Objective:To investigate the impact of limonin(LIM)on the necrotic apoptosis of myocardial cells in rats with myocardial infarction(MI)by regulating the receptor-interacting protein 1(RIP1)/receptor-interacting protein 3(RIP3)/mixed-lineage kinase domain-like protein(MLKL)signaling pathway.Methods:A total of 60 Sprague-Dawley rats were randomly divided into sham-operation group(Sham group),MI model group(Model group),low-dose LIM group(LIM-L group,25 mg/kg LIM),high-dose LIM group(LIM-H group,50 mg/kg LIM),and high-dose LIM+RIP1 inhibitor Nec-1 group(LIM-H+Nec-1 group,50 mg/kg LIM+0.6 mg/kg Nec-1),with 12 rats in each group.A rat model of MI was established by ligation of the coronary artery.The changes of cardiac func-tion were examined for each group;HE staining was used to observe the pathological changes of myocardial tissue;the 2,3,5-triphen-yltetrazolium chloride-Evans blue method was used to measure myocardial infarct area;the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method was used to observe the necrotic apoptosis of myocardial cells;quantitative real-time PCR and Western blot were used to measure the expression levels of mRNAs and proteins associated with the RIP1/RIP3/MLKL signaling pathway and the expression of apoptosis-related proteins.Results:Compared with the Sham group,the Model group had significant re-ductions in left ventricular ejection fraction,left ventricular systolic pressure,and the expression level of B-cell lymphoma-2(Bcl-2)in myocardial tissue(all P<0.001)and significant increases in left ventricular end-systolic volume,left ventricular end-diastolic pres-sure,left ventricular end-diastolic volume,myocardial infarct area,cell apoptosis rate,the mRNA and protein expression levels of RIP1,RIP3,and MLKL in myocardial tissue,and the protein expression levels of Bcl-2 associated X protein and cysteinyl aspartate-specific proteinase 3 in myocardial tissue(all P<0.001),as well as swelling and disordered arrangement of myocardial cells with ne-crosis and massive inflammatory cell infiltration on HE pathological sections.Compared with the Model group,the LIM-H group showed reverse changes in the above indicators(RIP1 mRNA:P=0.002,RIP3 mRNA:P=0.008,and the other indexes P were all<0.001),with alleviations of myocardial histopathological injury and inflammatory cell infiltration.Nec-1 promoted the effect of LIM in alleviating the necrotic apoptosis of myocardial cells in MI rats.Conclusion:LIM may alleviate the necrotic apoptosis of myocardial cells in MI rats by downregulating the RIP1/RIP3/MLKL signaling pathway.

Objective:To develop genetic testing quality control material which has good biological compatibility that can simulate the real sample properties.Methods:Study of the quality control material. The quality control matertial was prepared by embedding the cells and DNA containing specific gene mutations in a biological matrix formed by cross-linking γ-polyglutamic acid and gelatin, followed by formalin fixation and paraffin embedding. Fourier infrared spectroscopy and scanning electron microscopy were used to analyze the morphology and structure of the quality control material. The repeatability and homogeneity were verified by detecting the DNA extraction amount and mutation frequency at different parts of the quality control material. The quality control material was stored at 25 ℃ for 16 weeks, and the stability was verified by detecting the DNA extraction amount, mutation frequency and DNA fragment distribution.Results:The results of Fourier infrared spectroscopy and scanning electron microscopy analysis indicated that the quality control material prepared in this study was formed by cross-linking and had a dense three-dimensional framework structure. The amount of DNA extraction and mutation frequency from different parts of the quality control material varied within a small range ( CV<10%). After storage at 25 ℃ for 16 weeks, the DNA extraction amount and mutation frequency of the quality control material varied within a small range ( CV<5%), and the distribution range of the DNA fragments did not show significant change compared to the day of preparation of the quality control material. Conclusion:This study successfully prepared a genetic testing quality control material based on cross-linked biological matrix, which exhibited good repeatability, homogeneity and stability.

