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 observe the clinical efficacy of continuous theta burst stimulation in the treatment of post-stroke aphasia (PSA).Methods:Forty-six patients with post-stroke aphasia were randomly divided into an observation group and a control group, with 23 cases in each group. In addition to conventional rehabilitation and speech-language therapy, the observation group received daily theta burst stimulation (cTBS) targeting the contralesional inferior frontal gyrus, the superior temporal gyrus, and the superior frontal gyrus. The control group received sham stimulation over the same areas. The treatment was 5 days per week for 3 weeks. Before and after the treatment, both groups were evaluated using the China Rehabilitation Research Center′s aphasia examination method (CRRCAE), the Boston diagnostic aphasia examination (BDAE), the brief mental scale (MMSE), and the Stroke Aphasia Quality of Life scale (SAQOL-39).Results:After the treatment, intra-group comparison showed significantly greater improvements among the observation group in terms of their average CRRCAE listening comprehension (112.74±8.935), repetition (115.74±16.015), speaking (99.91±6.273), oral reading (98.22±14.087), and reading (86.57±8.101) sub-scores and their average BDAE [3.00 (3.00, 4.00)], MMSE [21.00 (20.00, 24.00)] and SAQOL-39 (112.96±21.995) scores.Conclusions:Continuous theta burst transcranial magnetic stimulation can effectively improve the speech, cognition and life quality of persons with post-stroke aphasia. This therapy is worthy of clinical application and promotion.

Objective:To analyze serum inflammatory factors associated with antihistamine resistance in patients with chronic spontaneous urticaria (CSU) .Methods:A total of 88 CSU patients were enrolled from Guangzhou Dermatology Hospital from January 2022 to December 2024. All patients received antihistamine treatment according to the "Guideline for diagnosis and treatment of urticaria in China (2022) " . Based on the 7-day urticaria activity score (UAS7) after 4-week treatment, these patients were divided into an antihistamine-sensitive group and an antihistamine-resistant group. Serum levels of inflammatory factors at the initial visit were analyzed using the Olink-targeted proteomics technology. Specific biomarkers associated with antihistamine resistance were identified, and Spearman correlation analysis was carried out to analyze correlations among differentially expressed proteins. A logistic regression model was constructed based on the Olink proteomics data, and the predictive performance of the model was evaluated using receiver operating characteristic (ROC) curve analysis. Measurement data were expressed as mean ± standard deviation or median (lower quartile, upper quartile) .Results:The 88 CSU patients aged 12 to 81 (38.78 ± 13.89) years, with the disease duration being 18 (7.00, 60.00) months. There were 32 patients in the antihistamine-sensitive group and 56 in the antihistamine-resistant group. No significant differences were found between the two groups in terms of age, disease duration, gender, or history of allergic diseases (all P > 0.05) . After 4 weeks of antihistamine treatment, the UAS7 score was significantly higher in the antihistamine-resistant group (25.00 [15.25, 31.00] points) than in the antihistamine-sensitive group (0.50 [0.00, 4.00] points; Z = -7.08, P < 0.001) . The Olink-targeted proteomics identified 5 differentially expressed proteins between the two groups: compared with the antihistamine-sensitive group, the antihistamine-resistant group showed > 2-fold higher expression of fibroblast growth factor 19 (FGF19) , interleukin-15 receptor subunit alpha (IL-15RA) , eotaxin (CCL11) , and monocyte chemoattractant protein-1 (MCP-1) ; in contrast, the expression of sulfotransferase 1A1 (ST1A1) in the antihistamine-sensitive group was 2.54 times that in the antihistamine-resistant group. Among the differentially expressed proteins, MCP-1 showed the highest specificity (1.00) for predicting antihistamine resistance, followed by CCL11 (0.97) . Correlation analysis revealed a significant positive correlation between MCP-1 and CCL11, and a significant negative correlation between IL-15RA and ST1A1. ROC curve analysis showed that MCP-1 and CCL11 had area under the curve values of 0.603 and 0.630, respectively, in predicting antihistamine resistance. Conclusion:MCP-1 and CCL11 may be potential biomarkers for predicting antihistamine resistance in CSU patients.

