OBJECTIVES: The purinergic receptor P2X2 (P2RX2) encodes an ATP-gated ion channel permeable to Na⁺, K⁺, and especially Ca²⁺. Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41 (DFNA41), which manifests as high-frequency hearing loss, accelerated presbycusis, and increased susceptibility to noise-induced damage. Zebrafish, owing to their small size, rapid development, high fecundity, transparent embryos, and high gene conservation with humans, provide an ideal model for studying human diseases and developmental mechanisms. This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system, providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies. METHODS: Two CRISPR targets (sgRNA1 and sgRNA2) spaced 47 bp apart were designed within the zebrafish p2rx2 gene. Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage Tübingen (TU)-strain zebrafish embryos. PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization (hpf). Surviving embryos were raised to adulthood (F0), tail-clipped, genotyped, and screened for positive mosaics. F1 heterozygotes were generated by outcrossing, and F2 homozygous mutants were obtained by intercrossing. Polymerase chain reaction (PCR) combined with sequencing verified mutation type and heritability. At 5 days post-fertilization (dpf), YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region. Auditory evoked potential (AEP) thresholds at 600, 800, 1 000, and 2 000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group. RESULTS: A stable p2rx2 knockout zebrafish line was successfully established. Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon (TAA), leading to early termination of protein translation and loss of function. Embryos developed normally with no gross malformations. At 5 dpf, mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type, although lateral line neuromasts were unaffected. AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type (all P<0.001), indicating reduced hearing sensitivity. CONCLUSIONS We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology. p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies, indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception. The phenotype's restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs. This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.
Animals ; Zebrafish/genetics* ; Receptors, Purinergic P2X2/deficiency* ; CRISPR-Cas Systems/genetics* ; Gene Knockout Techniques ; Phenotype ; Zebrafish Proteins/genetics* ; Disease Models, Animal

Objective To investigate the efficacy of noninvasive matrix RF technology in promoting functional rehabilitation of the pelvic floor in postpartum women who have undergone natural delivery.Methods A prospective study was conducted on 80 nulliparous women who underwent spontaneous vaginal delivery at the hospital from January 2022 to April 2023.Participants were randomly allocated into a control group and an observation group,each comprising 40 individuals,using the random number table method.The control group received biofeedback electrical stimulation therapy,while the observation group was treated with non-invasive matrix radiofrequency(RF)technology.Both groups were assessed for pelvic floor muscle strength,pelvic floor structural parameters(levator hiatus area,levator muscle thickness),and levator ani muscle[measured by the maximum value of Young's modulus(Emax)in both resting and contracting states]before treatment,after one session,and at the end of the treatment period.At the conclusion of the treatment,vaginal lubrication and vaginal sensitivity were evaluated using the Female Sexual Function Index(FSFI).Additionally,the incidence of adverse events was compared between the two groups before and after the completion of treatment.Results After one treatment,there were no significant changes in pelvic floor muscle strength,levator ani muscle hiatus area,and levator ani muscle thickness in the control group compared to pre-treatment levels(P>0.05).In contrast,the observation group showed significant improvements in pelvic floor muscle strength and levator ani muscle thickness compared to pre-treatment levels,while the levator ani muscle hiatus area was significantly reduced(P<0.05).Additionally,at this point,the pelvic floor muscle strength and levator ani muscle thickness in the observation group were significantly higher than those in the control group,whereas the levator ani muscle hiatus area was significantly smaller(P<0.05).By the end of the treatment,both groups demonstrated further improvements in pelvic floor muscle strength and levator ani muscle thickness compared to pre-treatment and post-first-treatment levels,with a continued reduction in the levator ani muscle hiatus area.Notably,the observation group consistently exhibited superior outcomes in all three parameters compared to the control group(P<0.05).At the end of treatment,the Emax differences in the subpubic branch,abdominal muscles,and posterior hypothalamus were significantly greater than those before and after a single treatment session in both groups(P<0.05).Moreover,the Emax differences in these regions were significantly higher in the observation group compared to the control group(P<0.05).Post-treatment,both groups showed increased scores for vaginal lubrication,vaginal sensitivity,and FSFI,with more pronounced improvements observed in the observation group(P<0.05).Following treatment,no significant differences were noted between the two groups regarding the incidence of urinary incontinence,lumbosacral pain,uterine prolapse,and vaginal wall bulge(P>0.05).However,the observa-tion group exhibited a lower frequency of adverse events compared to the control group(P<0.05).Additionally,maternal rehabilitation satisfaction was 100%(40/40)in the observation group,which was significantly higher than 85.00%(34/40)in the control group(P<0.05).Conclusions The application of noninvasive matrix RF technology in the rehabilitation of pelvic floor muscle function in postpartum women who have undergone vaginal delivery demonstrates rapid efficacy.It can enhance pelvic floor muscle strength within a short period,restore pelvic floor structure,and significantly improve vaginal lubrication and sensitivity,thereby enhancing sexual well-being.