Objective To explore the effect of transcutaneous auricular vagus nerve stimulation(taVNS)combined with dual-task training(DTT)on upper limb function recovery in patients with ischemic stroke.Methods From January to October,2024,60 ischemic stroke patients with upper limb dysfunction admitted to Zhejiang Rehabilitation Medical Center were selected and randomly divided into control group(n=30)and observation group(n=30).Both groups received conventional rehabilitation training.The control group underwent sham-taVNS combined with DTT,while the observation group received active taVNS combined with DTT,for four weeks.Before and after treatment,they were assessed with Fugl-Meyer Assessment-Upper Extremities(FMA-UE),Action Research Arm Test(ARAT),modified Barthel Index(MBI)and motor-evoked potential(MEP)tests.Results The FMA-UE scores,ARAT scores,MBI scores,MEP amplitude and MEP latency improved in both groups af-ter treatment(|t|>3.670,P<0.01),and were superior in the observation group than in the control group(|t|>3.081,P<0.01).Conclusion The combination of taVNS and DTT can significantly improve upper limb function,enhance activities of daily living,and promote neurological recovery in patients with ischemic stroke.

OBJECTIVE: To investigate the safety and prognostic factors influencing the treatment of upper urinary tract urothelial carcinoma (UTUC) combined with bladder cancer (BCa) by laparoscopic simultaneous radical cystectomy and nephroureterectomy (RCNU). METHODS: The clinical data of patients admitted to Peking University Third Hospital for laparoscopic RCNU surgery from January 2009 to September 2023 were analyzed retrospectively. Based on the same gender, age (±5 years), history of uroepithelial tumors, underlying diseases, T-stage, N-stage, M-stage, American Society of Anesthesiologists (ASA) score, Charlson comorbidity index, and body mass index (BMI) (±5), 34 patients with RCNU were matched 1 ∶1 with patients with bladder cancer who underwent laparoscopic radical cystectomy (RC) alone. Kaplan-Meier survival analysis was used to calculate patient survival, and Cox proportional regression risk model was used to analyze clinical factors affecting prognosis. RESULTS: Of the 68 patients enrolled, the follow-up rate was 100% with a median follow-up time of 27.0 (11.7, 60.2) months. Comparison of intraoperative conditions (including operation time, estimated intraoperative bleeding, intra-operative blood transfusion, etc.) between the two groups of patients showed no significant difference (P>0.05). Comparison of preoperative creatinine and postoperative creatinine between the two groups of patients showed significant differences (P < 0.05). The perioperative Clavien grade Ⅲ-Ⅳ complication rates were 2.9% (1/34) in the RC group and 5.9% (2/34) in the RCNU group. There was no significant difference in terms of perioperative complications between the two groups. Overall survival was significantly lower in the patients receiving RCNU compared with the matched group receiving RC alone (P < 0.05). Cox regression analysis suggested that two factors, high N stage and high postoperative creatinine, were independent risk factors affecting the prognosis of patients in the 2 groups (P < 0.05). CONCLUSION The overall survival prognosis of patients undergoing RCNU surgery was worse compared with laparoscopic RC surgery alone during the same period. There was no clinically significant difference between the two groups in terms of operation time, intraoperative bleeding, and perioperative complications, and there were clinically significant differences in preoperative renal function and post-operative renal function.
Humans ; Laparoscopy/methods* ; Nephroureterectomy/methods* ; Cystectomy/methods* ; Prognosis ; Male ; Retrospective Studies ; Female ; Urinary Bladder Neoplasms/mortality* ; Middle Aged ; Aged

Objective To explore the effect of transcutaneous auricular vagus nerve stimulation(taVNS)combined with dual-task training(DTT)on upper limb function recovery in patients with ischemic stroke.Methods From January to October,2024,60 ischemic stroke patients with upper limb dysfunction admitted to Zhejiang Rehabilitation Medical Center were selected and randomly divided into control group(n=30)and observation group(n=30).Both groups received conventional rehabilitation training.The control group underwent sham-taVNS combined with DTT,while the observation group received active taVNS combined with DTT,for four weeks.Before and after treatment,they were assessed with Fugl-Meyer Assessment-Upper Extremities(FMA-UE),Action Research Arm Test(ARAT),modified Barthel Index(MBI)and motor-evoked potential(MEP)tests.Results The FMA-UE scores,ARAT scores,MBI scores,MEP amplitude and MEP latency improved in both groups af-ter treatment(|t|>3.670,P<0.01),and were superior in the observation group than in the control group(|t|>3.081,P<0.01).Conclusion The combination of taVNS and DTT can significantly improve upper limb function,enhance activities of daily living,and promote neurological recovery in patients with ischemic stroke.

