Researchers commonly use cyclization recombination enzyme/locus of X-over P1 (Cre/loxP) technology-based conditional gene knockouts of model mice to investigate the functional roles of genes of interest in Sertoli and Leydig cells within the testis. However, the shortcomings of these genetic tools include high costs, lengthy experimental periods, and limited accessibility for researchers. Therefore, exploring alternative gene silencing techniques is of great practical value. In this study, we employed adeno-associated virus (AAV) as a vector for gene silencing in Sertoli and Leydig cells. Our findings demonstrated that AAV serotypes 1, 8, and 9 exhibited high infection efficiency in both types of testis cells. Importantly, we discovered that all three AAV serotypes exhibited exquisite specificity in targeting Sertoli cells via tubular injection while demonstrating remarkable selectivity in targeting Leydig cells via interstitial injection. We achieved cell-specific knockouts of the steroidogenic acute regulatory ( Star ) and luteinizing hormone/human chorionic gonadotropin receptor (Lhcgr) genes in Leydig cells, but not in Sertoli cells, using AAV9-single guide RNA (sgRNA)-mediated gene editing in Rosa26-LSL-Cas9 mice. Knockdown of androgen receptor ( Ar ) gene expression in Sertoli cells of wild-type mice was achieved via tubular injection of AAV9-short hairpin RNA (shRNA)-mediated targeting. Our findings offer technical approaches for investigating gene function in Sertoli and Leydig cells through AAV9-mediated gene silencing.
Animals ; Male ; Leydig Cells/metabolism* ; Mice ; Dependovirus/genetics* ; Sertoli Cells/metabolism* ; Gene Silencing ; Genetic Vectors ; Testis/cytology*

p120-catenin(p120ctn)is one of the crucial members of armadillo family,which is well known as a core stabilizing factor for vascular endothelial cadherin(VE-cadherin).Throughout the entire process of vascular development,p120ctn plays multiple roles.From neovascularization to endothelial barrier,the presence of p120ctn is indispensable.It participates in regulating the normal development of vertebrate embryo vasculature and promoting endothelial cell proliferation.Additionally,p120ctn contributes to the maintenance and remodeling of adherens junctions and can modulate adhesion strength by altering cell morphology.This review summarized the latest research progress on p120ctn from neovascular development and endothelial barrier function.

p120-catenin(p120ctn)is one of the crucial members of armadillo family,which is well known as a core stabilizing factor for vascular endothelial cadherin(VE-cadherin).Throughout the entire process of vascular development,p120ctn plays multiple roles.From neovascularization to endothelial barrier,the presence of p120ctn is indispensable.It participates in regulating the normal development of vertebrate embryo vasculature and promoting endothelial cell proliferation.Additionally,p120ctn contributes to the maintenance and remodeling of adherens junctions and can modulate adhesion strength by altering cell morphology.This review summarized the latest research progress on p120ctn from neovascular development and endothelial barrier function.

