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Purpose The overall response of cisplatin-based chemotherapy in bladder urothelial carcinoma (BUC) remains unsatisfactory due to the complex pathological subtypes, genomic difference, and drug resistance. The genes that associated with cisplatin resistance remain unclear. Herein, we aimed to identify the cisplatin resistance associated genes in BUC. Experimental design The cytotoxicity of cisplatin was evaluated in six bladder cancer cell lines to compare their responses to cisplatin. The T24 cancer cells exhibited the lowest sensitivity to cisplatin and was therefore selected to explore the mechanisms of drug resistance. We performed genome-wide CRISPR screening in T24 cancer cells in vitro, and identified that the gene heterogeneous nuclear ribonucleoprotein U (HNRNPU) was the top candidate gene related to cisplatin resistance. Epigenetic and transcriptional profiles of HNRNPU-depleted cells after cisplatin treatment were analyzed to investigate the relationship between HNRNPU and cisplatin resistance. In vivo experiments were also performed to demonstrate the function of HNRNPU depletion in cisplatin sensitivity. Results Significant correlation was found between HNRNPU expression level and sensitivity to cisplatin in bladder cancer cell lines. In the high HNRNPU expressing T24 cancer cells, knockout of HNRNPU inhibited cell proliferation, invasion, and migration. In addition, loss of HNRNPU promoted apoptosis and S-phase arrest in the T24 cells treated with cisplatin. Data from The Cancer Genome Atlas (TCGA) demonstrated that HNRNPU expression was significantly higher in tumor tissues than in normal tissues. High HNRNPU level was negatively correlated with patient survival. Transcriptomic profiling analysis showed that knockout of HNRNPU enhanced cisplatin sensitivity by regulating DNA damage repair genes. Furthermore, it was found that HNRNPU regulates chemosensitivity by affecting the expression of neurofibromin 1 (NF1). Conclusions Our study demonstrated that HNRNPU expression is associated with cisplatin sensitivity in bladder urothelial carcinoma cells. Inhibition of HNRNPU could be a potential therapy for cisplatin-resistant bladder cancer.

Background: Urinary bladder cancer is one of the most common malignancies of the urinary tract; however, the mechanism of its high recurrence and responses to immunotherapy remains unclear, resulting in difficulties in clinical outcome prediction. Epigenetic alterations, especially DNA methylation, play important roles in bladder cancer development and are increasingly investigated as biomarkers for diagnostic or prognostic predictions. However, little is known regarding hydroxymethylation since previous studies based on bisulfite-sequencing approaches could not differentiate between 5mC and 5hmC signals, resulting in entangled methylation results. Methods: Here, we used a multi-omics approach to provide the genome, transcriptome, methylome, and hydroxymethylome landscape of both primary and recurrent bladder cancers. Results: By whole exome sequencing, we identified driver mutations that are involved in the UBC development, such as FGFR3, KDMTA and KDMT2C. However, few of these driver mutations are associated with the recurrence or the PD-L1 down-regulation in UBC. By integrating RRBS and oxRRBS-seq data, we identified fatty acid oxidation-related genes significantly enriched in 5hmC-associated transcription alterations in recurrent bladder cancers. We also observed a serial of 5mC hypomethylation DMRs in the gene body of NFATC1, a gene that is highly involved in the T-cell immune response, in bladder cancer samples with high expression of PD-L1. And since 5mC and 5hmC alternations are globally anti-correlated, RRBS-seq based markers which combine the 5mC and 5hmC signals, attenuate the cancer-related signals and therefore are not optimal to be used as clinical biomarkers. Conclusions: By multi-omics profiling of UBC samples, we showed that epigenetic alternations are more involved than genetic mutations in the recurrence and PD-L1 regulation of UBC. In addition, as a proof of principle, we demonstrated that the combined measurement of 5mC and 5hmC level by bisulfite-based method will compromise the prediction accuracy of epigenetic biomarkers.

Despite N6-methyladenosine (m6A) is functionally important in various biological processes, its role and the underlying regulatory mechanism in the liver remain largely unexplored. In the present study, we showed that fat mass and obesity-associated protein (FTO, an m6A demethylase) was involved in mitochondrial function during hepatic ischemia–reperfusion injury (HIRI). We found that the expression of m6A demethylase FTO was decreased during HIRI. In contrast, the level of m6A methylated RNA was enhanced. Adeno-associated virus-mediated liver-specific overexpression of FTO (AAV8-TBG-FTO) ameliorated the HIRI, repressed the elevated level of m6A methylated RNA, and alleviated liver oxidative stress and mitochondrial fragmentation in vivo and in vitro. Moreover, dynamin-related protein 1 (Drp1) was a downstream target of FTO in the progression of HIRI. FTO contributed to the hepatic protective effect via demethylating the mRNA of Drp1 and impairing the Drp1-mediated mitochondrial fragmentation. Collectively, our findings demonstrated the functional importance of FTO-dependent hepatic m6A methylation during HIRI and provided valuable insights into the therapeutic mechanisms of FTO.

Solasonine, a steroidal alkaloid extracted from Solanum nigrum L., has been found to exert inhibitory effect on cancers. However, the underlying anticancer mechanisms of solasonine, particularly in urinary bladder cancer (BC), remain unclear. In this study, we identified the potential targets and biological functions associated with solasonine activity using a bioinformatics approach. Ingenuity pathway analysis revealed that neuropilin-1 (NRP1) and other signaling pathways, such as PI3K/AKT and ERK/MAPK pathways, were potentially involved in the therapeutic effects of solasonine. The ability of solasonine in inducing apoptosis and inhibiting proliferation in BC cells was confirmed experimentally, and the inhibition of ERK/MAPK, P38/MAPK, and PI3K/AKT pathways was validated by Western blot. Mechanistically, solasonine suppressed the expression of NRP1 protein, but not that of mRNA. Further results of molecular docking and molecular dynamics simulation analysis indicated that solasonine could directly bind to the b1 domain of NRP1 protein with a reasonable and stable docking conformation. We previously found that targeting NRP1 is a potential antitumor strategy. Combined with these findings, it can be speculated that the binding of solasonine with NRP1 on the cell membrane could prevent the formation of NRP1/VEGFA/VEGFR2 and NRP1/EGFR complexes, resulting in the inhibition of downstream signaling, including ERK/MAPK, P38/MAPK, and PI3K/AKT pathways. Additionally, intracellular solasonine could inhibit the membrane localization of NRP1 and provoke its cytoplasmic retention, facilitating the degradation of NRP1 protein in the cytoplasm. The dual effects induced by the binding of solasonine to NRP1 extracellularly and intracellularly could account for the antiproliferative and proapoptotic effects of solasonine on BC.