Objective:To explore the clinical characteristics and prognostic factors of primary bladder adenocarcinoma（ACB）.Methods:The clinical data of 33 patients with primary ACB who underwent surgical treatment in the Department of Urology of Peking University Third Hospital from July 2003 to January 2024 were retrospectively analyzed. There were 5 females and 28 males，with an average age of（61.3 ± 11.5）years. Twelve patients had comorbidities（6 hypertension，2 coronary heart disease，5 diabetes mellitus，and 3 cerebrovascular diseases）and 12 had a smoking history. The mean body mass index（BMI）was（24.8 ± 3.2）kg/m 2. The maximum tumor diameter measured by enhanced computed tomography（CT）was（29.7 ± 12.7）mm. The preoperative neutrophil-to-lymphocyte ratio（NLR）was 3.4 ± 3.2，and the systemic immune-inflammation index（SII）was（582 ± 496）× 10 9/L.Patients were divided into two groups according to the surgical approach：the radical cystectomy group（ n = 23）and the bladder-sparing group（ n = 10）. For the radical cystectomy group，there were 19 males and 4 females，with a mean age of（59.9 ± 12.6）years. Five patients had comorbidities（3 hypertension，1 coronary heart disease，1 diabetes mellitus，and 2 cerebrovascular diseases）. Eight patients had a smoking history，with a mean BMI of（25.2 ± 3.5）kg/m 2. The maximum tumor diameter was（33.6 ± 10.9）mm，the preoperative NLR was 3.5 ± 3.5，and the SII was（618 ± 558）× 10 9/L. For the bladder-sparing group，there were 9 males and 1 female，with a mean age of（64.5 ± 8.2）years. Seven patients had comorbidities（3 hypertension，1 coronary heart disease，4 diabetes mellitus，and 1 cerebrovascular diseases）. Four patients had a smoking history，with a mean BMI of（23.5 ± 2.3）kg/m 2. The maximum tumor diameter was（20.7 ± 12.5）mm，the preoperative NLR was 3.1 ± 2.2，and the SII was（501 ± 323）× 10 9/L. Statistically significant differences were observed between the two groups in terms of comorbidities（ P = 0.008）and maximum tumor diameter（ P = 0.006），while no significant differences were found in other data（ P > 0.05）. The Kaplan-Meier survival curve was drawn，and Cox regression was used to analyze the prognostic factors of progression-free survival（PFS）and overall survival（OS）of patients. Results:Among the 33 patients，low-grade adenocarcinoma and high-grade adenocarcinoma accounted for 60.6% and 39.4% respectively according to the postoperative pathology，and 3 patients had positive surgical margins. There were 22 cases of muscle-invasive bladder adenocarcinoma，5 cases of lymph node metastasis，and 1 case of distant metastasis. The patients in tumor stages Ⅰ—Ⅳ were 9 cases（27.3%），8 cases（24.2%），7 cases（21.2%），and 9 cases（27.3%）respectively. Nine patients received postoperative adjuvant therapy，including 6 with adjuvant chemotherapy，2 with adjuvant chemotherapy combined with radiotherapy，and 1 with adjuvant immunotherapy. In the radical cystectomy group（ n = 23），there were 13 cases of low-grade and 10 cases of high-grade pathological grading，2 cases with positive margins，19 cases of muscle-invasive bladder adenocarcinoma，5 cases of lymph node metastasis，1 case of distant metastasis，and 5 patients received adjuvant therapy（4 cases of adjuvant chemotherapy，and 1 case of adjuvant immunotherapy）. In the bladder-sparing group（ n = 10），there were 7 cases of low-grade，3 cases of high-grade pathological grading，1 case with positive margins，3 cases of muscle-invasive bladder adenocarcinoma，zero lymph node or distant metastasis，and 4 patients received adjuvant therapy（2 cases of adjuvant chemotherapy，and 2 cases of combined adjuvant chemotherapy and radiotherapy）. A statistically significant difference was found in the proportion of muscle-invasive bladder adenocarcinoma between the two groups（ P = 0.006），while no significant differences were observed in other data（ P > 0.05）.The median follow-up duration of the patients was 28.0 months，the median PFS was 86.0 months，and the