Based on whole-genome identification of the TPS gene family in Perilla frutescens and screening, cloning, bioinformatics, and expression analysis of the synthetic enzyme for the insect-resistant component germacrene D, this study lays the foundation for understanding the biological function of the TPS gene family and the insect resistance mechanism in P. frutescens. This study used bioinformatics tools to identify the TPS gene family of P. frutescens based on its whole genome and predicted the physicochemical properties, systematic classification, and promoter cis-elements of the proteins. The relative content of germacrene D was detected in both normal and insect-infested leaves of P. frutescens, and the germacrene D synthase was screened and isolated. Gene cloning, bioinformatics analysis, and expression profiling were then performed. The results showed that a total of 99 TPS genes were identified in the genome, which were classified into the TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g subfamilies. Conserved motif analysis showed that the TPS in P. frutescens has conserved structural characteristics within the same subfamily. Promoter cis-element analysis predicted the presence of light-responsive elements, multiple hormone-responsive elements, and stress-responsive elements in the TPS family of P. frutescens. Transcriptome data revealed that most of the TPS genes in P. frutescens were highly expressed in the leaves. GC-MS analysis showed that the relative content of germacrene D significantly increased in insect-damaged leaves, suggesting that it may act as an insect-resistant component. The germacrene D synthase gene was screened through homologous protein binding gene expression and was found to belong to the TPS-a subfamily, encoding a 64.89 kDa protein. This protein was hydrophilic, lacked a transmembrane structure and signal peptide, and was predominantly expressed in leaves, with significantly higher expression in insect-damaged leaves compared to normal leaves. In vitro expression results showed that germacrene D synthase tended to form inclusion bodies. Molecular docking showed that farnesyl pyrophosphate(FPP) fell into the active pocket of the protein and interacted strongly with six active sites. This study provides a foundation for further research on the biological functions of the TPS gene family in P. frutescens and the molecular mechanisms underlying its insect resistance.
Perilla frutescens/chemistry* ; Plant Proteins/chemistry* ; Multigene Family ; Sesquiterpenes, Germacrane/metabolism* ; Alkyl and Aryl Transferases/chemistry* ; Phylogeny ; Gene Expression Regulation, Plant

To investigate the impact of preoperative serum follicle-stimulating hormone (FSH) levels on the probability of testicular sperm retrieval, we conducted a study of nonobstructive azoospermic (NOA) men with different testicular volumes (TVs) who underwent microdissection testicular sperm extraction (micro-TESE). A total of 177 NOA patients undergoing micro-TESE for the first time from April 2019 to November 2022 in Shenzhen Zhongshan Obstetrics and Gynecology Hospital (formerly Shenzhen Zhongshan Urology Hospital, Shenzhen, China) were retrospectively reviewed. The subjects were divided into four groups based on average TV quartiles. Serum hormone levels in each TV group were compared between positive and negative sperm retrieval subgroups. Overall sperm retrieval rate was 57.6%. FSH levels (median [interquartile range]) were higher in the positive sperm retrieval subgroup compared with the negative outcome subgroup when average TV was <5 ml (first quartile [Q1: TV <3 ml]: 43.32 [17.92] IU l -1 vs 32.95 [18.56] IU l -1 , P = 0.048; second quartile [Q2: 3 ml ≤ TV <5 ml]: 31.31 [15.37] IU l -1 vs 25.59 [18.40] IU l -1 , P = 0.042). Elevated serum FSH levels were associated with successful micro-TESE sperm retrieval in NOA men whose average TVs were <5 ml (adjusted odds ratio [OR]: 1.06 per unit increase; 95% confidence interval [CI]: 1.01-1.11; P = 0.011). In men with TVs ≥5 ml, larger TVs were associated with lower odds of sperm retrieval (adjusted OR: 0.84 per 1 ml increase; 95% CI: 0.71-0.98; P = 0.029). In conclusion, elevated serum FSH levels were associated with positive sperm retrieval in micro-TESE in NOA men with TVs <5 ml. In men with TV ≥5 ml, increases in average TVs were associated with lower odds of sperm retrieval.
Humans ; Male ; Azoospermia/surgery* ; Sperm Retrieval/statistics & numerical data* ; Adult ; Follicle Stimulating Hormone/blood* ; Retrospective Studies ; Testis/pathology* ; Microdissection ; Organ Size