To investigate the effect of corosolic acid treatment on erectile function in metabolic syndrome induced rat model. Fifty male 3-week-old SD rats were fed a high fat and high sugar diet. Six months later, metabolic variables were determined. Metabolic syndrome induced erectile function (MED) rats were confirmed by an apomorphine test. Then MED rats were treated with corosolic acid daily by oral gavage for 4 weeks. To evaluate erectile function, intracavernosal pressure (ICP)/mean arterial blood pressure (MAP) ratio was measured. Thiobarbituric acid reactive substances assay and dihydroethidium staining were used to assess reactive oxygen species (ROS) level. Protein expressions of gp91phox and eNOS were examined by western blotting and immunohistochemistry. Fasting blood glucose, body weight, total cholesterol and insulin were markedly increased in metabolic syndrome rats compared with those of the control rats (p < 0.05). The ratios of max ICP/MAP and area under curve (AUC)/MAP was markedly reduced in MED rats compared with the control rats (p < 0.05). The concentration of cyclic guanosine mono-phosphate (cGMP) and the expression of eNOS were significantly decreased in MED rats compared with the control group (p < 0.05). Moreover, ROS level and the expression of gp91phox were significantly increased in MED rats. Treatment with corosolic acid reversed these changes (each p < 0.05). Corosolic acid reduces the level of ROS, ameliorating endothelial dysfunction and improvement of erectile function in MED rats.

BACKGROUND: Melatonin is a hormone naturally produced by the pineal gland in the brain. In addition to modulating circadian rhythms, it has pleiotropic biological effects including antioxidant, immunomodulatory, and anti-cancer effects. Herein, we report that melatonin has the ability to decrease the growth and metastasis of androgen-dependent prostate cancer. METHODS: To evaluate the anti-cancer effect of melatonin on androgen-sensitive prostate cancer in vitro or in vivo, the effects of cell proliferation, apoptosis, migration and invasion were analyzed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, flow cytometry, Transwell assay, and immunohistochemistry (IHC), respectively. Next, the interaction between androgen receptor (AR) and SUMO specific protease 1 (SENP1) was detected by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting, and confirmed by luciferase reporter assay. Furthermore, the Small Ubiquitin-like Modifier (SUMO) proteins are a group of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. (SUMOylation) of histone deacetylases 1 (HDAC1) was measured by proximity ligation assay (PLA). RESULTS: The treatment of melatonin cripples the transcriptional activity of AR, which is essential for the growth of the androgen-dependent prostate cancer cell, LNCaP. The lower activity of AR was dependent on melatonin induced SUMOylation of HDAC1, which has been established as a key factor for the transcriptional activity of AR. Mechanistically, the effect of melatonin on AR was due to the decreased SENP1 protein level and the subsequent increased HDAC1 SUMOylation level. The overexpression of SENP1 abrogated the anti-cancer ability of melatonin on LNCaP cells. CONCLUSIONS: These findings indicate that melatonin is a suppressor of androgen-dependent prostate cancer tumorigenesis.

Background Clear cell renal cell carcinoma (ccRCC) is one of the most lethal urological malignancies, but the pathogenesis and prognosis of ccRCC remain obscure, which need to be better understand. Methods Differentially expressed genes were identified and function enrichment analyses were performed using three publicly available ccRCC gene expression profiles downloaded from the Gene Expression Omnibus database. The protein-protein interaction and the competing endogenous RNA (ceRNA) networks were visualized by Cytoscape. Multivariate Cox analysis was used to predict an optimal risk mode, and the survival analysis was performed with the Kaplan-Meier curve and log-rank test. Protein expression data were downloaded from Clinical Proteomic Tumor Analysis Consortium database and Human Protein Atlas database, and the clinical information as well as the corresponding lncRNA and miRNA expression data were obtained via The Cancer Genome Atlas database. The co-expressed genes and potential function of candidate genes were explored using data exacted from the Cancer Cell Line Encyclopedia database. Results Of the 1044 differentially expressed genes shared across the three datasets, 461 were upregulated, and 583 were downregulated, which significantly enriched in multiple immunoregulatory-related biological process and tumor-associated pathways, such as HIF-1, PI3K-AKT, P53 and Rap1 signaling pathways. In the most significant module, 36 hub genes were identified and were predominantly enriched in inflammatory response and immune and biotic stimulus pathways. Survival analysis and validation of the hub genes at the mRNA and protein expression levels suggested that these genes, particularly complement component 3 (C3) and fibronectin 1 (FN1), were primarily responsible for ccRCC tumorigenesis and progression. Increased expression of C3 or FN1 was also associated with advanced clinical stage, high pathological grade, and poor survival in patients with ccRCC. Univariate and multivariate Cox regression analysis qualified the expression levels of the two genes as candidate biomarkers for predicting poor survival. FN1 was potentially regulated by miR-429, miR-216b and miR-217, and constructed a bridge to C3 and C3AR1 in the ceRNA network, indicating a critical position of FN1. Conclusions The biomarkers C3 and FN1 could provide theoretical support for the development of a novel prognostic tool to advance ccRCC diagnosis and targeted therapy.

