ObjectiveTo investigate the effect and underlying mechanism of the Wulao Qisun prescription on pathological new bone formation in ankylosing spondylitis （AS）. MethodsSynovial fibroblasts were isolated from the hip joints of AS patients and observed under a microscope to assess cell morphology. The cells were identified using immunofluorescence staining. The isolated AS fibroblasts were divided into blank group， low drug-containing serum group， medium drug-containing serum group， high drug-containing serum group， and positive drug group. After drug intervention， cell proliferation was measured using the cell counting kit-8 （CCK-8） assay to observe fibroblast growth and determine the optimal intervention time. Alkaline phosphatase （ALP） activity was measured using the alkaline phosphatase assay. Protein expression of osteocalcin （OCN）， osteopontin （OPN）， and runt-related transcription factor 2 （Runx2） was detected by Western blot. The mRNA expression levels of Wnt5a， β-catenin， and Dickkopf-1 （DKK-1） were measured by real-time quantitative polymerase chain reaction （Real-time PCR）. ResultsCompared with the blank group， each drug-containing serum group of Wulao Qisun prescription and the positive drug group inhibited the proliferation of AS fibroblasts and reduced ALP expression （P<0.01）. Compared with the blank group， the low drug-containing serum group of Wulao Qisun prescription downregulated β-catenin mRNA expression （P<0.05）. The medium and high drug-containing serum groups and the positive drug group significantly downregulated Wnt5a and β-catenin mRNA expression （P<0.05， P<0.01）， with the positive drug group showing the most pronounced effect （P<0.01）. The high drug-containing serum group and the positive drug group significantly upregulated DKK-1 mRNA expression （P<0.01）. Compared with the blank group， the low drug-containing serum group of Wulao Qisun prescription inhibited the expression of OPN and Runx2 proteins （P<0.05， P<0.01）， while the medium and high drug-containing serum groups and the positive drug group inhibited the expression of OCN， OPN， and Runx2 proteins （P<0.05， P<0.01）. ConclusionThe Wulao Qisun prescription can inhibit the proliferation and osteogenic differentiation of AS fibroblasts， thereby delaying the formation of pathological new bone in AS. The possible mechanism involves the regulation of Wnt/β-catenin-related gene expression， further inhibiting the transcription of downstream target genes.

Objective:To investigate the impact of postoperative complications on adverse outcomes following curative-intent resection for gallbladder cancer (GBC).Methods:The multi-center real-world study was conducted. The clinicopathological data of 629 patients with GBC, who were admitted to 14 medical centers including The First Affiliated Hospital of Army Medical University from the national multicenter database of Biliary Surgery Group of Elite Group of Chinese Journal of Digestive Surgery, from April 2020 to April 2024 were collected. There were 225 males and 404 females, aged (64±10)years. Patients underwent open curative-intent resection for GBC. Observation indicators: (1)surgery, postoperative complica-tions and adverse outcomes; (2) analysis of risk factors affecting postoperative adverse outcomes in patients and population attributable fraction (PAF). Missing data in predictor variables were addressed using multiple imputation with chained equations, while cases with missing outcome variables were addressed using the "multiple imputation then deletion (MID)" strategy. The severity of multicollinearity among independent variables was assessed using the variance inflation factor (VIF) test. Multivariable possion regression models with log link and robust error variance were construc-ted incorporating restricted cubic splines (3 knots) to address nonlinear relationships in continuous variables, calculating adjusted relative risk ( RR) with corresponding 95% confidence interval ( CI). Adjusted PAF was calculated for each imputed dataset using the AF package of R software, with subsequent pooling performed according to Rubin's rules. Results:(1) Surgery, postoperative complications and adverse outcomes. All 629 patients underwent curative-intent resection for GBC, of which 143 cases had postoperative complications, including 68 cases of intra-abdominal ascites, 39 cases of pulmonary infection, 21 cases of bile leakage, 12 cases of intra-abdominal hemorrhage, 11 cases of liver failure, 10 cases of pan-creatic fistula, 10 cases of wound infection, 10 cases of gastroparesis, 7 cases of cholangitis, 7 cases of sepsis. The same patient could have more than one kind of complication. Of 629 patients, there were 19 cases of postoperative 90-day death and 11 cases of missing data, 42 cases with post-operative 90-day reoperation and 7 cases with missing data, 44 cases with postoperative 90-day readmission and 3 cases with missing data, 155 cases with prolonged postoperative hospital stay and 3 cases with missing data. (2) Analysis of risk factors affecting the postoperative adverse outcomes in patients and PAF. Results of multivariate analysis showed that pulmonary infection and liver failure were independent risk factors for postoperative 90-day mortality ( RR=3.74, 12.15, 95% CI as 1.18-11.83, 1.98-74.48, P<0.05). Pulmonary infection demons-trated the highest PAF as 4.61% (95% CI as 3.94%-5.28%, P<0.05). Intra-abdominal ascites, pulmonary infection, bile leakage, and intra-abdominal hemorrhage were independent risk factors for post-operative 90-day reoperation ( RR=4.80, 3.62, 3.46, 4.99, 95% CI as 2.49-9.26, 1.42-9.21, 1.34-8.92, 1.55-16.06, P<0.05). Intra-abdominal ascites demonstrated the highest PAF as 8.65% (95% CI as 8.22%-9.08%, P<0.05). Intra-abdominal ascites, bile leakage, and liver failure were independent risk factors for postoperative 90-day readmission ( RR=6.20, 3.33, 14.33, 95% CI as 3.21-11.95, 1.33-8.35, 3.72-55.28, P<0.05). Intra-abdominal ascites demonstrated the highest PAF as 9.11% (95% CI as 8.85%-9.37%, P<0.05). Intra-abdominal ascites, pulmonary infection, bile leakage, liver failure, and wound infection were independent risk factors for prolonged postoperative hospital stay ( RR=2.29, 2.21, 2.26, 2.14, 3.35, 95% CI as 1.63-3.23, 1.41-3.46, 1.32-3.86, 1.11-4.13, 1.70-6.60, P<0.05). Intra-abdominal ascites demonstrated the highest PAF as 6.03% (95% CI as 5.71%-6.35%, P<0.05). Conclusion:Pulmonary infection is the most significant risk factor for postoperative 90-day mortality after curative-intent resection for GBC, while intra-abdominal ascites is the most significant risk factor for postoperative 90-day reoperation, postoperative 90-day readmission, and prolonged postoperative hospital stay.