This study investigated the specific immune response of BALB/c mice that was induced by a circular RNA (circRNA) vaccine expressing the herpes simplex virus type II (HSV-2) glycoprotein D (gD). The aim was to evaluate the immunological potential of this vaccine and lay a foundation for developing an mRNA vaccine against HSV-2. PCR and homologous recombination were employed to integrate the gD gene obtained from the pT7AMP-gD ectodomain plasmid into pUC57 to generate the recombinant plasmid pUC57-circ-gD, which was then sequenced and characterized. In vitro transcription and cyclization were performed on the template DNA to generate pUC57-circ-gD mRNA. To validate the formation of circular RNA, we cleaved the pUC57-circ-gD mRNA with RNase R and employed RT-PCR to validate the cyclization. The pUC57-circ-gD mRNA was then transfected into 293T cells. After 72 h, the cell supernatant was collected, and Western blotting was employed to measure the protein level of gD. Subsequently, the mRNA was encapsulated in lipid nanoparticles (LNPs) by microfluidic encapsulation. BALB/c mice were administrated with the encapsulated mRNA, and blood was collected from the fundus venous plexus after 21 and 35 days, and from the enucleated eyeballs after 49 days. Enzyme-linked immunosorbent assay was employed to measure the titers of antibodies, including virus-neutralizing antibodies. After 49 days, spleens were harvested and assessed for secretion of interferon-gamma (IFN-γ) by solid-phase enzyme-linked immunospot. The results showed successful construction and sequencing of the recombinant plasmid. RNase R digestion confirmed the presence of circular RNAs. Western blotting of the 293T cells transfected with the mRNA showed clear specific bands. The quality of the vaccine was tested by size exclusion chromatography-high performance liquid chromatography, which showed that the purity of the vaccine was about 90%. The mRNA-LNP showcased the particle size of 82.76 nm and an encapsulation rate of approximately 98%. Following three-dose vaccination, all immunized mice exhibited steady weight gain with 100% survival rate throughout the 28-day observation period, indicating no significant acute toxicity associated with the vaccine formulation. The immunized mice showed dose-dependent increases in serum IgG antibody titer and IFN-γ secretion by splenocytes and they were resistant to virus attacks. These findings indicate good immunogenicity and persistence of the pUC57-circ-gD mRNA vaccine, providing a reference for further studies on circRNA vaccines.
Animals ; Mice, Inbred BALB C ; RNA, Circular ; Mice ; Humans ; Herpesvirus 2, Human/genetics* ; Viral Envelope Proteins/genetics* ; Antibodies, Viral/blood* ; HEK293 Cells ; Female ; Nanoparticles ; Plasmids

Objective:To investigate the effectiveness and clinical efficacy of tension-free vaginal tape(TVT)surgery in treating female recurrent stress urinary incontinence(rSUI).Methods:A retrospec-tive analysis was conducted on 24 patients who experienced recurrence of mid-urethral sling failure and were treated with TVT surgery at Beijing Chao-Yang Hospital from January 2016 to June 2020.Basic pa-tient information was collected.The International Consultation on Incontinence questionnaire-short form(ICI-Q-SF)was used to record urinary incontinence symptom scores preoperatively,1-year postoperative-ly,and more than three years postoperatively.The changes in various ICI-Q-SF scores and total scores were compared.Additionally,clinical symptom severity of urinary incontinence was recorded and com-pared preoperatively and more than three years postoperatively.Results:Among the 24 patients included in the follow-up,one patient was lost to follow-up due to death from a cerebrovascular accident one year postoperatively,leaving 23 patients with a follow-up period ranging from 3.9 to 7.3 years,with an ave-rage follow-up time of(5.2±1.1)years.Preoperatively,the median ICI-Q-SF total score was 20.0(16.0,21.0);at the 1-year follow-up,the median ICI-Q-SF total score was 5.0(1.5,7.8)(P＜0.001);at an average follow-up of five years,the median ICI-Q-SF total score was 6.0(3.0,9.0),still showing a statistically significant difference compared with preoperative scores(P＜0.001).The in-dividual ICI-Q-SF scores were significantly lower at the 1-year and average five-year follow-ups compared with preoperative scores(P＜0.001).Regarding the severity of urinary incontinence,all the patients had moderate to severe urinary incontinence preoperatively;Five years postoperatively,87.0％(20/23)of the patients had no or only mild urinary incontinence,and 13.0％(3/23)had recurrent moderate to severe urinary incontinence(P＜0.001).Conclusion:TVT surgery is effective in treating female recur-rent stress urinary incontinence,with an average 5-year cure and improvement rate of 87.0％.

