGC), and the differentially expressed proteins of obtained sperm were identified by the Sequential Window Acquisition of All Theoretical Mass Spectra Mass Spectrometry (SWATH-MS) and performed GO and KEGG analysis. RESULTS: A total of 2186 proteins were identified and 1591 proteins were quantified, of which 252 proteins were identified as differentially expressed proteins, including 124 upregulated and 128 downregulated. These differentially expressed proteins were involved in metabolic pathways, replication/recombination/repair, acrosomal vesicles, kinase regulators, fertilization, tyrosine metabolism, etc. Western blotting results showed that the expression levels of RAD23B and DFFA proteins and the levels of posttranslational ubiquitination and acetylation modifications in the experimental group were significantly higher than those in the control group, which was consistent with the results of proteomics analysis. CONCLUSIONS: Proteomic markers of sperm with high DNA fragmentation can be identified by the SWATH-MS and bioinformatic analysis, and new protein markers and posttranslational modifications related to sperm DNA damage are expected to be intensively explored. Our findings may improve our understanding of the basic molecular mechanism of sperm DNA damage.

Investigation of the mechanisms leading to human sperm DNA damage based on transcriptome analysis by RNA-seq techniques.

RESEARCH QUESTION: What are the molecular mechanisms leading to human sperm DNA damage? DESIGN: Semen samples were collected and the sperm DNA fragmentation index (DFI) was assessed. Differentially expressed RNA in spermatozoa with a high (DFI ≥30%, experimental group) or normal (DFI <30%, control group) DFI were identified by RNA-sequencing (RNA-seq) technology, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed. Three differentially expressed RNA related to sperm DNA damage and repair, namely PMS1, TP53BP1 and TLK2, were validated using real-time quantitative (RT-qPCR). RESULTS: A total of 19,970 expressed RNA were detected in the two groups. Compared with the control group, the expression levels of 189 RNA in the experimental group were significantly increased and those of 163 genes decreased. Gene Ontology enrichment analysis showed that these RNA were mainly concentrated in the ATPase-dependent transmembrane transport complex, extracellular exosome, somatic cell DNA recombination, protein binding, cytoplasm and regulation of localization. KEGG pathway analysis showed that these RNA were mainly related to the PI3K-Akt signalling pathway, endocytosis, p53 signalling pathway and cGMP-PKG signalling pathway. The RT-qPCR results showed that the expression levels of PMS1, TP53BP1 and TLK2 in the experimental group were significantly lower than in the control group (P = 0.01, 0.015 and 0.004, respectively), which was identical to the results of RNA sequencing. CONCLUSIONS: Differentially expressed RNA related to sperm DNA damage and repair may be identified by RNA-seq technology, which provides new insights into the understanding of sperm DNA damage and repair, and will help to discover new biomarkers related to sperm DNA damage.

[Clinical observation of dapoxetine combined with percutaneous neuromuscular electrical stimulation in the treatment of primary premature ejaculation].

OBJECTIVE: To investigate the clinical efficacy of dapoxetine combined with transcutaneous neuromuscular electrical stimulation (TNES) in the treatment of primary premature ejaculation. METHODS: A total of 60 patients who met the diagnostic criteria for primary premature ejaculation were selected as study subjects and randomly divided into a dapoxetine group (control group) and a dapoxetine combined with percutaneous neuromuscular electrical stimulation group (observation group).30 patients in each group were treated for 4 weeks. Intravaginal ejaculatory latency time (IELT), the score of Premature Ejaculation Diagnostic Tool (PEDT), sympathetic skin response located in the penis (PSSR), Patient Health Questionnaire (PHQ-9), and Generalized Anxiety Disorder Questionnaire (GAD-7) before and after treatment were recorded in the two groups. Before and after treatment, the difference in observed indexes in the two groups and the comparison of effective rates between the two groups were analyzed. RESULTS: The latency of IELT and PSSR was prolonged and the PEDT score was decreased in both the observation group and the control group, the difference was statistically significant (P<0.01). Compared with the control group, the observation group had statistically significant differences in extending IELT and PSSR latency and reducing PEDT score (P<0.05). The effective rates of the observation group and control group were 90% and 63.33%, respectively, and the difference was statistically significant (P<0.05). There was no significant difference in the improvement of depression and anxiety levels between the two groups (P> 0.05). CONCLUSION: Dapoxetine combined with TNES has a better clinical effect than dapoxetine alone in the treatment of primary premature ejaculation, and can be used as an effective option for clinical treatment of primary premature ejaculation.