Objective To explore the significance of the prostate central gland to total gland volume ratio (PVc/PV) in the diagnosis of prostate cancer (PCa) in patients with prostate specific antigen (PSA) levels in the grey zone (4–10 ng/ml). Methods This retrospective study enrolled patients that had undergone prostate biopsy. The volume of the prostate and the central prostate gland were measured. The differences in PSA, the ratio of free to total PSA (f/tPSA), PSA density (PSAD) and PVc/PV between the PCa and non-PCa groups were compared. Receiver operating characteristic curve analysis for PCa and clinically significant PCa (csPCa) diagnosis were calculated according to PSA (reference), f/tPSA, PSAD and PVc/PV. Results This study enrolled 136 patients. There was no significant difference in PSA and f/tPSA between the PCa and non-PCa groups, while there were significant differences in PSAD and PVc/PV. The area under the curve values of PVc/PV for PCa or csPCa diagnosis were 0.876 and 0.933, respectively; and for PSAD, they were 0.705 and 0.790, respectively. These were significantly different compared with the PSA curve, whereas f/tPSA showed no significant difference from the PSA curve. Conclusion PVc/PV could be a predictor of PCa when PSA is between 4–10 ng/ml.

Renal ischemia-reperfusion injury (IRI) is one of the most common causes of acute kidney injury (AKI), which is closely related to high morbidity and mortality. However, the pathogenesis underlying renal IRI is complex and not fully defined. N6-methyladenosine (m6A) was recently found to be an abundant modification in mammalian messenger RNAs. It is implicated in various biological processes, while the role of m6A in IRI is not illustrated. Here we show that the m6A-methylated RNA level and its methyltransferase METTL14 are elevated in human AKI renal tissues and IRI HK-2 cells. Moreover, METTL14 knockdown protects the kidney against IRI in vitro and in vivo. Mechanistically, we identified that YAP1 is a direct target of METTL14 in AKI progression. Inhibition of YAP1-TEAD signaling by peptide 17 abrogates the protective effect of METTL14 against IRI in vitro and in vivo. Taken together, these results reveal that the N6-methyladenosine mRNA methylase METTL14 promotes the renal IRI via suppressing YAP1. The discovery of the METTL14-YAP1 pathway provides an important new perspective for understanding AKI and is conducive to revealing new therapeutic strategies and targets.

Prostate cancer (PCa) is one of the major cancers affecting males with high mortality around the world. Recent studies have found that some long noncoding RNAs play a critical part in the cellular processes of PCa. In our study, aberrant expressed lymphoid enhancer-binding factor-1 antisense RNA 1 (LEF1-AS1), microRNA-330-5p (miR-330-5p), and lymphoid enhancer-binding factor-1 (LEF1) were screened out from a microarray database, the role of the novel noncoding RNA regulatory circuitry in the initiation and development of PCa was investigated. LEF1-AS1 and LEF1 were highly expressed while miR-330-5p was poorly expressed in PCa. Following that, the PCa PC-3 cell line was adopted for subsequently experiments, in which the expression of LEF1-AS1 and miR-330-5p was subsequently altered by means of exogenous transfection. After that, the effects of up- or downregulation of LEF1-AS1 and miR-330-5p on epithelial-mesenchymal transition (EMT) and the cell ability for proliferation, invasion, migration in vitro, and tumorigenesis and lymph node metastasis (LNM) in vivo were evaluated. RNA crosstalk revealed that LEF1-AS1 bound to miR-330-5p and LEF1 was the target gene of miR-330-5p. Silenced LEF1-AS1 or elevated miR-330-5p exhibited inhibited EMT processes, reduced ability of proliferation, invasion and migration, coupling with decreased tumorigenesis and LNM in nude mice. The key findings of this study collectively propose downregulation of LEF1-AS1 competing with miR-330-5p to inhibit EMT, invasion and migration of PCa by LEF1 repression.

Objective: To explore the significance of the prostate central gland to total gland volume ratio in the diagnosis of PSA 4-10ng/ml prostate cancer (PCa) patients. Method: A retrospective analysis was performed on patients who had undergone prostate biopsy in our hospital from July 2015 to December 2020.The anteroposterior, transverse and axial diameters of the prostate and the central prostate gland were measured using multiparametric magnetic resonance imaging (mpMRI). The differences in PSA, f/tPSA, PSAD and PVc/PV between the PCa group and the non-PCa group were compared. ROC curves for PCa and clinically significant PCa (csPCa) diagnosis were drawn according to PSA, f/tPSA, PSAD and PVc/PV respectively. Corresponding PSA was used as the reference standard for comparison.Results: There was no statistically significant difference in PSA and f/tPSA between the two groups (P>0.05), while there were statistically significant differences in PSAD and PVc/PV between the two groups (PP0.05).Conclusion: Low volume ratio of central prostate gland PVc/PV has a higher incidence of PCa and csPCa, which can be used as an important reference index for the diagnosis of PCa in PSA 4-10 ng/ml patients.

The supplementary materials of article "Bioinformatics-based identification of critical biomarkers for pathogenesis and prognosis of non–muscle-invasive bladder cance".Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Normalisation of gene expression, Figure S2: DElncRNAs and DEmiRNAs in patients with NMIBC, Table S1: The clinical information of the samples in each dataset, Table S2: Information of the integrated DEGs, Table S3: GO terms enrichment analysis of DEGs, Table S4: GO terms analysis of hub genes, Table S5: KEGG enrichment of genes in the hub modules, Table S6: Genes correlated with CDH1 based on CCLE database, Table S7: Representative signalling pathways enriched in phenotype of high and low expression of CDH1 in BC cell lines, Table S8: The miRNAs related to hub genes, Table S9: The miRNAs paired with DElncRNAs and hub genes.

