A detection model of testis-derived circular RNAs in serum for predicting testicular sperm retrieval rate in non-obstructive azoospermia patients.

BACKGROUND: Microdissection testicular sperm extraction is an effective method to retrieve sperm from non-obstructive azoospermia patients. However, its successful rate is less than 50%. OBJECTIVES: To identify the predictive value of circular RNAs in serum for sperm retrieval rate in non-obstructive azoospermia patients. MATERIALS AND METHODS: 180 non-obstructive azoospermia patients were recruited in this study, including 84 individuals with successful sperm retrieval and 96 individuals with failed sperm retrieval. Our study contained two phases. First, 20 patients, selected from the 180 patients, were included in screening cohort. In this cohort, the top 20 circular RNAs from our previous testicular circRNA profiles were verified between successful and failed sperm retrieval groups using real-time polymerase chain reaction. Six circular RNAs with the most significantly different expressions were selected for further verification. Second, the 180 patients were included as discovery cohort to verify the six circular RNAs. Circular RNAs were extracted from serum in each participant. Logistic regression analysis was further performed to identify the predictive value and the area under the curve analysis was used to evaluate diagnostic efficiency, sensitivity, and specificity. RESULTS: Six circular RNAs including hsa_circ_0058058, hsa_circ_0008045, hsa_circ_0084789, hsa_circ_0000550, hsa_circ_0007422, and hsa_circ_0004099 showed aberrant expressions between the successful and failed sperm retrieval group. In addition, both single-circular RNA panels and multi-circular RNA panels were finally verified to be significant in predicting sperm retrieval rate. Notably, multi-circular RNAs panels demonstrated better predictive abilities compared with single-circRNA panels, and the combined panel of six-circular RNAs (risk score = 1.094×hsa_circ_0058058+0.697×hsa_circ_0008045+0.718×hsa_circ_0084789-0.591×hsa_circ_0000550-0.435×hsa_circ_0007422-1.017×hsa_circ_0004099-1.561) exhibited the best predictive ability in the present study with an AUC of 0.977, a sensitivity of 91.7% and a specificity of 86.5%. A higher risk score indicated a higher risk of failure in sperm retrieval. DISCUSSION AND CONCLUSION: Our study was the first to report that testis-derived circular RNAs in serum have the ability to predict sperm retrieval rate in non-obstructive azoospermia patients, whether it is a single-circular RNA or a combination of multi-circular RNAs.

GRPR down-regulation inhibits spermatogenesis through Ca2+ mediated by PLCß/IP3R signaling pathway in long-term formaldehyde-exposed rats.

Formaldehyde (FA), which is known as an air pollutant, has been proven to induce male infertility. However, the underlying mechanism of FA-induced male infertility remains elusive. In this study, 24 male SD rats were exposed to different levels of FA (0, 0.5, 2.46, and 5 mg/m3) for eight consecutive weeks. Through HE staining and sperm smear, we observed that FA exposure resulted in spermatogenic injury and the sperm quality decreased in rats. The qRT-PCR and Western blot analysis further revealed that GRPR was down-regulated in testicular tissues of FA-exposed rats as well as primary spermatogenic cells. Meanwhile, ZDOCK uncovered an interaction between GRPR and PLCß. In addition, the CCK8, Fluo 3-AM and Flow cytometry results showed that FA exposure suppressed the expression of GRPR, PLCß and IP3R, consequently reducing the Ca2+ concentration in spermatogenic cells, inducing apoptosis and inhibiting proliferation of spermatogenic cells. Moreover, rescue experiments confirmed that promoting GRPR could improve intracellular Ca2+ concentration by upregulating PLCß and IP3R, partially reducing the apoptosis and promoting the proliferation of FA-treated spermatogenic cells. These findings revealed that GRPR participates in spermatogenesis through Ca2+ mediated by the PLCß/IP3R signaling pathway in FA-exposed rats.

Semen Quality Following Long-term Occupational Exposure to Formaldehyde in China.