Objective:To explore the prevalence and independent associated factors of vascular calcification (VC) in non-dialysis chronic kidney disease (CKD) patients of stage 3-5.Methods:It was a single-center cross-sectional observational study. Non-dialysis stage 3-5 CKD patients ≥18 years old who were admitted to the Department of Nephrology, the First Affiliated Hospital of Sun Yat-sen University from May 1, 2022 to December 31, 2022 with VC evaluation were enrolled. The patients' general information, laboratory examination and imaging data were collected. Coronary artery calcification (CAC), thoracic aorta calcification (TAC), abdominal aorta calcification (AAC), carotid artery calcification and aortic valve calcification (AVC) were evaluated by cardiac-gated electron-beam CT (EBCT) scans, lateral lumbar x-ray, cervical macrovascular ultrasound and echocardiography, respectively. The differences in clinical data and the prevalence of VC at different sites of patients with different CKD stages were compared, and the prevalence of VC at different sites of patients in different age groups [youth group (18-44 years old), middle-aged group (45-64 years old) and elderly group (≥65 years old)] and patients with or without diabetes were compared. Multivariate logistic regression analysis was used to analyse the independent associated factors of VC for different areas.Results:A total of 206 patients aged (51±14) years were included, including 129 (62.6%) males. There were 44 patients with CKD stage 3 (21.4%), 51 patients with CKD stage 4 (24.8%), and 111 patients with CKD stage 5 (53.9%). CKD was caused by chronic glomerulonephritis [104 cases (50.5%)], diabetic kidney damage [35 cases (17.0%)], hypertensive kidney damage [29 cases (14.1%)] and others [38 cases (18.4%)]. Among 206 patients, 131 (63.6%) exhibited cardiovascular calcification, and the prevalence of CAC, TAC, AAC, carotid artery calcification, and AVC was 37.9%, 43.7%, 37.9%, 35.9% and 9.7%, respectively. The overall prevalence of VC in young, middle-aged and elderly patients was 24.6%, 73.6% and 97.4%, respectively. With the increase of age, the prevalence of VC in each site gradually increased, and the increasing trend was statistically significant (all P<0.001). The overall prevalence of VC in CKD patients with diabetes was 92.5% (62/67), and the prevalence of VC at each site in the patients with diabetes was significantly higher than that in the patients without diabetes (all P<0.001). Multivariate logistic regression analysis revealed that age (every 10 years increase, OR=2.51, 95% CI 1.77-3.56, P<0.001), hypertension ( OR=5.88, 95% CI 1.57-22.10, P=0.009), and diabetes ( OR=4.66, 95% CI 2.10-10.35, P<0.001) were independently correlated with CAC; Age (every 10 years increase, OR=6.43, 95% CI 3.64-11.36, P<0.001) and hypertension ( OR=6.09, 95% CI 1.33-27.84, P=0.020) were independently correlated with TAC; Female ( OR=0.23, 95% CI 0.07-0.72, P=0.011), age (every 10 years increase, OR=3.90, 95% CI 2.42-6.29, P<0.001), diabetes ( OR=5.37, 95% CI 2.19-13.19, P<0.001) and serum magnesium ( OR=0.01,95% CI 0-0.35, P=0.014) were independently correlated with AAC. Moreover, age and diabetes were independently correlated with carotid artery calcification, AVC and overall VC Conclusions:The prevalence of VC in non-dialysis CKD patients of stage 3-5 is 63.59%, of which CAC reaches 37.9%, TAC is the most common one (43.7%), while AVC is the least one (9.7%). Age and diabetes are the independent associated factors for VC of all sites except TAC, while hypertension is an independent associated factor for both CAC and TAC.

Objective:To analyze the clinical phenotype, sperm morphological characteristics and assisted reproductive therapy efficiency in patients with total globozoospermia.Methods:Four male patients with total globozoospermia were collected during November 2019 to May 2022 from Reproductive Medical and Genetic Center, the People's Hospital of Guangxi Zhuang Autonomous Region. Peripheral blood samples were collected for genetic detection and the whole exome sequencing to explore the pathogenic genes. Semen characteristics, sperm morphology and ultrastructure were analyzed. Four patients were treated with intracytoplasmic sperm injection (ICSI) combined with artificial oocyte activation (AOA). Conventional ICSI cycles ( n=9) were selected as control group, and the development dynamic parameters were monitored by Time-lapse. The fertilization and embryo development parameters, developmental dynamic parameters and clinical outcomes were analyzed between the two groups. Results:Four patients were complicated with low sperm motility and increased sperm DNA fragmentation. Sperm morphology analysis and acrosome fluorescence staining represented that all the spermatozoas were with a small round head lacked of acrosome. By the transmission electron microscope, it was observed that round-headed spermatozoas were lacked of acrosome completely, loose chromatin structure, vacuolation and other abnormal changes in the head, mitochondrial sheath in neck were reduced arranged in disorder, and the structure of "9+2" of the flagellar axial filament was incomplete. Of the 4 patients, 1 was homozygous deletion of DPY19L2 gene and 1 was homozygous frameshift mutation of DPY19L2 gene. There were no significance differences in fertilization rate, two pronuclei fertilization rate, day 3 high-quality embryo rate, day 6 blastocyst formation rate and day 6 high-quality blastocyst formation rate between total globozoospermia group and control group (all P>0.05). The developmental dynamic parameters as the time at which the second polar body is extruded, the time when both (or the last) PN disappear, two to six discrete cells in the total globozoospermia group were significantly earlier than those in control group, and the difference was statistically significant (all P<0.05). There was no significant difference in the embryo cleavage patterns between the two groups ( P>0.05). Among the 4 patients with total globozoospermia, 2 live births with signal healthy male baby were achieved by fresh embryo transfer, and 2 live births with one signal healthy male baby and one healthy female baby were achieved by frozen-thawed embryo transfer respectively. Conclusion:Abnormal morphology characteristics of spermatozoas from patients with total globozoospermia are obvious, patients with total globozoospermia could have favorable clinical outcomes following ICSI combined with AOA.