Additional file 1: Table A1. Thirty-nine components of Solanum nigrum and their corresponding molecular weight (MW), predicted oral bioavailability (OB) and drug likeness (DL) scores. Table A2. Bladder cancer (BC) related targets in Solanum nigrum. By combining the related targets of active ingredients screened from S. nigrum and the disease-related targets, 100 overlapping targets (involving 23 duplicates) were selected as the key targets involved in the treatment of BC. Table A3. Gene ontology (GO) terms of therapeutic target genes and their corresponding counts, p-value, and FDR. Through the GO enrichment of the key targets, 86 remarkably enriched (p-value ≤0.01) GO terms were obtained, indicating that some targets were involved in tumorigenesis. Table A4. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of therapeutic target genes and their corresponding counts, p-value, and FDR. Through KEGG enrichment of the key targets, 89 remarkably enriched (p-value ≤0.01) pathways were obtained, indicating that numerous targets are involved in the processes of tumorigenesis and tumor progression.

To gain a deeper insight into the local anatomic structures of the seminal vesicle and ejaculatory duct and provide some anatomic guidance in seminal vesiculoscopy.We analyzed the clinical data on 48 cases of seminal vesiculoscopy and recorded the surgical approaches to the seminal vesicle and clinical effects after operation. At the same time, we made an anatomic study of the seminal vesicle, ejaculatory duct and prostatic utricle and simulated the surgical approaches in 12 adult pelvis specimens.The anatomical results accorded well with the surgical findings. The ejaculatory ducts failed to be revealed for 52.1%（25/48）, but 93.8%(45/48) of the operations were completed through different approaches into the seminal vesicle.Transurethral seminal vesiculoscopy can be applied in the treatment of seminal tract and seminal vesicle diseases, but has its obvious limitations and has to be used with other strategies to achieve better results.

In the present study, the clinical effects of flexible ureteroscopy lithotripsy (FURL) and percutaneous nephrolithotomy (PCNL) for the treatment of kidney stones of ≤2 cm was studied. Seventy-two patients with kidney stones were randomly divided into the FURL group (n=39) under ureteroscope lithotripsy with holmium laser and PCNL group (n=33) under PCNL with holmium laser and compared their clinical effects. At 3 months after the operation, the stone removal rate of the FURL group was significantly higher than that of the PCNL group. The subgroup analysis revealed that the difference in the lower kidney calyx was more obvious (P0.05). The incidence of complications of the FURL group was significantly lower than that of the PCNL group (P0.05). Additionally, 3 and 7 days after operation, the cystatin C levels of the FURL group were significantly higher than those of the PCNL group, and the KIM-1 levels were significantly lower than the PCNL group (P

We aim to investigate the correlations between hemodynamic parameters, penile rigidity grading, and the therapeutic effects of phosphodiesterase type 5 inhibitors using color Doppler flow imaging after intracavernosal injection in patients with erectile dysfunction. This study involved 164 patients. After intracavernosal injection with a mixture of papaverine (60 mg), prostaglandin E1 (10 μg), and lidocaine (2%, 0.5–1 ml), the penile vessels were assessed using color Doppler flow imaging. Penile rigidity was classified based on the Erection Hardness Score system as Grades 4, 3, 2 or 1 (corresponding to Schramek Grades V to II). Then, the patients were given oral sildenafil (50–100 mg) and scored according to the International Index of Erectile Function (IIEF-5) questionnaire. The number of patients with penile rigidities of Schramek Grades II to V was 14, 18, 21, and 111, respectively. The IIEF-5 score was positively correlated with the refilling index of the penile cavernosal artery (r = 0.79, P < 0.05), the peak systolic velocity (r = 0.45, P < 0.05), and penile rigidity (r = 0.75, P < 0.05), and was negatively correlated with the end diastolic velocity (r = −0.74, P < 0.05). For patients with erectile dysfunction, both the IIEF-5 score after sildenafil administration, which is correlated with penile rigidity, and the hemodynamic parameters detected using color Doppler flow imaging may predict the effects of phosphodiesterase type 5 inhibitor treatment and could provide a reasonable model for the targeted-treatment of erectile dysfunction.

To investigate the clinical effect of plaque excision plus autologous perididymal patch grafting in the treatment of Peyronie's disease.This study included 10 patients with Peyronie's disease received in our Department of Urology between January 2013 and December 2015, who had failed to respond to over 12 months of expectant drug therapy and remained stable for more than 6 months, none able to perform sexual intercourse due to penile curvature (60°). All the patients underwent plaque excision plus autologous perididymal patch grafting.Postoperative follow-up ranged from 6 to 24 months. All the patients achieved normal penile erection, without testicular atrophy, torsion or necrosis at the surgery side and all were satisfied with the surgical results without complaining about obvious penile shortening.Plaque excision plus autologous perididymal patch grafting is a safe, simple, economic and effective method for the treatment of Peyronie's disease.目的: 探讨斑块切除+自体睾丸鞘膜移植术治疗阴茎硬结症的临床疗效。方法: 选取2013年1月至2015年12月收治的10例经药物保守治疗12个月以上无效、处于稳定期6个月以上、阴茎弯曲60°、不能完成性交的阴茎硬结症患者，行阴茎硬结切除+自体睾丸鞘膜移植修补缺损。 结果: 10例患者术后随访期6~24个月，所有患者术后阴茎均能正常勃起，无手术侧睾丸萎缩、扭转或坏死。患者对阴茎外形满意，未觉阴茎有明显缩短。 结论: 斑块切除+自体睾丸鞘膜移植术是治疗阴茎硬结症造成的阴茎勃起畸形的一种安全、简便、经济和有效的手术方式。.