Importance: The potential effects of long-term occupational exposure to formaldehyde (FA) on human semen quality is not clear. Objective: To assess whether long-term occupational exposure to FA is associated with semen quality. Design, Setting, and Participants: This population-based cohort study was conducted from June 1 to June 30, 2021, in Xi'an, China. Participants were adults aged 23 to 40 years who had lived in the study area for 24 months or longer. Data analysis was performed from September 1 to October 1, 2021. Exposures: Long-term occupational exposure to FA was measured using a formaldehyde detector, and the FA exposure index (FEI) was calculated as follows: FEI = final concentration of FA (mg/m3) × work time during a workday (hour) × cumulative workdays (year). Main Outcomes and Measures: Semen samples were collected by masturbation after 3 to 7 days of abstinence and were then assessed by the computer-automated semen analysis system, Baso-Papanicolaou staining, and sperm-chromatin structure assay. Results: A total of 205 men (mean [SD] age, 29.49 [3.64] years), with 124 individuals in the FA exposure group (mean [SD] FEI, 73.72 [54.86]) and 81 age-matched controls, were included in the final analysis. Long-term personal occupational exposure to FA was significantly associated with poor semen quality. Specifically, a 1-unit increase in FEI was associated with a change of -0.99% (95% CI, -1.00% to -0.98%) in total sperm motility, -0.99% (95% CI, -0.99% to -0.97%) in progressive sperm motility, -0.05% (95% CI, -0.08% to -0.02%) in curvilinear velocity, -0.07% (95% CI, -0.10% to -0.04%) in straight line velocity, -0.07% (95% CI, -0.10% to -0.04%) in time-average velocity, -0.98% (95% CI, -0.99% to -0.93%) in normal sperm morphology, -0.24% (95% CI, -0.35% to -0.11%) in seminal neutral glucosidase, -0.61% (95% CI, -0.66% to -0.56%) in seminal plasma zinc, 0.52% (95% CI, 0.15% to 1.02%) in beat cross frequency, and 0.10% (95% CI, 0.06% to 0.14%) in the DNA fragmentation index. These associations remained significant after adjusting for confounding factors. Furthermore, subgroup analysis found that high levels of oxidative stress might promote the associations between FA exposure and semen quality. Conclusions and Relevance: This study found an association between long-term occupational exposure to FA and semen quality. This deterioration was dose and time dependent and might be induced by oxidative stress.

Rno_circRNA_008646 regulates formaldehyde induced lung injury through Rno-miR-224 mediated FOXI1/CFTR axis.

Formaldehyde (FA) serves as a prevailing air pollutant, which has seriously threatened public health in recent years. Of all the known health effects, lung injury is one of the most severe risks. However, little is known about the circRNAs related molecular mechanism in the development of lung injury induced by FA. This study was designed to explore the potential roles of dysregulated circRNAs as well as its mechanism in FA-induced lung injury. In the present study, 24 male SD rats were exposed to formaldehyde (control, 0.5, 2.46 and 5 mg/m3) for 8 h per day for 8 weeks to induce lung injury. We used H&E staining to evaluate the histopathological changes of lung injury indifferent groups. The expression of circRNAs in lung tissue was detected by real-time PCR. Meanwhile, circRNA/miRNA/mRNA interaction networks were predicted by bioinformatics analysis. Our study revealed that formaldehyde exposure resulted in abnormal histopathological changes in lung tissues. Moreover, the expression of rno_circRNA_008646 was significantly higher in lung tissues of formaldehyde exposure rats than in control. Bioinformatics analysis showed that one potential target miRNA/mRNA for rno_circRNA_008646 was rno-miR-224/Forkhead Box I1 (FOXI1). Besides, luciferase report gene confirmed that there was targeted binding relationship between rno_circRNA_008646 and rno-miR-224, rno-miR-224 and FOXI1. Further verification experiments indicated that the expression of rno_circRNA_008646 was negatively correlated rno-miR-224, while it was positively correlated with FOXI1. JASPAR database showed transcription factor FOXI1 located in promotor of CF Transmembrane Conductance Regulator (CFTR). Both FOXI1 and CFTR were up-regulated in lung tissues after formaldehyde exposure. In conclusion, our findings suggested that formaldehyde may induce lung injury, and this may be caused by up-regulatedrno_circRNA_008646, which medicated rno-miR-224/FOXI1/CFTR axis.

Effect of naturally derived surgical hemostatic materials on the proliferation of A549 human lung adenocarcinoma cells.