Aim To clarify the role of sirtuin 1(SIRT1)/dynamin-related protein 1(DRP1)in genistein inhibi-ting oxidized low density lipoprotein(ox-LDL)-induced apoptosis of human umbilical vein endothelial cells(HUVEC).Methods HUVECs were cultured in vitro.The levels of mitochondrial fission factor(MFF),mitochondrial fission pro-tein 1(FIS1),mitofusin 1(MFN1),mitofusin 2(MFN2),optic atrophy 1(OPA1),Bcl-2,Bax,cytochrome c(Cyt c),apoptotic protease activating factor 1(APAF1),cleaved Caspase-9,cleaved Caspase-3,p-DRP1(Ser616)and acety-lation-DRP1(ac-DRP1),as well as the interaction between p-DRP1 and Bax were examined.Results Compared with ox-LDL treatment,genistein pretreatment suppressed the activity of DRP1 through diminishing acetylation and phos-phorylation,which was associated with activating SIRT1(all P＜0.05).Genistein pretreatment inhibited mitochondrial fission by enhancing MFN1,MFN2 and OPA1,and reducing MFF and FIS1(all P＜0.05).Genistein pretreatment sup-pressed apoptosis through inhibiting the interaction between p-DRP1 and Bax,accompanied with increasing Bcl-2 and de-creasing Bax,Cyt c,APAF1,cleaved Caspase-9 and Caspase-3(all P＜0.05).Conclusion Genistein suppressed ox-LDL-induced mitochondrial fission and apoptosis through activating SIRT1 and inhibiting DRP1 in HUVEC.Therefore,SIRT1/DRP1 had a crucial role in genistein inhibiting ox-LDL-induced apoptosis in HUVEC.

In recent years,the Ross procedure has been increasingly applied in the treatment of aortic valve disease in children and young patients.However,right ventricular outflow tract(RVOT)reconstruction in this procedure often relies on allogeneic or artificial materials,which may lead to complications such as calcification and valve dysfunction.This article reports a case of protective Ross procedure using completely autologous tissue to construct a right ventricular-pulmonary artery(RV-PA)conduit.The patient was an 11-year-old male who presented with severe aortic stenosis combined with regurgitation.During the operation,his dilated ascending aortic wall and fresh pericardium were used to construct an autologous valved conduit for RV-PA reconstruction.This innovative technique achieves RV-PA reconstruction without allogeneic tissue,provides a new technical approach for the Ross procedure.Short-term results are satisfactory and the medium-and long-term outcomes require further follow-up verification.

Objective:To analyze serum inflammatory factors associated with antihistamine resistance in patients with chronic spontaneous urticaria (CSU) .Methods:A total of 88 CSU patients were enrolled from Guangzhou Dermatology Hospital from January 2022 to December 2024. All patients received antihistamine treatment according to the "Guideline for diagnosis and treatment of urticaria in China (2022) " . Based on the 7-day urticaria activity score (UAS7) after 4-week treatment, these patients were divided into an antihistamine-sensitive group and an antihistamine-resistant group. Serum levels of inflammatory factors at the initial visit were analyzed using the Olink-targeted proteomics technology. Specific biomarkers associated with antihistamine resistance were identified, and Spearman correlation analysis was carried out to analyze correlations among differentially expressed proteins. A logistic regression model was constructed based on the Olink proteomics data, and the predictive performance of the model was evaluated using receiver operating characteristic (ROC) curve analysis. Measurement data were expressed as mean ± standard deviation or median (lower quartile, upper quartile) .Results:The 88 CSU patients aged 12 to 81 (38.78 ± 13.89) years, with the disease duration being 18 (7.00, 60.00) months. There were 32 patients in the antihistamine-sensitive group and 56 in the antihistamine-resistant group. No significant differences were found between the two groups in terms of age, disease duration, gender, or history of allergic diseases (all P > 0.05) . After 4 weeks of antihistamine treatment, the UAS7 score was significantly higher in the antihistamine-resistant group (25.00 [15.25, 31.00] points) than in the antihistamine-sensitive group (0.50 [0.00, 4.00] points; Z = -7.08, P < 0.001) . The Olink-targeted proteomics identified 5 differentially expressed proteins between the two groups: compared with the antihistamine-sensitive group, the antihistamine-resistant group showed > 2-fold higher expression of fibroblast growth factor 19 (FGF19) , interleukin-15 receptor subunit alpha (IL-15RA) , eotaxin (CCL11) , and monocyte chemoattractant protein-1 (MCP-1) ; in contrast, the expression of sulfotransferase 1A1 (ST1A1) in the antihistamine-sensitive group was 2.54 times that in the antihistamine-resistant group. Among the differentially expressed proteins, MCP-1 showed the highest specificity (1.00) for predicting antihistamine resistance, followed by CCL11 (0.97) . Correlation analysis revealed a significant positive correlation between MCP-1 and CCL11, and a significant negative correlation between IL-15RA and ST1A1. ROC curve analysis showed that MCP-1 and CCL11 had area under the curve values of 0.603 and 0.630, respectively, in predicting antihistamine resistance. Conclusion:MCP-1 and CCL11 may be potential biomarkers for predicting antihistamine resistance in CSU patients.