Bladder cancer is one of the most common cancers. Approaches that block tumor angiogenesis are a new therapeutic strategy for locally advanced or metastatic BC. VEGF/VEGFR signaling has been obviously and negatively correlated with the progression and invasion of cancer. In this study, we constructed the recombinant adenovirus vAd-VEGFR-3 to investigate its antitumor effector in vitro/vivo. First, we used the recombinant adenovirus vAd-VEGFR-3 to infect bladder cancer cells and then collected the cell culture supernatant to treat human umbilical vein endothelial cells (HUVECs). The proliferation, migration and apoptosis of HUVECs were respectively detected by MTT, transwell and Annexin V-FITC/PI double staining. In addition, mouse bladder mucosa was injured by trypsin, and the orthotopic transplantation model of human bladder cancer was successfully constructed to clarify the anti-tumor effect of Ad-VEGFR in vivo. The results showed that Ad-VEGFR could inhibit the cancer’s proliferation and migration, while promoting the apoptosis of HUVECs in vitro. Moreover, Ad-VEGFR could significantly promote the apoptosis of bladder cancer cells and then prevent tumor growth in vivo. In addition, it also down-regulated the expression levels of CD31, an endothelial cell marker which is closely related to the angiogenesis. Taken together, it suggests that the infection of adenovirus-carrying VEGFR in bladder cancer cells may inhibit blood vessel formation and prevent tumor progression.

To investigate the effects of cynomorium songaricum (CS) decoction on the testis weight, serum testosterone level, and sperm parameters of rats with oligoasthenospermia (OAS), explore its action mechanism of improving the proliferation of undifferentiated spermatogonial cells, and provide some experimental and theoretical evidence for the development of new Chinese drugs for OAS.Thirty 8-week-old male SD rats were randomly divided into five groups of equal number: blank control, model control, high-dose CS, medium-dose CS, and low-dose CS. OAS models were established by intraperitoneal injection of cyclophosphamide and, a month later, treated intragastrically with normal saline or CS at 2, 1, and 0.5 g per kg of the body weight per day, all for 4 weeks. Then, the testes of the animals were harvested to obtain the testicular weight, sperm concentration and motility, and the level of serum testosterone (T), detect the expressions of the transcription factor 1 (Oct4), Thy-1 cell surface antigen (Thy1), promyelocytic leukemia zinc finger (PLZF), KIT proto-oncogene receptor tyrosine kinase (C-kit) and glial cell-derived neurotrophic factor (GDNF) in the testis tissue of the rats in the low-dose CS group by real-time PCR.The testis weights in the blank control, model control, high-dose CS, medium-dose CS, and low-dose CS groups were (1.52±0.06), (1.55±0.06), (1.43±0.30), (1.35±0.40) and (1.34±0.04) g, respectively, not significantly different in the blank and model controls from those in the CS groups (P0.05). The visual field sperm count per 10 HP was significantly increased in the high-, medium-, and low-dose CS groups (202±20, 196±5 and 216±25) as compared with the blank and model controls (200±15 and 134±30) (P0.05). The mRNA expressions of the Oct4, Thy1, PLZF and GDNF genes were remarkably higher in the low-dose CS group than in the controls (P0.05), but that of the C-kit gene showed no significant difference from the latter (P0.05). The visual field sperm motility per 10 HP was markedly increased in the blank control (［52.1±5.5］%), model control (［38.1±2.5］%), high-dose CS (［59.1±9.5］%), medium-dose CS (［58.7±9.5］%), and low-dose CS (［49.6±1.0］%) groups, and so was the level of serum testosterone (［190±87.5］, ［82.5±25.8］, ［229±75.6］, ［331±86.7］ and ［185±82.4］ mmol/L), both remarkably higher in the CS groups than in the model controls (P0.05) but with no statistically significant difference between the CS groups and the blank controls (P0.05).CS can significantly improve sperm concentration, sperm motility and serum T level in OAS rats, probably by inducing the expression of GDNF in the rat Sertoli cells, promoting the proliferation of undifferentiated spermatogonial cells, and enhancing spermatogenesis.目的： 探究锁阳对少、弱精子症大鼠睾丸重量、血清睾酮浓度、以及精子数量、活动率等精子参数的影响，及促进未分化精原细胞增殖的机制，为开发新的治疗男性少、弱精子症的中药提供实验和理论依据。方法： 选取8周龄，体重约(220±10) g的SD大鼠30只，随机分为5组（n=6）：空白对照组、模型对照组、3组实验组（低、中、高锁阳浓度剂量）；将模型对照组与锁阳实验组予以环磷酰胺［30 mg/（kg·d）］腹腔注射，连续5 d，构建大鼠少、弱精子症模型；将3组实验组分别用低浓度［0.5 g/（kg·d）］、中浓度［1 g/（kg·d）］及高浓度［2 g/（kg·d）］锁阳水煎液灌胃，每天1次，连续4周后观察各组精子数量、活动率，测量各组大鼠血清睾酮浓度，并取各组大鼠睾丸称重；采用Real-time PCR方法检测低浓度组睾丸组织中精原干细胞标志物（Oct4、Thy1、PLZF、C-kit）表达情况，采用β-actin作为内参；采用Real-time PCR方法检测低浓度组睾丸组织中胶质细胞源性神经营养因子(GDNF)表达情况。结果： 空白对照组、模型对照组及低、中、高浓度锁阳实验组的大鼠睾丸重量分别为(1.52±0.06) g、(1.55±0.06) g、(1.34±0.04) g、(1.35±0.40) g、(1.43±0.30) g，不同浓度锁阳实验组大鼠睾丸重量与空白对照组、模型对照组比较未见明显差异（P0.05）。空白对照组、模型对照组及低、中、高浓度实验组在每10个高倍镜视野精子数（精子数/10HP）分别为200±15、134±30、216±25、196±5、202±20个,锁阳实验组与模型对照组比较可显著提高大鼠的精子数量（P0.05）；低浓度锁阳水提物组睾丸组织中Oct4、Thy1、PLZF、GDNF基因mRNA表达水平较模型对照组增加，差异有统计学意义（P0.05），C-kit基因mRNA表达水平未见明显差异（P0.05）; 空白对照组、模型对照组及低、中、高浓度锁阳实验组的大鼠每10个高倍镜视野精子活动率（精子活动率/10HP）分别为(52.1±5.5)%、(38.1±2.5)%、(49.6±1.0)%、(58.7±9.5)%、(59.1±9.5)%；大鼠血清睾酮浓度分别为(190.0±87.5)、(82.5±25.8)、(185.0±82.4)、(331.0±86.7)、(229.0±75.6) mmol/L，锁阳实验组与模型对照组比较可显著提高大鼠的精子活动率及血清睾酮浓度（P均0.05），但与空白对照组比较无明显差异(P0.05)。结论： 锁阳能显著提高少、弱精子症大鼠的精子数量、精子活动能力、血清睾酮水平，其改善机制可能是：①通过诱导睾丸Sertoli细胞中GDNF表达，促进未分化精原细胞增殖，从而促进精子发生过程，增加附睾尾部精子数目；②通过促进睾酮分泌，提高血清睾酮水平，进而改善精子活动率。.