median OS was 90.0 months. The 2-year PFS and OS were 65.4% and 73.1% respectively. The 5-year PFS and OS were 54.2% and 56.5% respectively. The Kaplan-Meier survival analysis showed that there were no significant differences in PFS（ P = 0.777）and OS（ P = 0.585）between the radical cystectomy group and the bladder-preserving group. Female（ P = 0.011），BMI < 25 kg/m2（ P = 0.038），and positive surgical margins（ P < 0.01）were associated with poorer PFS. Aged ≥ 70 years（ P = 0.003），lymph node metastasis（ P = 0.041），and positive surgical margins（ P = 0.025）were associated with poorer OS，and patients in the adjuvant therapy group had better OS（ P = 0.005）. Multivariate Cox regression analysis indicated that positive surgical margins（HR 10.2， P = 0.012）were an independent impact factor for PFS，and positive surgical margins（HR 39.8， P = 0.001）and adjuvant therapy（HR 0.12， P = 0.021）were independent impact factors for OS. Conclusions:Positive surgical margins and adjuvant therapy are independent impact factors for the prognosis of patients with primary ACB.

Objective:To explore the clinical characteristics and prognostic factors of primary bladder adenocarcinoma（ACB）.Methods:The clinical data of 33 patients with primary ACB who underwent surgical treatment in the Department of Urology of Peking University Third Hospital from July 2003 to January 2024 were retrospectively analyzed. There were 5 females and 28 males，with an average age of（61.3 ± 11.5）years. Twelve patients had comorbidities（6 hypertension，2 coronary heart disease，5 diabetes mellitus，and 3 cerebrovascular diseases）and 12 had a smoking history. The mean body mass index（BMI）was（24.8 ± 3.2）kg/m 2. The maximum tumor diameter measured by enhanced computed tomography（CT）was（29.7 ± 12.7）mm. The preoperative neutrophil-to-lymphocyte ratio（NLR）was 3.4 ± 3.2，and the systemic immune-inflammation index（SII）was（582 ± 496）× 10 9/L.Patients were divided into two groups according to the surgical approach：the radical cystectomy group（ n = 23）and the bladder-sparing group（ n = 10）. For the radical cystectomy group，there were 19 males and 4 females，with a mean age of（59.9 ± 12.6）years. Five patients had comorbidities（3 hypertension，1 coronary heart disease，1 diabetes mellitus，and 2 cerebrovascular diseases）. Eight patients had a smoking history，with a mean BMI of（25.2 ± 3.5）kg/m 2. The maximum tumor diameter was（33.6 ± 10.9）mm，the preoperative NLR was 3.5 ± 3.5，and the SII was（618 ± 558）× 10 9/L. For the bladder-sparing group，there were 9 males and 1 female，with a mean age of（64.5 ± 8.2）years. Seven patients had comorbidities（3 hypertension，1 coronary heart disease，4 diabetes mellitus，and 1 cerebrovascular diseases）. Four patients had a smoking history，with a mean BMI of（23.5 ± 2.3）kg/m 2. The maximum tumor diameter was（20.7 ± 12.5）mm，the preoperative NLR was 3.1 ± 2.2，and the SII was（501 ± 323）× 10 9/L. Statistically significant differences were observed between the two groups in terms of comorbidities（ P = 0.008）and maximum tumor diameter（ P = 0.006），while no significant differences were found in other data（ P > 0.05）. The Kaplan-Meier survival curve was drawn，and Cox regression was used to analyze the prognostic factors of progression-free survival（PFS）and overall survival（OS）of patients. Results:Among the 33 patients，low-grade adenocarcinoma and high-grade adenocarcinoma accounted for 60.6% and 39.4% respectively according to the postoperative pathology，and 3 patients had positive surgical margins. There were 22 cases of muscle-invasive bladder adenocarcinoma，5 cases of lymph node metastasis，and 1 case of distant metastasis. The patients in tumor stages Ⅰ—Ⅳ were 9 cases（27.3%），8 cases（24.2%），7 cases（21.2%），and 9 cases（27.3%）respectively. Nine patients received postoperative adjuvant therapy，including 6 with adjuvant chemotherapy，2 with adjuvant chemotherapy combined with radiotherapy，and 1 with adjuvant immunotherapy. In