Objective To investigate the efficacy of noninvasive matrix RF technology in promoting functional rehabilitation of the pelvic floor in postpartum women who have undergone natural delivery.Methods A prospective study was conducted on 80 nulliparous women who underwent spontaneous vaginal delivery at the hospital from January 2022 to April 2023.Participants were randomly allocated into a control group and an observation group,each comprising 40 individuals,using the random number table method.The control group received biofeedback electrical stimulation therapy,while the observation group was treated with non-invasive matrix radiofrequency(RF)technology.Both groups were assessed for pelvic floor muscle strength,pelvic floor structural parameters(levator hiatus area,levator muscle thickness),and levator ani muscle[measured by the maximum value of Young's modulus(Emax)in both resting and contracting states]before treatment,after one session,and at the end of the treatment period.At the conclusion of the treatment,vaginal lubrication and vaginal sensitivity were evaluated using the Female Sexual Function Index(FSFI).Additionally,the incidence of adverse events was compared between the two groups before and after the completion of treatment.Results After one treatment,there were no significant changes in pelvic floor muscle strength,levator ani muscle hiatus area,and levator ani muscle thickness in the control group compared to pre-treatment levels(P>0.05).In contrast,the observation group showed significant improvements in pelvic floor muscle strength and levator ani muscle thickness compared to pre-treatment levels,while the levator ani muscle hiatus area was significantly reduced(P<0.05).Additionally,at this point,the pelvic floor muscle strength and levator ani muscle thickness in the observation group were significantly higher than those in the control group,whereas the levator ani muscle hiatus area was significantly smaller(P<0.05).By the end of the treatment,both groups demonstrated further improvements in pelvic floor muscle strength and levator ani muscle thickness compared to pre-treatment and post-first-treatment levels,with a continued reduction in the levator ani muscle hiatus area.Notably,the observation group consistently exhibited superior outcomes in all three parameters compared to the control group(P<0.05).At the end of treatment,the Emax differences in the subpubic branch,abdominal muscles,and posterior hypothalamus were significantly greater than those before and after a single treatment session in both groups(P<0.05).Moreover,the Emax differences in these regions were significantly higher in the observation group compared to the control group(P<0.05).Post-treatment,both groups showed increased scores for vaginal lubrication,vaginal sensitivity,and FSFI,with more pronounced improvements observed in the observation group(P<0.05).Following treatment,no significant differences were noted between the two groups regarding the incidence of urinary incontinence,lumbosacral pain,uterine prolapse,and vaginal wall bulge(P>0.05).However,the observa-tion group exhibited a lower frequency of adverse events compared to the control group(P<0.05).Additionally,maternal rehabilitation satisfaction was 100%(40/40)in the observation group,which was significantly higher than 85.00%(34/40)in the control group(P<0.05).Conclusions The application of noninvasive matrix RF technology in the rehabilitation of pelvic floor muscle function in postpartum women who have undergone vaginal delivery demonstrates rapid efficacy.It can enhance pelvic floor muscle strength within a short period,restore pelvic floor structure,and significantly improve vaginal lubrication and sensitivity,thereby enhancing sexual well-being.

Objective To investigate the changes in blood microbiota in patients with high altitude polycythemia(HAPC)and the correlation with the risk of HAPC.Methods A cross-sectional trial was carried out among 41 HAPC patients(HAPC group)and 41 healthy plateau individuals(control group)who took physical examination in the Health Management Department of No.953 Hospital of PLA Army in 2021.High-throughput sequencing technology was used to detect the V3-V4 variable region of the 16S ribosomal RNA(16S rRNA)gene in the blood smaples,and the composition and difference of the blood microbiota were compared and analyzed between the 2 groups.Results All the participants were male and Han people,and there were no significant differences in baseline data such as age,body mass index and plateau migration time between the 2 groups(P＞0.05).The α-diversity of blood microbiota in the HAPC group,including the Simpson index(0.931±0.005 vs 0.907±0.008,P＜0.05),Goods Coverage index(0.998±0.001 vs 0.997±0.001,P＜0.001)and Pielou index(0.597±0.011 vs 0.567±0.009,P＜0.05)were significantly higher than those in the control group.Meanwhile,obvious difference was observed in the β diversity between the 2 groups(P＜0.01).The relative abundance analysis of bacteria showed that Pelomonas(0.046±0.004 vs 0.033±0.003,P＜0.05),Azospirillum(0.046±0.006 vs 0.021±0.003,P＜0.01),Acidovorax(0.032± 0.003 vs 0.019±0.002,Azospirillum(0.046±0.006 vs 0.021±0.003,P＜0.01)and Acidovorax(0.032± 0.003 vs 0.019±0.002,P＜0.05)were statistically higher in the HAPC group than the control group.LEfSe analysis showed that the characteristic blood microbiota of the HAPC group were α-Proteobacteria,and those of control group were Trichospiridae.Conclusion Significant changes are found in diversity,relative abundance and characteristic bacteria of the blood microbiota between the HAPC patients and healthy people at the high altitude,which might be closely associated with the occurrence and development of HAPC.