Hemostatic materials are generally applied in surgical operations for cancer, but their effects on the growth and recurrence of tumors are unclear. Herein, three commonly used naturally derived hemostatic materials, gelatin sponge, Surgicel (oxidized regenerated cellulose), and biopaper (mixture of sodium hyaluronate and carboxymethyl chitosan), were cocultured with A549 human lung adenocarcinoma cells in vitro. Furthermore, the performance of hemostatic materials and the tumorigenicity of the materials with A549 â€‹cells were observed after subcutaneous implantation into BALB/c mice. The in vitro results showed that biopaper was dissolved quickly, with the highest cell numbers at 2 and 4 days of culture. Gelatin sponges retained their structure and elicited the least cell infiltration during the 2- to 10-day culture. Surgicel partially dissolved and supported cell growth over time. The in vivo results showed that biopaper degraded rapidly and elicited an acute Th1 lymphocyte reaction at 3 days after implantation, which was decreased at 7 days after implantation. The gelatin sponge resisted degradation and evoked a hybrid M1/M2 macrophage reaction at 7-21 days after implantation, and a protumor M2d subset was confirmed. Surgicel resisted early degradation and caused obvious antitumor M2a macrophage reactions. Mice subjected to subcutaneous implantation of A549 â€‹cells and hemostatic materials in the gelatin sponge group had the largest tumor volumes and the shortest overall survival (OS), while the Surgicel and the biopaper group had the smallest volumes and the longest OS. Therefore, although gelatin sponges exhibited cytotoxicity to A549 â€‹cells in vitro, they promoted the growth of A549 â€‹cells in vivo, which was related to chronic M2d macrophage reaction. Surgicel and biopaper inhibited A549 â€‹cell growth in vivo, which is associated with chronic M2a macrophage reaction or acute Th1 lymphocyte reaction.

Pregabalin does not decrease acute pain or postoperative nausea and vomiting after hysterectomy: a meta-analysis.

OBJECTIVE: Hysterectomy is associated with severe postoperative pain. The relative efficacy of pregabalin compared with other treatments for post-hysterectomy pain is unclear. METHODS: We searched the PubMed, Cochrane Library, and Web of Science databases for studies that compared the use of pregabalin and placebo for reducing pain in patients undergoing hysterectomy. RESULTS: This meta-analysis showed that pregabalin had limited pain-relieving effects at 2, 6, 24, and 48 hours after hysterectomy compared with placebo. Pregabalin significantly reduced postoperative nausea and vomiting. However, there was no significant difference in postoperative sedation or visual disturbances between patients treated with pregabalin and placebo. CONCLUSIONS: Pregabalin is not clinically superior to placebo in terms of reducing pain intensity and morphine consumption in patients undergoing hysterectomy. However, the limitations of this meta-analysis mean that more high-quality randomized controlled trials are necessary to verify our pooled results.

Critically dysregulated signaling pathways and clinical utility of the pathway biomarkers in lymphoid malignancies.

Accumulating evidence confirmed that many dysregulated signaling pathways and aberrant genetic alterations contribute to the oncogenesis and heterogeneity of lymphoid malignancies. Therapeutically targeting dysregulating signaling pathways and their hidden oncogenic biomarkers are becoming available, but did not show desired therapeutic effect in current clinical practice. It is meaningful to further understand the underlying mechanisms of the dysregulated signaling pathways and to address the potential utility of pathway-related biomarkers. To precisely identify the dysregulation of signaling pathways and the "driver" oncogenic biomarkers, as well as to develop reliable and reproducible risk-stratification based on biomarkers will be challenging. Nevertheless, pathway-based targeted therapy will raise the hope to improve the outcomes of the patients with lymphoid malignancies, especially with aggressive types, and the efficient utility of pathway-related biomarkers in diagnosis, prognosis, prediction of lymphoid malignancies may also be able to power precision medicine.

Photooxidatively crosslinked acellular tumor extracellular matrices as potential tumor engineering scaffolds.