[Transcutaneous neuromuscular electrical stimulation for the treatment of erectile dysfunction: A clinical observation].

OBJECTIVE: To observe the clinical efficacy of transcutaneous neuromuscular electrical stimulation (TNES) in the treatment of ED. METHODS: According to the inclusion and exclusion criteria, we included and studied 25 cases of ED treated by TNES in Northern Jiangsu People's Hospital from June 2021 to February 2022 using the self-matched pre- and post-control method. Before and after treatment, we conducted RigiScan penile hardness test under audiovisual sexual stimulation (AVSS) for all the patients and obtained their scores on the Erectile Hardness Scale (EHS), IIEF-5, Premature Ejaculation Diagnostic Tool (PEDT), Patient Health Questionnaire 9 (PHQ-9) and Generalized Anxiety Disorder Questionnaire 7 (GAD-7). RESULTS: No adverse reactions such as pain, allergy, skin burn, and subcutaneous congestion were observed in any of the patients. There were statistically significant differences after treatment in erection time, average and maximum hardness of the penile tip, mean hardness of the penile root, and circumference of the penile tip (P < 0.05), but not in the circumference and maximum hardness of the penile root during erection compared with the baseline (P > 0.05). Significant improvement was observed after treatment in the IIEF-5 score (P < 0.05), with a total effectiveness rate of 68%, as well as in the PEDT score (P < 0.05) GAD-7 anxiety score (P < 0.05), but not in the PHQ-9 depression score (P > 0.05). CONCLUSION: TNES, as a safe and non-invasive therapy, can improve penile hardness under AVSS and the erectile function and anxiety symptoms of ED patients, and can be used as a new option for the treatment of ED.

Relationship of inflammatory indices with left atrial appendage thrombus or spontaneous echo contrast in patients with atrial fibrillation.

BACKGROUND: Inflammatory indices derived from complete blood tests have been reported to be associated with poor outcomes in patients with atrial fibrillation (AF). The data about the relationship between inflammatory indices and left atrial appendage thrombus (LAAT) or dense spontaneous echo contrast (SEC) are limited. AIM: To explore the value of inflammatory indices for predicting the presence of LAAT or dense SEC in nonvalvular AF patients. METHODS: A total of 406 patients with nonvalvular AF who underwent transesophageal echocardiography were included and divided into two groups based on the presence (study group) or absence (control group) of LAAT or dense SEC. Inflammatory indices, including the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR), were calculated from complete blood analysis. The associations of inflammatory indices with LAAT/dense SEC were analyzed using logistic regression. RESULTS: LAAT and dense SEC were detected in 11 (2.7%) and 42 (10.3%) patients, respectively. The PLR only showed an association with LAAT/dense SEC in the univariate model. Elevated NLR (odds ratio [OR] = 1.48, 95% confidence interval [CI]: 1.11-1.98, P = 0.007) and reduced LMR (OR = 0.59, 95%CI: 0.41-0.83, P = 0.003) were found to be independent risk factors for the presence of LAAT/dense SEC. The areas under the NLR and LMR curves for predicting LAAT/dense SEC were 0.73 (95%CI: 0.66-0.80, P < 0.001) and 0.73 (95%CI: 0.65-0.81, P < 0.001), respectively, while the cutoff values were 2.8 (sensitivity: 69.8%; specificity: 64.0%) and 2.4 (sensitivity: 71.7%; specificity: 60.6%), respectively. CONCLUSION: Increased NLR and decreased LMR may predict LAAT/dense SEC in patients with nonvalvular AF.

Levels of trace metals and their impact on oocyte: A review.

Trace metals play a vital role in a variety of biological processes, but excessive amounts can be toxic and are receiving increasing attention. Trace metals in the environment are released from natural sources, such as rock weathering, volcanic eruptions, and other human activities, such as industrial emissions, mineral extraction, and vehicle exhaust. Lifestyle, dietary habits and environmental quality are the main sources of human exposure to trace metals, which play an important role in inducing human reproductive infertility. The purpose of this review is to summarize the distribution of various trace metals in oocyte and to identify the trace metals that may cause oocyte used in the design and execution of toxicological studies.

In vivo anti-melanoma efficacy of allo-restricted CTLs specific for melanoma expanded by artificial antigen-presenting cells.