the radical cystectomy group（ n = 23），there were 13 cases of low-grade and 10 cases of high-grade pathological grading，2 cases with positive margins，19 cases of muscle-invasive bladder adenocarcinoma，5 cases of lymph node metastasis，1 case of distant metastasis，and 5 patients received adjuvant therapy（4 cases of adjuvant chemotherapy，and 1 case of adjuvant immunotherapy）. In the bladder-sparing group（ n = 10），there were 7 cases of low-grade，3 cases of high-grade pathological grading，1 case with positive margins，3 cases of muscle-invasive bladder adenocarcinoma，zero lymph node or distant metastasis，and 4 patients received adjuvant therapy（2 cases of adjuvant chemotherapy，and 2 cases of combined adjuvant chemotherapy and radiotherapy）. A statistically significant difference was found in the proportion of muscle-invasive bladder adenocarcinoma between the two groups（ P = 0.006），while no significant differences were observed in other data（ P > 0.05）.The median follow-up duration of the patients was 28.0 months，the median PFS was 86.0 months，and the median OS was 90.0 months. The 2-year PFS and OS were 65.4% and 73.1% respectively. The 5-year PFS and OS were 54.2% and 56.5% respectively. The Kaplan-Meier survival analysis showed that there were no significant differences in PFS（ P = 0.777）and OS（ P = 0.585）between the radical cystectomy group and the bladder-preserving group. Female（ P = 0.011），BMI < 25 kg/m2（ P = 0.038），and positive surgical margins（ P < 0.01）were associated with poorer PFS. Aged ≥ 70 years（ P = 0.003），lymph node metastasis（ P = 0.041），and positive surgical margins（ P = 0.025）were associated with poorer OS，and patients in the adjuvant therapy group had better OS（ P = 0.005）. Multivariate Cox regression analysis indicated that positive surgical margins（HR 10.2， P = 0.012）were an independent impact factor for PFS，and positive surgical margins（HR 39.8， P = 0.001）and adjuvant therapy（HR 0.12， P = 0.021）were independent impact factors for OS. Conclusions:Positive surgical margins and adjuvant therapy are independent impact factors for the prognosis of patients with primary ACB.

Objective:To develop genetic testing quality control material which has good biological compatibility that can simulate the real sample properties.Methods:Study of the quality control material. The quality control matertial was prepared by embedding the cells and DNA containing specific gene mutations in a biological matrix formed by cross-linking γ-polyglutamic acid and gelatin, followed by formalin fixation and paraffin embedding. Fourier infrared spectroscopy and scanning electron microscopy were used to analyze the morphology and structure of the quality control material. The repeatability and homogeneity were verified by detecting the DNA extraction amount and mutation frequency at different parts of the quality control material. The quality control material was stored at 25 ℃ for 16 weeks, and the stability was verified by detecting the DNA extraction amount, mutation frequency and DNA fragment distribution.Results:The results of Fourier infrared spectroscopy and scanning electron microscopy analysis indicated that the quality control material prepared in this study was formed by cross-linking and had a dense three-dimensional framework structure. The amount of DNA extraction and mutation frequency from different parts of the quality control material varied within a small range ( CV<10%). After storage at 25 ℃ for 16 weeks, the DNA extraction amount and mutation frequency of the quality control material varied within a small range ( CV<5%), and the distribution range of the DNA fragments did not show significant change compared to the day of preparation of the quality control material. Conclusion:This study successfully prepared a genetic testing quality control material based on cross-linked biological matrix, which exhibited good repeatability, homogeneity and stability.