To understand lncRNAs expression profiling and their potential functions in bladder cancer, we investigated the lncRNA and coding RNA expression on human bladder cancer and normal bladder tissues. Bioinformatic analysis revealed thousands of significantly differentially expressed lncRNAs and coding mRNA in bladder cancer relative to normal bladder tissue. Co-expression analysis revealed that 50% of lncRNAs and coding RNAs expressed in the same direction. A subset of lncRNAs might be involved in mTOR signaling, p53 signaling, cancer pathways. Our study provides a large scale of co-expression between lncRNA and coding RNAs in bladder cancer cells and lays biological basis for further investigation.

We describe a method for solid-phase extraction of biogenic thiols using multi-walled carbon nanotubes as adsorbent, and their subsequent determination via HPLC and fluorescence detection. The fluorogenic reagent N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl) iodoacetamide was applied to derivatizate the thiols. The type of eluent and its volume, the sample pH, extraction time and sample volume were optimized. The calibration curves of the thiols are linear in the range from 0.5 to 200 nM (for glutathione), 0.02 to 5 nM (for cysteine), and 2 to 500 nM (for acetylcysteine), and the correlation coefficients range between 0.9955 and 0.9997. The respective limits of detection are 20 pM, 4 pM and 80 pM (at an SNR of 3), and the limits of quatification are 67 pM, 13 pM, and 267 pM (at an SNR of 10). Recoveries range from 85.0% to 113.1% for human urine and plasma samples spiked with the three thiols, and relative standard deviations are in the range from 2.1 to 7.4%.

Solid-phase extraction (SPE) coupled with high-performance liquid chromatography–fluorescence detection (LC–FL) was developed for the determination of three thiol compounds including glutathione, cysteine and acetylcysteine. 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole was used for derivatization of thiols. Factors affecting derivatization and extraction efficiency were optimized. Sample solution (2 mL) was extracted on a SPE column for 2 min and then eluted with 400 μL methanol. The analytes were injected onto the LC system for separation on a C18 column, and eluted with methanol–acetate buffer. The analytes were detected by fluorescence at an emission wavelength of 515 nm with excitation at 385 nm. The linearity of the method was in the range of 0.1–60 μM, with correlation coefficients ranging from 0.9979 to 0.9990. The detection limits of the method were in the range of 5–20 nM. The proposed method was applied to the analysis of human plasma samples with recoveries of 86–112.9%.

A nano-material carboxylic acid functionalized graphene (graphene-COOH) was prepared and used to construct a novel biosensor for the simultaneous detection of adenine and guanine. The direct electrooxidation behaviors of adenine and guanine on the graphene-COOH modified glassy carbon electrode (graphene-COOH/GCE) were carefully investigated by cyclic voltammetry and differential pulse voltammetry. The results indicated that both adenine and guanine showed the increase of the oxidation peak currents with the negative shift of the oxidation peak potentials in contrast to that on the bare glassy carbon electrode. The electrochemical parameters of adenine and guanine on the graphene-COOH/GCE were calculated and a simple and reliable electroanalytical method was developed for the detection of adenine and guanine, respectively. The modified electrode exhibited good behaviors in the simultaneous detection of adenine and guanine with the peak separation as 0.334 V. The detection limit for individual determination of guanine and adenine was 5.0 × 10 −8 M and 2.5 × 10 −8 M (S/N = 3), respectively. Furthermore, the measurements of thermally denatured single-stranded DNA were carried out and the value of (G + C)/(A + T) of single-stranded DNA was calculated as 0.80. The biosensor exhibited some advantages, such as simplicity, rapidity, high sensitivity, good reproducibility and long-term stability.

Solid-phase extraction coupled with LC-fluorescence detection was used for the extraction and determination of three thiol compounds including glutathione (GSH), cysteine (Cys) and acetylcysteine (NAc). Fluorescence probe N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl)iodoacetamide (BODIPY Fl-C1-IA) was applied for derivatization of thiols. The derivatization and extraction conditions have been investigated in detail. The calibration graph of the proposed method was linear in the range of 1–200 nM (GSH and Cys) and 20–2,000 nM (NAc). The limits of detection were in the range of 0.2–4.0 nM. The usefulness of the proposed method has been validated by measuring thiol-containing compounds in human urine and blood samples from healthy persons, with recoveries of 92.5–110%.