Aim To clarify the role of sirtuin 1(SIRT1)/dynamin-related protein 1(DRP1)in genistein inhibi-ting oxidized low density lipoprotein(ox-LDL)-induced apoptosis of human umbilical vein endothelial cells(HUVEC).Methods HUVECs were cultured in vitro.The levels of mitochondrial fission factor(MFF),mitochondrial fission pro-tein 1(FIS1),mitofusin 1(MFN1),mitofusin 2(MFN2),optic atrophy 1(OPA1),Bcl-2,Bax,cytochrome c(Cyt c),apoptotic protease activating factor 1(APAF1),cleaved Caspase-9,cleaved Caspase-3,p-DRP1(Ser616)and acety-lation-DRP1(ac-DRP1),as well as the interaction between p-DRP1 and Bax were examined.Results Compared with ox-LDL treatment,genistein pretreatment suppressed the activity of DRP1 through diminishing acetylation and phos-phorylation,which was associated with activating SIRT1(all P＜0.05).Genistein pretreatment inhibited mitochondrial fission by enhancing MFN1,MFN2 and OPA1,and reducing MFF and FIS1(all P＜0.05).Genistein pretreatment sup-pressed apoptosis through inhibiting the interaction between p-DRP1 and Bax,accompanied with increasing Bcl-2 and de-creasing Bax,Cyt c,APAF1,cleaved Caspase-9 and Caspase-3(all P＜0.05).Conclusion Genistein suppressed ox-LDL-induced mitochondrial fission and apoptosis through activating SIRT1 and inhibiting DRP1 in HUVEC.Therefore,SIRT1/DRP1 had a crucial role in genistein inhibiting ox-LDL-induced apoptosis in HUVEC.

In recent years,the Ross procedure has been increasingly applied in the treatment of aortic valve disease in children and young patients.However,right ventricular outflow tract(RVOT)reconstruction in this procedure often relies on allogeneic or artificial materials,which may lead to complications such as calcification and valve dysfunction.This article reports a case of protective Ross procedure using completely autologous tissue to construct a right ventricular-pulmonary artery(RV-PA)conduit.The patient was an 11-year-old male who presented with severe aortic stenosis combined with regurgitation.During the operation,his dilated ascending aortic wall and fresh pericardium were used to construct an autologous valved conduit for RV-PA reconstruction.This innovative technique achieves RV-PA reconstruction without allogeneic tissue,provides a new technical approach for the Ross procedure.Short-term results are satisfactory and the medium-and long-term outcomes require further follow-up verification.

OBJECTIVE To observe the detection and drug resistance of carbapenem-resistant Klebsiella pneumoni-ae(CRKP)strains causing hospital-acquired infections(HAI)and community-acquired infections(CAI)in recent years so as to provide bases for prevention and control of CRKP infection and reasonable clinical use of antibiotics.METHODS A total of 3444 patients who were diagnosed with Klebsiella pneumonia infection and were hospitalized in the 2nd Affiliated Hospital of Fujian Medical University from Jan.1,2017 to Dec.31,2023 were recruited as the research subjects.Totally 230 patients with CRKP infection were chosen based on the result of drug susceptibility testing,73 of whom had HAI,and 157 had CAI.The isolation rate of CRKP strains,popula-tion distribution,specimens sources and drug resistance rates were observed and compared between the patients with HAI and the patients with CAI.RESULTS The total isolation rate of CRKP strains was 6.68％(230/3444).There was no difference in the sex of the patients with CRKP infection between the HAI patients and the CAI patients,however,the isolation rate of the CRKP strains from the patients aged between 18 and 45 years old was higher in the HAI group than in the CAI group(P＜0.05).The isolation rates of CRKP strains causing the two types of infections increased year by year,showing a remarkable increasing amplitude in 2022-2023,with the HAI increasing from 9.33％to 20.67％,the CAI increasing from 5.54％to 15.03％.The lower respiratory tract,urinary tract and bacteremia were the most common infection sites,the detection rate of soft tissue infec-tions was higher among the patients with HAI than among the patients with CAI(P=0.047).CRKP strains cau-sing HAI showed the highest isolation rate(33.33％)in catheter specimens,and the isolation rate of CRKP strains in pus specimens was higher among the HAI patients than among the CAI patients(P=0.011).The isola-tion rate of CRKP strains in sputum specimens of the CAI patients raised four times in 2023 as compared with that in 2022.The drug resistance rates of the CRKP strains to 25 types of antibiotics were relatively high and showed upward trends;the drug resistance rate of the HAI-KPN strains to imipenem was 48.78％,higher than 7.09％of the CAI-KPN strains(P＜0.001),and there were no significant differences in the drug resistance rates to other carbapenems between the CAI-KPN strains and the HAI-KPN strains.CONCLUSIONS The isolation rates of the CRKP strains causing the HAI and CAI are increasing year by year.The clinical invasive procedures and community-acquired respiratory tract infections are the key points for prevention and control.It is necessary to in-tensify the hospital-community cooperative prevention and control system based on the isolation rates and drug re-sistance rates of the CRKP strains,and take comprehensive prevention and control measures so as to curb the transmission of the drug-resistant strains.