Objective:To investigate the effects of apolipoprotein E (APOE) ε4 allele on β-amyloid (Aβ) deposition in subcortical structures and functional connectivity (FC) between brain regions in patients with Alzheimer′s disease (AD). Methods:Forty-three patients with probable mild/moderate AD were prospectively enrolled from the First Medical Centre, Chinese PLA General Hospital between January 2023 and October 2023, including 23 APOE ε4+ patients (12 males and 11 females, age (74.8±8.4) years), 20 APOE ε4- patients (14 males and 6 females, age (77.6±8.9) years) and 20 normal cognitive volunteers (NC) (15 males and 5 females, age (75.3±6.2) years). All subjects underwent 11C-Pittsburgh compound B (PIB) PET/MR brain imaging. The differences of gray matter volume (GMV) in subcortical structures (hippocampus, amygdala) among the three groups were analyzed by one-way analysis of variance and least significant difference (LSD) t test. Independent-sample t test and Pearson correlation analysis were used to analyze difference in Aβ deposition between APOE ε4+ patients and APOE ε4- patients, and the correlation between subcortical structure and brain FC. Results:The GMV of bilateral amygdala between NC group and APOE ε4+ gene carrier group, and between APOE ε4+ and APOE ε4- gene carrier groups were significantly different ( F=6.43, P=0.002; P values: 0.002, 0.003). Significant difference of GMV was observed in the bilateral hippocampus among three groups ( F=5.34, P=0.030). Abnormal PIB uptake was detected in both the hippocampus and amygdala of both APOE ε4+ and APOE ε4- gene carrier groups, with a more pronounced effect observed in the APOE ε4+ group ( t values: 3.14, 2.19, P values: 0.032, 0.009). Taking the hippocampus as the seed point, there was no obvious abnormality in the whole brain connectivity map among APOE ε4+, APOE ε4- carriers and NC groups. With the amygdala as the seed point, the whole brain connectivity in the APOE ε4+ gene carrier group was significantly reduced, and the connectivity between the amygdala and the cingulate gyrus, parietal lobe and temporal lobe was significantly reduced in the APOE ε4+ gene carrier group compared with NC group, while the connectivity between the amygdala and the whole brain was not significantly reduced in the APOE ε4- gene carrier group. Aβ deposition in amygdala was positively correlated with FC coefficients of frontal brain regions, gyrus rectus, right middle occipital gyrus and left temporal lobe ( r values: 0.56-0.70, all P<0.05). Conclusion:APOE influences GMV and Aβ deposition of hippocampus and amygdala, and FC of amygdala, and may be involved in the pathological mechanism of cognitive impairment.

Objective:To investigate the positive rate and isolate Bordetella pertussis in children with suspected whooping cough and their close family members in Zhejiang Province, and further explore the susceptibility and resistant mechanism of Bordetella pertussis to antibiotics. Methods:A total of 273 nasopharynx swabs specimens from children with suspected whooping cough in Hangzhou Children′s Hospital from May 2022 to October 2022 were collected. The strains were isolated and cultured using charcoal select agar plate. Pertussis target genes were detected by RT-PCR. E-test method was used to detect the sensitivity of Bordetella pertussis strains to different antibiotics. The mechanism of resistance of Bordetella pertussis to macrolides was analyzed by whole genome sequencing. The phylogenetic analysis of isolated strains was based on core genome multilocus sequence typing(cgMLST). Results:Among 273 clinical samples of children with suspected pertussis and their close family members, 168 samples were positive by fluorescence quantitative PCR, accounting for 61.54%, and 30 pertussis strains were successfully isolated with a positive rate of 10.98%. In addition, among the 143 samples of close family members, 54.55% (78/143) samples were positive by RT-PCR and 9.79% (14/143) samples were positive by culture, suggesting that the close family member are important in family transmission of pertussis. Besides, most of the positive samples were from mothers. The results of E-test showed that 96.67%(29/30) strains showed high resistance to azithromycin with MIC value>256 mg/L, and the resistant mechanism of azithromycin was A2047G mutation in 23S rRNA. The phylogenetic analysis based on the cgMSLT showed that the isolated strains were clustered into two new different clades.Conclusions:The positive rate of Bordetella pertussis in close family members is at a high level and the mother may be the main source of infection, which is of great significance for monitoring and laboratory detection of suspected children′s family members. Bordetella pertussis shows high resistance to macrolides in Zhejiang Province, so monitoring of the antimicrobial resistance should be further strengthened to provide theoretical basis for clinical treatment and drug guidance.