Objective To investigate the expression level of circular RNA circ-TNRC6A in bladder cancer tissues and its mechanism of regulating the proliferation and migration of bladder cancer cells.Methods The expression level of circ-TNRC6A in bladder cancer tissues and its relationship with clinical stage of patients with bladder cancer were analyzed using the Cancer Genome Atlas database.The expression levels of circ-TNRC6A in human normal bladder epithelial cell SV-HUC-1 and bladder cancer cell lines(MGH-U3,5637,RT-4,T24,J82)were analyzed by real-time fluorescence quantitative PCR(qPCR).The circ-TNRC6A plas-mid(circ-TNRC6A group)and the control plasmid(NC group)were transfected into 5637 bladder cancer cells,respectively.The effects of circ-TNRC6A on the proliferation and migration of bladder cancer cells were detected by colony formation assay and cell scratch assay,respectively.The targeting relationship between circ-TNRC6A and microRNA(miR)-494-3p was predicted by bioinformatics technology and confirmed by lu-ciferase reporter gene assay.qPCR was used to detect the effect of circ-TNRC6A on miR-494-3p expression.Western blot was used to detect the effect of circ-TNRC6A on the expression of key proteins in Wnt/β-catenin signaling pathway.Results circ-TNRC6A was down-regulated in bladder cancer tissues compared with adja-cent tissues(P＜0.01).The expression level of circ-TNRC6A was correlated with the clinical stage of bladder cancer(P＜0.05).Compared with SV-HUC-1 cells,the expression of circ-TNRC6A was lower in bladder cancer cell lines(all P＜0.05),and the expression level of circ-TNRC6A was the lowest in 5637 cells(P＜0.01).Compared with the NC group,overexpression of circ-TNRC6A inhibited the proliferation of 5637 cells(P＜0.01)and reduced the migration ability of 5637 cells(P＜0.01).circ-TNRC6A could target miR-494-3p(P＜0.01).Compared with NC group,overexpression of circ-TNRC6A significantly reduced the expression level of miR-494-3p(P＜0.01)and inhibited the activation of Wnt/β-catenin signaling pathway(P＜0.01).Conclusion circ-TNRC6A inhibits the proliferation and migration of bladder cancer cells by down-regulating miR-494-3p.circ-TNRC6A may be a new therapeutic target for bladder cancer.

The human oral microbiome harbors one of the most diverse microbial communities in the human body,playing critical roles in oral and systemic health.Recent technological innovations are propelling the characterization and manipulation of oral microbiota.High-throughput sequencing enables comprehensive taxonomic and functional profiling of oral microbiomes.New long-read platforms improve genome assembly from complex samples.Single-cell genomics provides insights into uncultured taxa.Advanced imaging modalities including fluorescence,mass spectrometry,and Raman spectroscopy have enabled the visualization of the spatial organization and interactions of oral microbes with increasing resolution.Fluorescence techniques link phylogenetic identity with localization.Mass spectrometry imaging reveals metabolic niches and activities while Raman spectroscopy generates rapid biomolecular fingerprints for classification.Culturomics facilitates the isolation and cultivation of novel fastidious oral taxa using high-throughput approaches.Ongoing integration of these technologies holds the promise of transforming our understanding of oral microbiome assembly,gene expression,metabolites,microenvironments,virulence mechanisms,and microbe-host interfaces in the context of health and disease.However,significant knowledge gaps persist regarding community origins,developmental trajectories,homeostasis versus dysbiosis triggers,functional biomarkers,and strategies to deliberately reshape the oral microbiome for therapeutic benefit.The convergence of sequencing,imaging,cultureomics,synthetic systems,and biomimetic models will provide unprecedented insights into the oral microbiome and offer opportunities to predict,prevent,diagnose,and treat associated oral diseases.