Objective:To investigate the prognostic factors for all-cause mortality in patients with muscle-invasive bladder cancer(MIBC)with intermediate-to-high-risk primary prostate cancer.Methods:From January 2012 to October 2023,the clinical data of the patients with MIBC with intermediate-to-high-risk primary prostate cancer in Peking University Third Hospital were retrospectively analyzed.All the patients were monitored and the occurrence of all-cause death was documented as the outcome event in the prognostic study.Univariate and multivariate Cox proportional risk regression analysis models were implemented to search for independent influences on the prognosis of patients.For significant influencing factors(pathological T stage,M stage and perineural invasion of bladder cancer),survival curves were plotted before and after multifactorial Cox regression adjusting for confounding factors.Results:A total of 32 patients were included in this study.The mean age was(72.5±6.6)years;the median preoperative total prostate specific antigen(tPSA)was 6.68(2.47,6.84)μg/L;the mean preoperative creatinine was(95±36)μmol/L,and the median survival time was 65 months.The majority of the patients(87.5％)had high-grade bladder cancer,53.1％had lymphatic invasion,and 31.3％had perineural invasion.Prostate involvement was observed in 25.0％of the cases,and the positive rate of soft-tissue surgical margin was 37.5％.Multivariate Cox analysis revealed that preoperative creatinine level(HR=1.02,95％CI:1.01-1.04),pathological stage of bladder cancer T3(HR=11.58,95％CI:1.38-97.36)and T4(HR=19.53,95％CI:4.26-89.52)metastasis of bladder cancer(HR=9.44,95％CI:1.26-70.49)and perineural invasion of bladder cancer(HR=6.26,95％CI:1.39-28.27)were independent prognostic factors(P＜0.05).Survival curves with Log-rank test after adjusting for confounding factors demonstrated that bladder cancer pathology T3,T4,M1,and perineural invasion were unfavorable factors affecting the patients'survival prognosis(P＜0.05).Conclusion:Patients with MIBC with intermediate-to-high risk primary prostate cancer generally portends a poor prognosis.High preoperative serum creatinine,T3 or T4 pathological stage of bladder cancer,metastasis of bladder cancer and bladder cancer perineural invasion are poor prognostic factors for patients with MIBC with intermediate-to-high risk primary prostate cancer.

Purpose BRCA1/2 gene germline variants of the uncertain significance(VUS)are categorized into five clas-ses based on their risk levels,and three classes require regular periodic analysis due to their unclear clinical significance.The aim of this study was to investigate the influence of updating var-iation evidences on the VUS sites and guide clinical diagnosis and treatment.Methods The VUS sites in BRCA1/2 gene were analyzed.971 samples(breast or ovarian cancer)that un-derwent BRCA1/2 germline testing were stored.The VUS sites in BRCA1/2 gene were further reinterpreted by integrating the following evidences,including population frequency database,disease database,computer software prediction,co-segregation evidence,allelic evidence and population cohort evidence to determine whether the variation classification was changed.Results The number of the patients with VUS sites was 142,accounting for 14.6％(142/971).The total number of VUS sites was 128,among which the proportions of missense muta-tion,synonymous mutation,in-frame non-shifted mutation and non-coding region mutation were 70.3％,4.7％,3.1％and 21.9％,respectively.Reinterpretation of VUS sites discovered that 11.7％(15/128)of VUS sites could be downgraded to Class 2,likely benign.Conclusion With the continuous in-creasing evidences of germline variation,the variation classifica-tion of VUS sites will be changed after periodic analysis.