Acellular tumor extracellular matrices (ECMs) have limitations when employed as three-dimensional (3D) scaffolds for tumor engineering. In this work, methylene blue-mediated photooxidation was used to crosslink acellular tumor ECMs. Photooxidative crosslinking greatly increased the stiffness of acellular tumor ECM scaffolds but barely altered the Amide III band of the secondary structure of polypeptides and proteins. MCF-7, HepG2 and A549 cells cultured on photooxidatively crosslinked acellular tumor ECM scaffolds exhibited greater cell number per scaffold, more IL-8 and VEGF secretion, and increase migration and invasion abilities than cells cultured on uncrosslinked acellular tumor ECM scaffolds. The three tumor cell lines cultured on the stiffer photooxidatively crosslinked acellular matrices acquire mesenchymal properties (mesenchymal shift) and dedifferentiated phenotypes. Furthermore, the malignant phenotypes induced in vitro when cultured on the crosslinked scaffold promoted the in vivo tumor growth of BALB/c nude mice. Finally, the dedifferentiated cancer cells, including MCF-7, HepG2 and A549 cells, were less sensitive to chemotherapeutics. Thus, photooxidatively crosslinked acellular tumor ECMs have potentials as 3D tumor engineering scaffolds for cancer research. STATEMENT OF SIGNIFICANCE: Natural material scaffolds have been successfully used as 3D matrices to study the in vitro tumor cell growth and mimic the in vivo tumor microenvironment. Acellular tumor ECMs are developed as 3D scaffolds for tumor engineering but have limitations in terms of elastic modulus and cell spheroid formation. Here we use methylene blue-mediated photooxidation to crosslink acellular tumor ECMs and investigate the influence of photooxidative crosslinking on structural, mechanical and biological characteristics of acellular tumor ECM scaffolds. It is the first study to evaluate the feasibility of photooxidatively crosslinked acellular tumor ECMs as 3D scaffolds for cancer research and the results are encouraging. Moreover, this study provides new research areas in regard to photodynamic therapy (PDT) for Cancer.

[Genetic differentiation and patterns of gene flow of ten minorities in Yunnan province].

OBJECTIVE: To explore the features of genetic differentiation and gene flow of ten minorities in Yunnan province according to nine CODIS short tandem repeat(STR) loci (CSF1PO, FGA, THO1, TPOX, v WA, D3S1358, D5S818, D7S820 and D13S317). METHODS: Heterozygosity and parameters of population differentiation such as F, theta, f and Gst at each locus were calculated. DA genetic distance and fixation index Fst were calculated by Phylip 3.6 and Arlequin 3.0 software, respectively. Phylogenetic trees were constructed by Mega 3.0, and the patterns of gene flow were analyzed by R-matrix model. RESULTS: It showed that average genetic heterogeneity in ten minorities was above 0.7. Significant difference was found for most of the loci in genetic differentiation. Phylogenetic tree analysis showed that the ten minorities were divided into two clusters. The results of the R-matrix analysis showed that the gene flow of Yi and Dai minorities were higher than that of other minorities, while the pattern of gene flow of Dulong minority demonstrated some of the isolation. CONCLUSION: Nine STR loci commonly used in forensic identification show a high polymorphism. Heterozygosity can be used for investigating genetic differentiation and gene flow of minority. The ten minorities in Yunnan are independent populations, while the level of differentiation is not high. The relationship in evolution is not far from each other and shows a widely gene flow among the minorities.

[Correlation of Bcl-2 with immunological subtype and prognosis in diffuse large B-cell lymphoma.].

OBJECTIVE: To investigate the Bcl-2 protein and gene expression in diffuse large B-cell lymphoma (DLBCL), and analyze its correlation with immunosubtype and prognosis. METHODS: Seventy-three cases of DLBCL were performed immunohistochemistry analysis with a panel of antibodies CD3, CD10, CD20, Bcl-6, Bcl-2 and MUM-1, and classified into germinal center B-cell (GCB) type and non-GCB type. Fluorescence in situ hybridization (FISH) was employed to detect bcl-2 gene expression in 57 cases with chromosome translocation t (14;18). RESULTS: The percentages of tumor cells expressed CD10, Bcl-6, MUM-1 and Bcl-2 were 15.1%, 38.4%, 71.2% and 79.2%, respectively. 16 cases (21.9%) were GCB type and the rest (78.1%) were non-GCB type. 16 of 57 cases (28.1%) were t (14; 18), including 5 of GCB type (31.2%) and 11 of non-GCB type (68.2%). The expression of Bcl-2 protein was correlated with immunological subtype (P = 0.035), but not with survival time (P = 0.253). Between the t(14;18) positive and negtive groupes, there was significant difference for survival time (P = 0.022), but no difference for immunological subtype (P = 0.340). There was no correlation between Bcl-2 protein and t(14;18). CONCLUSIONS: GCB type DBLBCL with expression of Bcl-2 protein had a poor prognosis. t(14; 18) positive BLBCL had poor prognosis. The expression of Bcl-2 protein and t(14; 18) are usually discordant.