Cytotoxic CD8(+) T cells are key effectors in the immunotherapy of malignant and viral diseases. However, autologous T cell responses to tumor antigens presented by self-MHC are usually weak and ineffective. Allo-restricted T cells represent a potent source of tumor-specific T cells for adoptive immunotherapy. This study reports in vivo anti-melanoma efficacy of the pTRP2-specific allo-restricted CTLs expanded from the BALB/c splenocytes by multiple stimulations with aAPCs made by coating H-2K(b)-Ig/pTRP2 dimeric complexes, anti-CD28 antibody, 4-1BBL molecules and CD83 molecules to cell-sized latex beads. The induced allo-restricted CTLs exhibited specific lysis against RMA-S cells pulsed with the peptide pTRP2 and H-2K(b+) melanoma cells expressing TRP2, while a murine Lewis lung carcinoma cell line 3LL could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-H-2K(b) monoclonal antibody Y3. Adoptive transfer of the allo-restricted CTLs specific for malignant melanoma expanded by the aAPCs can mediate effective anti-melanoma response in vivo. These results suggested that the specific allo-restricted CTLs expanded by aAPCs coated with an MHC-Ig/peptide complex, anti-CD28 antibody, 4-1BBL and CD83 could be a potential option of specific immunotherapy for patients with malignant melanoma.

Shear wave elastography and contrast-enhanced ultrasonography in the diagnosis of thyroid malignant nodules.

The aim of the present study was to evaluate the value of shear wave elastography (SWE) and contrast-enhanced ultrasonography (CEUS) in the diagnosis of thyroid malignant nodules. A total of 253 patients with 319 thyroid nodules were subjected to two-dimensional ultrasound (2DUS) and CEUS examinations prior to thyroidectomy between March, 2014 and December, 2015. Young's modulus value for each nodule on 2DUS and CEUS images were recorded. The sensitivity, specificity and accuracy of 2DUS, SWE and CEUS in the diagnosis of thyroid malignant nodules were assessed. The results demonstrated that, of the 319 nodules that were pathologically confirmed, 183 were malignant and 136 were benign. The area under the receiver operating characteristic curve as a result of SWE diagnosis was 0.77. When the threshold of the Young's modulus value was ≥27.65 kPa in the diagnosis of malignant thyroid nodules, SWE exhibited a sensitivity of 84.55% (115/136), a specificity of 84.15% (154/183) and an accuracy of 84.32% (269/319). US contrast imaging of malignant thyroid nodules revealed a major tendency for early hypoenhancement and hypoenhancement. CEUS exhibited a sensitivity of 87.5% (119/136), a specificity of 86.33% (158/183) and an accuracy of 86.83% (277/319) in the diagnosis of malignant thyroid nodules. Compared with 2DUS, SWE, CEUS and their combined use exhibited statistically significant differences in the diagnosis of thyroid malignant nodules in terms of sensitivity, specificity and accuracy (χ2=9.220,15.310 and 40.296, respectively; P=0.000); SWE or CEUS did not differ significantly in the diagnosis of thyroid malignant nodules in terms of sensitivity, specificity or accuracy (χ2=0.737;P=0.542); Compared with the use of SWE or CEUS alone, their combination exhibited statistically significant differences in the diagnosis of malignant thyroid nodules in terms of sensitivity, specificity and accuracy (χ2=12.264 and 6.939, respectively; P=0.000,0.005). In conclusion, the high accuracy of the combined use of SWE and CEUS in the diagnosis of malignant thyroid nodules is of great clinical value.

Mastermind-like Transcriptional Coactivator 1 Overexpression Predicts Poor Prognosis in Human with Hepatocellular Carcinoma.

BACKGROUND: Mastermind-like transcriptional coactivator 1(MAML1) is a transcriptional coregulator of activators in various signaling pathways. High MAML1 was associated with tumorigenesis, progression, and aggressiveness in various tumors. The role of MAML in Hepatocellular Carcinoma (HCC), however, has not been directly addressed. The present study was to determine its association with clinicopathological characteristics and prognosis of HCC patients. MATERIALS AND METHODS: MAML1 expression at protein level in human HCC and normal liver tissues was detected by immunohistochemistry analysis, which was further validated by high-throughput sequencing data TCGA dataset at mRNA level. Then, the association of MAML1 expression with clinicopathological features of HCC patients was statistically analyzed. RESULTS: Immunohistochemistry analysis found that MAML1 expression was significantly increased in HCC tissues compared with those in normal tissues (P=0.005). High MAML1 was dramatically associated with advanced clinical stage (P=0.019) and enhanced tumor invasion (P=0.019). The TCGA mRNA expression data showed that MAML1 was upregulated in HCC with young age (P=0.005). Kaplan-Meier survival curves revealed that HCC patients with high MAML1 levels had shorter survival (P=0.040). Furthermore, high MAML1 expression was an independent prognostic factor for HCC patients (HR 1.841, 95% CI 1.045-3.243; P=0.035). CONCLUSIONS: Our data suggests that MAML1 may play an important role in tumor progression of HCC. The increased expression of MAML1 may efficiently predict poor overall survival in HCC patients, and it may be a potential prognostic marker of this malignancy.