The human oral microbiome harbors one of the most diverse microbial communities in the human body,playing critical roles in oral and systemic health.Recent technological innovations are propelling the characterization and manipulation of oral microbiota.High-throughput sequencing enables comprehensive taxonomic and functional profiling of oral microbiomes.New long-read platforms improve genome assembly from complex samples.Single-cell genomics provides insights into uncultured taxa.Advanced imaging modalities including fluorescence,mass spectrometry,and Raman spectroscopy have enabled the visualization of the spatial organization and interactions of oral microbes with increasing resolution.Fluorescence techniques link phylogenetic identity with localization.Mass spectrometry imaging reveals metabolic niches and activities while Raman spectroscopy generates rapid biomolecular fingerprints for classification.Culturomics facilitates the isolation and cultivation of novel fastidious oral taxa using high-throughput approaches.Ongoing integration of these technologies holds the promise of transforming our understanding of oral microbiome assembly,gene expression,metabolites,microenvironments,virulence mechanisms,and microbe-host interfaces in the context of health and disease.However,significant knowledge gaps persist regarding community origins,developmental trajectories,homeostasis versus dysbiosis triggers,functional biomarkers,and strategies to deliberately reshape the oral microbiome for therapeutic benefit.The convergence of sequencing,imaging,cultureomics,synthetic systems,and biomimetic models will provide unprecedented insights into the oral microbiome and offer opportunities to predict,prevent,diagnose,and treat associated oral diseases.

Objective To investigate the expression of pepsin in vocal cord polyps and vocal cord cancer,and to compare the difference of pepsin expression.Methods From May 2020 to December 2021,27 patients with vocal cord polyp,27 patients with vocal cord cancer and 23 healthy volunteers were selected.RSI and RFS scoring scales were used for scoring,pepsin detection kit was used for saliva pepsin detection,and immunohistochemical methods were used to detect the expression of pepsin in vocal cord tissues of patients with vocal cord polyps and vocal cord cancer.Results The RSI score,RFS score and pepsin test kit results of vocal cord polyp group and vocal cord canc-er group were higher than those of non-vocal cord disease group,and the differences of the three indexes were statis-tically significant(P＜0.05).RSI score,pepsin detection kit results and pepsin immunohistochemistry results of vocal cord polyp group showed no significant difference compared with vocal cord cancer group(P＞0.05).The RFS score of vocal cord polyp group was significantly different from that of vocal cord cancer group(P＜0.05).Conclusion Pepsin may be an important pathogenic factor of vocal cord polyp and vocal cord cancer,and play an im-portant role in the occurrence of these two diseases.The difference of pepsin expression in vocal cord polyp and vo-cal cord cancer suggests that pepsin may have different pathogenesis.

Objective To evaluate the value of miniprobe endoscopic ultrasound(EUS)in guiding endoscopic treatment of small-diameter(maximum diameter less than 1 cm)and low-grade(G1 grade)rectum neuroendocrine neoplasm(R-NEN),and to provide evidence and clues for its clinical application and further research.Methods The clinical data of 85 cases of low-grade(G1 grade)R-NEN with a maximum diameter of less than 1 cm who underwent endoscopic treatment in our center from January 2014 to December 2020 were retrospectively analyzed.The patients were divided into the EUS group(37 cases)and control group(48 cases)according to whether EUS was performed before endoscopic treatment.The positive rate of incision margin,the incidence of complications,the recurrence rate,the hospital stay,the cost of hospitalization and endoscopic therapy were compared between the two groups.Results The positive rate of incision margin in the EUS group was significantly lower than that in control group(P<0.05).There was no significant difference in the incidence of complications,tumor recurrence rate,hospital stay or hospital costs between the two groups(P>0.05).There was statistically significant difference in the endoscopic therapy between the two groups(P<0.05).Conclusion Evaluating the lesion depth of small-diameter and low-grade(G1 grade)R-NEN before surgery by miniprobe EUS and selecting endoscopic surgery according to its results of can significantly reduce the residual risk of resection margin tumors.