Objective:To investigate the related genes, signaling pathways and possible mechanisms of malignant transformation of human papillomavirus type 16 (HPV-16) immortalized cervical epithelial cell line H8.Methods:The malignant transformed H8 cell model was constructed, and the changes of cell invasion ability and cell migration ability of H8 cells after malignant transformation were detected by Transwell assay, and the changes of clone formation ability of H8 cells after malignant transformation were detected by plate clone formation assay. Total RNA was extracted from malignant transformed H8 cells and H8 cells, and the two groups of cells were sequenced by transcriptome using Illumina novaseq 6000 sequencing platform, differentially expressed genes (DEGs) were identified and analyzed, and Gene Ontology (GO) function enrichment analysis, Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis and protein-protein interaction were performed.Results:The invasion ability, migration ability and clone formation ability of malignant transformed H8 cells significantly increased as compared to H8 cells. A total of 203 differentially expressed genes were identified in H8 cells before and after malignant transformation, of which 98 were up-regulated and 105 down-regulated. GO enrichment analysis showed that DEGs were mainly involved in biological processes such as cellular processes, biological regulation, and metabolic processes. KEGG pathway enrichment analysis showed that DEGs were mainly enriched in alanine, aspartate and glutamate metabolic pathway, glycine, serine and threonine metabolism pathway, p53 signaling pathway and TGF-β signaling pathway, PI3K-Akt signaling pathway. PPI analysis screened 10 hub genes including DDIT3, TRIB3 and ASNS.Conclusions:Compared with H8 cells, malignant transformed H8 cells have a large number of differentially expressed genes and pathways at the transcriptional level, which could further provide new ideas for the mechanism of malignant transformation and carcinogenesis as well as finding new targets for the prevention of malignant transformation.

Objective:To explore the prognostic value of tumor deposits (TD) by number and anatomical distribution in gastric cancer (GC) patients without lymph node metastasis.Methods:From Aug 2012 to Aug 2018 all 91 GC patients undergoing radical gastrectomy and without nodal metastasis at Yijishan Hospital of Wannan Medical College were enrolled in this study. Patients were divided into L1, L2, and L3 groups according to the number of TD and into Q1 and Q2 groups according to the anatomical regions of the TD.Results:The 3-year overall survival (OS) rates of groups L1, L2, and L3 were 58.9%, 52.1%, and 31.5%, respectively ( χ2=9.769, P=0.008). The 3-year OS rates of groups Q1 and Q2 were 58.9% and 7.1% ( χ2=46.310, P<0.001). The number of TD, their distribution, neural invasion, vascular invasion, tumor size, and pT stage were all related to prognosis by univariate analysis (all P<0.05). Tumor size>4 cm ( HR=2.460, 95% CI:1.307-4.629, P=0.005), distribution of TD (non-perigastric)( HR=3.959, 95% CI:2.077-7.545, P<0.001), neural invasion ( HR=4.299,95% CI:1.953-9.461, P<0.001), and pT 4 stage ( HR=2.283, 95% CI:1.250-4.171, P=0.007) were independent risk factors for prognosis by multivariate analysis. Conclusion:The distribution of TD (non-perigastric) is an independent risk factor for poor prognosis in gastric cancer patients after radical gastrectomy and with negative lymph node metastasis.

Humans ; Asthma/drug therapy* ; Biological Therapy

High Altitude Military Hygiene is the professional core course of high-altitude medicine, which is significant to the cultivation of military medical talents urgently needed by plateau troops. Under the background of "golden course" construction and army curriculum reform, aiming at the problems such as outdated content of course materials, single teaching mode and insufficient capacity of practical courses, we actively explored the effective path of "golden course" construction, including the renovation of the curriculum-construction concept, the optimization and reorganization of the teaching content, the expansion of case teaching and equipment teaching methods, and the implementation of curriculum ideological and political education and examination reforms. The reform has further improved the learning effect of students and the level of curriculum construction, and also provided beneficial reference for the construction of similar courses in military colleges and universities.