Immobilizing osteogenic growth peptide with and without fibronectin on a titanium surface: effects of loading methods on mesenchymal stem cell differentiation.

In this study, to improve the osseointegration of implants, osteogenic growth peptide (OGP) and fibronectin (FN) were loaded within mineral, which was formed on titanium, through adsorption and coprecipitation methods. The release profiles of OGP loaded by either adsorption or coprecipitation and the effects of the loading methods to immobilize OGP with and without FN on rat mesenchymal stem cell (rMSC) osteogenic differentiation were studied. The coprecipitation approach slightly reduced the initial burst release, while the adsorption approach provided a more sustained release. Dual loading of OGP and FN further improved cell attachments compared with either OGP or FN alone. Dually loaded OGP and FN also had a positive impact on rMSC proliferation and osteogenic differentiation. The difference in methods of loading OGP with and without FN also had some effects on osteogenic differentiation. Compared with coprecipitated OGP alone, adsorbed OGP enhanced later differentiation, such as osteocalcin secretion and matrix mineralization. Simultaneously adsorbed OGP and FN led to higher proliferation and higher osteogenic differentiation in both early and late stages compared with sequentially loaded OGP and FN. rMSC culture clearly indicated that simultaneously adsorbed OGP and FN could improve osseointegration, and this treatment represents a potential method for effective surface modification of dental and orthopedic implants.

Biomimetic apatite formation on calcium phosphate-coated titanium in Dulbecco's phosphate-buffered saline solution containing CaCl(2) with and without fibronectin.

Calcium phosphate (CaP) thin films with different degrees of crystallinity were coated on the surfaces of commercially pure titanium by electron beam evaporation. The details of apatite nucleation and growth on the coating layer were investigated in Dulbecco's phosphate-buffered saline solutions containing calcium chloride (DPBS) or DPBS with fibronectin (DPBSF). The surfaces of the samples were examined by field emission scanning electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. The concentrations of fibronectin and calcium ions (Ca(2+)) were monitored by the bicinchoninic acid method (BCA) and use of a calcium assay kit (DICA-500), respectively. Apatite initially formed at the fastest rate on the CaP-coated samples with the lowest degree of crystallinity and reached the maximum Ca(2+) concentration after immersion in DPBS solution for 15min. After 15min the concentration of Ca(2+) decreased with the growth of apatite on the coating layers. For all the samples the maximum Ca(2+) concentration in the DPBS solutions decreased with increasing crystallinity and immersion time to reach the maximum concentration increased. The presence of fibronectin in the DPBS solutions delayed the formation and affected the morphology of the apatite. Fibronectin incorporated into apatite deposited on the surface of titanium did not affect its biological activity in terms of promoting osteoblast adhesion.

The enhanced anti-angiogenic and antitumor effects of combining flk1-based DNA vaccine and IP-10.

The purpose of the present study was to evaluate the anti-vasculature effects and the anti-tumor effects of attenuated Salmonella typhimurium vaccine strain encoding murine vascular endothelial growth factor (VEGF) receptor-2 (flk1) in combination with plasmid DNA vector encoding the murine interferon-induced protein of 10kDa (IP-10 or CXCL10) gene. Mouse models of malignant melanoma (B16-F10) were treated with combining orally given attenuated S. typhimurium vaccine strain encoding flk1 with direct intratumoral injection of a non-viral plasmid DNA vector encoding the murine IP-10 gene. The volumes of tumors and survival of mice bearing B16-F10 tumors were observed. Cytolytic T lymphocyte (CTL) response was measured by cytotoxic assay, vessel density and tumor-cell proliferation were observed by immunostaining, and tumor apoptosis was determined by TUNEL staining. The results revealed the combination therapy groups showed more significantly inhibited tumor growth, apoptosis of tumor cells, and reduced neovascularization, cell proliferation, and developed a strong CTL response in these mice. In summary, the therapy of attenuated S. typhimurium vaccine strain encoding flk1 combined with the IP-10 gene has significant synergistic effect against tumors.