Objective:To analyze the clinical phenotype, sperm morphological characteristics and assisted reproductive therapy efficiency in patients with total globozoospermia.Methods:Four male patients with total globozoospermia were collected during November 2019 to May 2022 from Reproductive Medical and Genetic Center, the People's Hospital of Guangxi Zhuang Autonomous Region. Peripheral blood samples were collected for genetic detection and the whole exome sequencing to explore the pathogenic genes. Semen characteristics, sperm morphology and ultrastructure were analyzed. Four patients were treated with intracytoplasmic sperm injection (ICSI) combined with artificial oocyte activation (AOA). Conventional ICSI cycles ( n=9) were selected as control group, and the development dynamic parameters were monitored by Time-lapse. The fertilization and embryo development parameters, developmental dynamic parameters and clinical outcomes were analyzed between the two groups. Results:Four patients were complicated with low sperm motility and increased sperm DNA fragmentation. Sperm morphology analysis and acrosome fluorescence staining represented that all the spermatozoas were with a small round head lacked of acrosome. By the transmission electron microscope, it was observed that round-headed spermatozoas were lacked of acrosome completely, loose chromatin structure, vacuolation and other abnormal changes in the head, mitochondrial sheath in neck were reduced arranged in disorder, and the structure of "9+2" of the flagellar axial filament was incomplete. Of the 4 patients, 1 was homozygous deletion of DPY19L2 gene and 1 was homozygous frameshift mutation of DPY19L2 gene. There were no significance differences in fertilization rate, two pronuclei fertilization rate, day 3 high-quality embryo rate, day 6 blastocyst formation rate and day 6 high-quality blastocyst formation rate between total globozoospermia group and control group (all P>0.05). The developmental dynamic parameters as the time at which the second polar body is extruded, the time when both (or the last) PN disappear, two to six discrete cells in the total globozoospermia group were significantly earlier than those in control group, and the difference was statistically significant (all P<0.05). There was no significant difference in the embryo cleavage patterns between the two groups ( P>0.05). Among the 4 patients with total globozoospermia, 2 live births with signal healthy male baby were achieved by fresh embryo transfer, and 2 live births with one signal healthy male baby and one healthy female baby were achieved by frozen-thawed embryo transfer respectively. Conclusion:Abnormal morphology characteristics of spermatozoas from patients with total globozoospermia are obvious, patients with total globozoospermia could have favorable clinical outcomes following ICSI combined with AOA.

Objective To understand the distribution and antimicrobial resistance changes of bacteria isolated from pleural and peritoneal effusion in Hunan Province,and provide reference for correct clinical diagnosis and rational antimicrobial use.Methods Data reported by member units of Hunan Provincial Antimicrobial Resistance Survei-llance System from 2012 to 2021 were collected.Bacteria antimicrobial resistance surveillance method was imple-mented according to technical scheme of China Antimicrobial Resistance Surveillance System(CARSS),and WHO-NET 5.6 software was used to analyze the data of bacteria isolated from pleural and peritoneal effusion as well as antimicrobial susceptibility testing results.Results From 2012 to 2021,a total of 28 934 bacterial strains were iso-lated from specimens of pleural and peritoneal effusions from member units of Hunan Provincial Antimicrobial Re-sistance Surveillance System,with 5 752 strains from pleural effusion and 23 182 from peritoneal effusion.The top five bacteria isolated from pleural effusion were Escherichia coli(n=907,15.8％),Staphylococcus aureus(n=535,9.3％),Klebsiella pneumoniae(n=369,6.4％),Staphylococcus epidermidis(n=452,7.9％),and Staphy-lococcus haemolyticus(n=285,5.0％).The detection rate of methicillin-resistant Staphylococcus aureus(MR-SA)from pleural effusion was 24.3％-39.2％,and that of methicillin-resistant coagulase negative Staphylococcus(MRCNS)was 58.8％-77.1％.The top five bacteria isolated from peritoneal effusion were Escherichia coli(n=8 264,35.6％),Klebsiella pneumoniae(n=2 074,9.0％),Enterococcus faecium(n=1 458,6.3％),Staphylo-coccus epidermidis(n=1 383,6.0％),and Pseudomonas aeruginosa(n=1 152,5.0％).The detection rate of MRSA from peritoneal effusion was 22.1％-52.4％,which presented a decreasing trend(P=0.004).The detec-tion rate of MRCNS was 60.4％-79.4％.The resistance rates of Enterobacterales from peritoneal effusion to ce-fazolin,cefuroxime,ceftriaxone and cefepime all showed decreasing trends(all P＜0.05).Vancomycin-,linezo-lid-,and teicoplanin-resistant Staphylococcus strains were not found in pleural and peritoneal effusions.The resis-tance rates of Enterococcus faecium to most tested antimicrobial agents were higher than those of Enterococcus fae-calis.The resistance rates of Enterobacterales to imipenem and meropenem were ≤8.5％.The resistance rates of non-fermentative Gram-negative bacilli to imipenem and meropenem were ≤43.3％.Conclusion The data structure of Hunan Antimicrobial Resistance Surveillance System for pleural and peritoneal effusions from 2012 to 2021 is relatively complete.The constituent and antimicrobial susceptibility of isolated pathogenic bacteria vary in different years.