[Effect of tanshinone II on hepatic fibrosis in mice].

OBJECTIVE: To explore the mechanism of action of tanshinone II A for liver protection in hepatic fibrotic mice by observing its effects on signaling pathway of insulin-like growth factor binding protein 7 (IGFBP7) and TGFbeta1/Smad3. METHODS: Hepatic fibrosis model was induced by intraperitoneal injection of thioacetamide (TAA). Thirty-six male Kunming mice were divided into five groups: the normal control group (N), the 4-week model group (A), the 4-week tanshinone II A prevented group (B), the 6-week model group (C) and the 3-week tanshinone II A treated group (D). Changes of serum levels of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), histopathology of liver (HE staining), area density of collagen in liver (Masson staining), expressions of alpha-smooth muscle actin (alpha-SMA), collagen I , fibronectin (FN), transforming growth factor-beta1 (TGF-beta1), Smad3 and IGFBP7 in liver (by immunohiStochemistry), liver content of FN, Smad3 and IGFBP7 (by Western blot), and the hepatocyte apoptosis (by TUNEL) were observed. RESULTS: The serum levels of ALT and LDH, the expressions of alpha-SMA, collagen I , TGF-beta1 in liver, expressions and contents of FN, Smad3 and IGFBP7 in liver were significantly lower; the liver damage and the hepatic apoptosis index were lesser in Group B than in Group A, also in Group D than in Group C, respectively, all showing statistical significance (P < 0.05). CONCLUSION: Tanshinone II A could improve liver function, inhibit the activation of hepatic stellate cells, reduce the production of extracellular matrix, and protect the hepatocytes, and its of mechanisms of actions might be related with blocking TGF-beta1/Smad3 signaling pathway and down-regulating the expression of IGFBP7 in liver.

[Relationship among HPV16 infection and Expression of hTERT, p21waf1, Ki67 in cervical intraepithelial neoplasias and squamous cell carcinomas of cervix uteri].

BACKGROUND: To investigate the relationship among human papillomavirus (HPV)16 infection and the expression of telomerase catalytic protein subunit (hTERT), tumor suppressor gene p21waf1, proliferation antigen Ki67 in cervical intraepithelial neoplasias (CIN) and squamous cell carcinomas (SCC) of cervix uteri and their significance. METHODS: Tissue microarray combined with in situ hybridization (ISH) and immunohistochemical staining (EliVision plus method) was used to detect the expression of HPV16 RNA, hTERT, Ki67 and p21waf1 proteins in the cervix uteri specimens from 130 subjects, including normal cervical tissue (n=26), CIN (n=46) and SCC (n=58). RESULTS: (1) The positive rate of HPV16 hybridization signals and expression of hTERT, Ki67 in CINII-III, in situ carcinoma and invasive SCC were all significantly higher than in normal cervical tissue (P < 0.05 for all), and was also higher in SCC than in CIN (P < 0.05 for all). There was a significant difference among CIN I, II and III (P < 0.05 for all). (2) The positive expression of p21waf1 protein only in SCC was significantly lower than in normal cervical tissue (P < 0.05), but there was no significant differences among other groups (all P > 0.05). (3) The positive rate of HPV16 and the expression of Ki67 showed respectively positive being correlated with the expression of hTERT (P < 0.05, r=0.339; P < 0.05, r=0.398); HPV16, hTERT and Ki67 showed respectively negative correlation with the expression of p21waf1 (P < 0.05, r=-0.337; P < 0.05, r=-0.248; P < 0.05, r=-0.446); There was no significant relation between HPV16 and Ki67 (P > 0.05). CONCLUSION: The results suggest that in tissues of CIN and SCC changes in hTERT, p21waf1 and Ki67 expression may be associated with HPV16 infection and they interact with each other, which can influent the progression of CIN and carcinogenesis of SCC. These biomarkers may be analyzed comprehensively to reveal the detailed mechanism by which HPV16 participate in malignant transformation and to provide some informations on the diagnosis of patients with high risk for malignant progression. Tissue microarray is an efficient platform for high-throughput analysis of genes and their expression products in investigations.