Neuropilin-1 (NRP-1) overexpression has been reported in a variety of human cancers. However, the role of NRP-1 in bladder cancer (BC) remains unclear. The aim of present study was to analyze NRP-1 protein expression in BC tissues and to assess its prognostic significance for BC. NRP-1 messenger ribonucleic acid (mRNA) and protein expression were determined by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunohistochemistry in specimens of primary cancer and their adjacent noncancerous tissues in BC patients. Additionally, NRP-1 protein expression in 139 archived paraffin-embedded BC samples was analyzed by immunohistochemistry and correlated with clinicopathological characteristics and survival. Student’s t test, Spearman’s rank correlation, Kaplan-Meier plots, and Cox’s proportional hazards regression model were used to analyze the data. By qRT-PCR and immunohistochemistry, the levels of NRP-1 mRNA and protein were significantly higher in BC, compared to that in adjacent noncancerous tissues (P

To evaluate the effect of elective microscopic resection of dorsal penile nerves in the treatment of primary premature ejaculation (PPE).Seventy-eight PPE patients received elective microscopic resection of dorsal penile nerves, 5 branches in 9 cases, 6 in 17, 7 in 15, 8 in 14, 9 in 8, 10 in 6, 11 in 6, and 12 in 3. The patients were followed up for 12 months, and their intravaginal ejaculation latency time (IELT) and sexual intercourse satisfaction scores were recorded before and after treatment.Compared with the baseline, the IELT was significantly prolonged after surgery ([0.86 +/- 0.32] vs [6.65 +/- 3.9] min, P0.01), and the sexual intercourse satisfaction scores of the patients were dramatically increased (7.32 +/- 2.52 vs 12.32 +/- 3.76, P0.01), so were those of their sexual partners (4.46 +/- 1.36 vs 12.73 +/- 1.45, P0.01).Elective microscopic resection of dorsal penile nerves is safe and effective for the treatment of PPE.

This study aimed to investigate the depression status among high-risk pregnancy women, and to analyze its relevant social and psychological factors.A total of 42 high-risk pregnancy women and 40 normal pregnancy women in a teaching hospital in Harbin city were followed up at time points of 32 - 36 weeks pregnancy, one week before labor, one week postpartum, and six weeks postpartum, respectively. During follow-up, the basic situation, social psychosocial factors of pregnancy women were collected and the depression of pregnancy women was measured by self-designed questionnaire and self-rating depression scale. The Edinburgh Postnatal Depression Scale (EPDS) was applied at timepoint of one week postpartum. Single factor analysis and the unconditional multivariate logistic regression were applied for analyzing the on the related social-psychosocial factors among high-risk pregnancy women.The age of high-risk pregnancy women was (31.0±5.6), and the age of normal pregnancy women was (30.5±3.8) (t=0.169, P0.05). The results showed that the depression rate in high-risk pregnancy women was 45.2% (19/42), which was 25.0% (10/40) in normal pregnancy women, the difference was significant (χ2=3.671, P=0.045). The depression rates at different time points were 30.9% (13/42), 42.9% (18/42), 23.8% (10/42), 26.2% (11/42) in high-risk pregnancy women respectively, and 25.0% (10/40), 15.0% (6/40), 20.0% (8/40), 17.5% (7/40) in the control group respectively, the difference of the depression rates among groups at one week before labor was significant (χ2=7.680, P0.01), the difference among groups at 32-36 weeks pregnancy (χ2=0.133, P=0.80), at one week postpartum (χ2=0.174, P=0.79) and at six weeks postpartum (χ2=0.903, P=0.43) were not significant. At one week postpartum and six weeks postpartum periods, the EPDS depression rate were 12.5% (4/32), 30.4% (7/23) in case group respectively, 8.3% (3/36), 22.9% (8/35) in control group respectively, the difference were not significant (χ2=0.319, 0.416, P=0.573, 0.519). There were significantly associations between the depression mood of one week before labor and the depressive symptoms of six weeks postpartum in both groups (r=0.824, 0.677, both P values were0.05). The risk factors for maternal depression among high-risk pregnancy women were not ready for production (OR=2.73, P0.01) and fearing of childbirth safety (OR=2.89, P0.01).The depression date of high-risk pregnancy was high, especially at the time point one week before labor. Risk factors of maternal depression among high-risk pregnancy were "not ready for production" and "fear of childbirth safety".

To explore the inhibitory effect and anti-cancer mechanisms of adenovirus-mediated ING4 gene on the human bladder cancer T24 cells in vitro.The methods of reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of ING4 in human bladder urothelial carcinoma T24 line. The influence of Ad-ING4 transfection on cell proliferation was evaluated by MTT assay and cell apoptosis by Hochest33258 staining and flow cytometry. RT-PCR and Western blot were performed to detect the transcriptional level of such apoptosis-related genes as Bcl-2, Bax, p53, HIF-1α and caspase-3.Human ING4 was successfully transcribed in T24 cell. Apoptosis rate of T24 cell in Ad-ING4 group was significant higher than that in control groups (17.2% ± 4.1% vs 4.7% ± 1.2% and 4.8% ± 1.2%, P0.05). Ad-ING4 not only up-regulated the expression of p53 and Bax(1.40 ± 0.11 vs 0.27 ± 0.04, 1.50 ± 0.12 vs 0.60 ± 0.05) and down-regulated the expression of Bcl-2 and HIF-1α (0.19 ± 0.02 vs 1.20 ± 0.08, 0.33 ± 0.03 vs 0.98 ± 0.06, all P0.05), but also enhanced the caspase-3 activation and eventually led to apoptosis.Adenovirus-mediated ING4 gene exhibits anti-tumor capacity in T24 human bladder cancer cell and induces in vitro apoptosis. It may be related to the up-regulation of P53 and Bax/Bcl-2, and the down-regulation of HIF-1α. Thus the caspase-3 activation is enhanced so as to lead to apoptosis.