In this study, the underlying mechanism of Qiwei Guibao Granules(QWGB) in the treatment of premature ovarian fai-lure(POF) was explored by the proteomics technique. Firstly, the POF model was induced in mice by intragastric administration of Tripterygium wilfordii glycosides solution at 50 mg·kg~(-1) for 14 days. Ten days prior to the end of the modeling, the estrous cycle of mice was observed every day to evaluate the success of modeling. From the 1st day after modeling, the POF model mice were treated with QWGB by gavage every day and the treatment lasted four weeks. On the 2nd day after the end of the experiment, blood was collected from the eyeballs and the serum was separated by centrifugation. The ovaries and uterus were collected and the adipose tissues were carefully stripped. The organ indexes of the ovaries and uterus of each group were calculated. The serum estrogen(E_2) level of mice in each group was detected by ELISA. Protein samples were extracted from ovarian tissues of mice, and the differential proteins before and after QWGB intervention and before and after modeling were analyzed by quantitative proteomics using tandem mass tags(TMT). As revealed by the analysis of differential proteins, QWGB could regulate 26 differentially expressed proteins related to the POF model induced by T. wilfordii glycosides, including S100A4, STAR, adrenodoxin oxidoreductase, XAF1, and PBXIP1. GO enrichment results showed that the 26 differential proteins were mainly enriched in biological processes and cellular components. The results of KEGG enrichment showed that those differential proteins were involved in signaling pathways such as completion and coalescence cascades, focal adhesion, arginine biosynthesis, and terpenoid backbone biosynthesis. The complement and coalescence cascades signaling pathway was presumably the target pathway of QWGB in the treatment of POF. In this study, the proteomics technique was used to screen the differential proteins of QWGB in the treatment of POF in mice induced by T. wilfordii glycosides, and they were mainly involved in immune regulation, apoptosis regulation, complement and coagulation cascade reactions, cholesterol metabolism, and steroid hormone production, which may be the main mechanisms of QWGB in the treatment of POF.
Female ; Humans ; Mice ; Animals ; Primary Ovarian Insufficiency/chemically induced* ; Proteomics ; Signal Transduction ; Glycosides/adverse effects*

OBJECTIVES: Safe and effective anticoagulation is essential for hemodialysis patients who are at high risk of bleeding. The purpose of this trial is to evaluate the effectiveness and safety of two-stage regional citrate anticoagulation (RCA) combined with sequential anticoagulation and standard calcium-containing dialysate in intermittent hemodialysis (IHD) treatment. METHODS: Patients at high risk of bleeding who underwent IHD from September 2019 to May 2021 were prospectively enrolled in 13 blood purification centers of nephrology departments, and were randomly divided into RCA group and saline flushing group. In the RCA group, 0.04 g/mL sodium citrate was infused from the start of the dialysis line during blood draining and at the venous expansion chamber. The sodium citrate was stopped after 3 h of dialysis, which was changed to sequential dialysis without anticoagulant. The hazard ratios for coagulation were according to baseline. RESULTS: A total of 159 patients and 208 sessions were enrolled, including RCA group (80 patients, 110 sessions) and saline flushing group (79 patients, 98 sessions). The incidence of severe coagulation events of extracorporeal circulation in the RCA group was significantly lower than that in the saline flushing group (3.64% vs. 20.41%, P<0.001). The survival time of the filter pipeline in the RCA group was significantly longer than that in the saline flushing group ((238.34±9.33) min vs. (221.73±34.10) min, P<0.001). The urea clearance index (Kt/V) in the RCA group was similar to that in the saline flushing group with no statistically significant difference (1.12±0.34 vs. 1.08±0.34, P=0.41). CONCLUSIONS Compared with saline flushing, the two-stage RCA combined with a sequential anticoagulation strategy significantly reduced extracorporeal circulation clotting events and prolonged the dialysis time without serious adverse events.
Humans ; Citric Acid/adverse effects* ; Prospective Studies ; Sodium Citrate ; Hemorrhage/chemically induced* ; Citrates/adverse effects* ; Anticoagulants/adverse effects* ; Renal Dialysis/adverse effects*