To investigate the in vitro inhibitive effect and apoptotic mechanism of BDI-1 monoclonal antibody targeted SEA on human bladder cancer cell line BIU-87.Human peripheral blood mononuclear cells (PBMC) and human bladder cancer cell line BIU-87 were used as effector and target cells respectively in three experimental groups, control group, SEA group and monoclonal antibody targeted SEA group, treated with different concentrations of SEA and targeted SEA respectively in the latter two. The inhibition rates of target cells were measured by cell counting kit-8 (CCK-8) assay. Apoptosis was determined by fluorescent Hocehst 33258 staining and flow cytometry (FCM). The expression of Bax, Bcl-2 proteins and cytokine concentration of co-culture supernatants were detected by Western blot and ELISA respectively.Compared with those of control group (from 5.8% +/- 3.0% to 16.5% +/- 4.0%) and SEA group (from 20.7% +/- 2.1% to 68.6% +/- 4.0%), the inhibition rates in targeted SEA group increased significantly (from 31.2% +/- 2.6% to 88.7% +/- 3.0%, P0.05). Characteristic changes of apoptosis were observed in both experimental treatment groups, especially in targeted SEA group. FCM also showed more prominent apoptosis induced in targeted SEA group (from 14.6% +/- 0.7% to 66.5% +/- 3.3%), significantly higher than those of control group (from 4.6% +/- 0.7% to 6.5% +/- 0.3%) and SEA group (from 9.1% +/- 0.6% to 29.9% +/- 1.3%, P0.05). In addition, Western blot analysis indicated that the ratio of Bax/Bcl-2 significantly increased to (3.29 +/- 0.34) by targeted SEA treatment at an extremely low concentration, significantly higher than those of SEA group (1.21 +/- 0.05) and control group (0.45 +/- 0.15) (P0.05). While at the same time ELISA assay showed no significant difference of IL-2, TNF-alpha, IFN-gamma concentrations between SEA and targeted SEA groups.Monoclonal antibody targeted SEA significantly increases the inhibitive effect of BIU-87 cell line possibly through the up-regulation of Bax/Bcl-2 ratio to promote the cellular apoptosis.

The production of sperm is a very complicated and orderly process, which involves the proliferation and differentiation of cells. The in vitro culture of spermatogenic cells includes tissue culture and cell culture. This study focuses on the advances in the in vitro culture of spermatogenic cells, testicular tissue culture and their culture conditions, and sums up the advantages and disadvantages of various culture methods, in an attempt to find the best method for differentiating spermatogenic cells in vitro and make the culture of spermatogenic cells an important means of treating male infertility.

Sptrx-1, 2 and 3 are a series of thioredoxins specifically expressed in the testis/sperm. They play a significant role structurally and functionally in the process of spermiogenesis. The genesis and mutation of sptrx-1, 2 and 3 are correlated to male reproduction. Taking sptrx-1, 2 and 3 as the target of study and treatment will open up a new field in the clinical study of male reproduction.

To investigate the influence of mycophenolate mofetil (MMF) on the maturation and allostimulatory activity of cultured dendritic cell progenitors (DCPs) and to evaluate the efficacy of pretreatment of donor dendritic cells (DCs) with MMF in tolerance induction in allograft recipients and its possible mechanism.DCPs of Balb/c mice were cultured in culture fluid containing recombinant mouse granulocyte-macrophage colony stimulating factor (GM-CSF), and then divided into 4 groups: control group, MMF group, lipopolysaccharide (LPS) group, and MMF + LPS group. Seven days later, flow cytometry was used to analyze the phenotypes of the DCs. ELISA was used to examine the level of IL-12 in the supernatant. T cells from the spleens of C57/BL6 mice were cultured together with inactivated DCs from Balb/c mice, and added with [(3)H]-TdR. Mixed lymphocyte reaction (MLR) was analyzed. The DCs and MMF-DCs cultured for 5 days were co-cultured with T hybridoma cells of the line MF2.2D9 in culture fluid containing ovalbumin (OVA). Twenty-four hours later, the supernatant was collected and ELISA was used to measure the level of interleukin (IL)-12 so as to reflect the antigen-presenting ability of the DCs. OVA immunized C57/BL6 mice for 4 times. 21 days after T cells were collected from the spleens and co-incubated with DCs and MMF-DCs, [(3)H]-TdR was added and the values of counts per minute (cpm) were calculated so as to analyze the antigen-specific proliferation. Twenty-four Balb/c mice were randomly divided into 3 groups: group A (without treatment), group B (treatment with intravenous injection of untreated DCs of Balb/c mice), and group C (treatment with intravenous injection of DCs of Balb/c mice treated with MMF), and then transplanted with the hearts of C57/BL6 mice. The functions of the transplanted hearts were evaluated by touching the arterial pulse and histological examination. ELISA was used to detect the levels of Th1 cytokines, such as IL-12, IL-4, IL-10, IL-2, and IFN-gamma, in the cultured DCs and in the sera of the recipients 7 and 14 days after culture or transplantation.The immunophenotypic analysis showed that in comparison with those in the control group and the LPS group the expressions of the costimulatory molecules, Ia(d), CD80, and CD86, of the DCPs were significantly weaker in the MMF-group and MMP + LPS group. The IL-12 levels in the supernatant of the MMF and MMF + LPS groups of DCPs were significantly lower than those in the other groups (P0.01). The IL-12 level of the MF2.2D9 cells treated with MMF-treated DCs was significantly lower than control group (P0.01). The ability to stimulate proliferation of T cells of the same genotype in the MMF-DC group was significantly inhibited (P0.01). The survival time of the transplanted heart was 30.50 +/- 3.25 days in the C57/BL6 mice injected with untreated DCPs of Balb/c mice (21.25 +/- 2.12, P0.01) and that in the control C57/BL6 mice (8.63 +/- 1.06 days, P0.01) and with a significant difference between the latter 2 groups too (P0.01). The levels of, such as IL-2 and IFN-gamma, 7 and 14 days after heart transplantation of the group B were all significantly lower than those of the group A (both P0.05). The IL-2 and IFN-gamma levels 7 days after the heart transplantation were similar to those in the group B (both P0.05) and even lower than those of the group C (both P0.05). The IL-10 level in the groups B and C were all higher than those in the group A (all P0.05) with a significant difference between the group B and group C. The level of IL-4 was not significantly different among the 3 groups.MMF has a significant suppressive effect on the maturation and function of DCs, which leads to a donor-specific tolerance in transplant recipients.