Objective To explore the biological mechanisms of isorhamnetin(ISO)inhibition of breast cancer(BC)progression and effects on paclitaxel sensitivity by in vivo and in vitro experiments.Methods The effects of ISO on MDA-MB-231 cell viability,migration,invasion and apoptosis were explored by CCK-8,scratch,Transwell invasion and apoptosis assays.ISO biological functions were analyzed and core target genes were identified by genome-wide microarrays,functional enrichment and protein-protein interactions(PPI).CYP1B1 expression characterization and prognosis in BC were assessed by quantitative Real-time polymerase chain reaction(qRT-PCR),Western blotting(WB),and Kaplan-Meier plotter database.The effects of CYP1B1 overexpression on MDA-MB-231 cell viability,migration,invasion and apoptosis were explored by CCK-8,scratch,Transwell invasion and apoptosis assays.The effects of ISO and CYP1B1 on BC tumor growth were analyzed by establishing a subcutaneous graft tumor animal model and verified by histological analysis with immunohistochemistry(IHC)and hematoxylin and eosin(HE)staining.The effects of ISO and CYP1B1 on NF-κB signaling were analyzed by qRT-PCR and WB.The effect of ISO combined with paclitaxel on BC progression was analyzed by in vitro and in vivo experiments.Results The results of CCK-8 assay showed that ISO inhibited the viability of MDA-MB-231 cells with an IC50 value of 11.93 μmol/L;the scratch,Transwell invasion and apoptosis assays confirmed that ISO inhibited the migration and invasion of MDA-MB-231 cells and promoted apoptosis(P＜0.05).Volcano plot showed a total of 80 differentially expressed genes(DEGs),of which ISO promoted the expression of 27 genes and inhibited the expression of 53 genes.Gene set enrichment analyses showed that DEGs were mainly enriched in the signaling of organic hydroxyl compound metabolic process,inflammatory response,sterol metabolic process,and nuclear factor-κB(NF-κB).qRT-PCR,WB,and Kaplan-Meier plotter results showed that CYP1B1 mRNA and protein expression was increased in BC and mediated worse prognosis(P＜0.05).CCK-8,scratch,Transwell invasion and apoptosis assays showed that CYP1B1 overexpression enhanced MDA-MB-231 cell viability,migration and invasion,and inhibited apoptosis,which was reversed by the addition of ISO(P＜0.05).The results of in vivo experiments showed that ISO downregulated CYP1B1 expression and attenuated NF-κB signaling(P＜0.05).The results of in vitro and in vivo experiments showed that ISO combined with paclitaxel significantly inhibited BC cell viability and tumor growth(P＜0.05).Conclusion ISO inhibits BC malignant biological behavior by modulating the CYP1B1/NF-κB signaling axis,and ISO enhances paclitaxel sensitivity.

Objective To elucidate the anti-aging effect of β-sitosterol(BS),an important component in the fruits of Alpinia oxyphylla Miq.,in C.elegans and its regulatory effect on ETS-5 gene to modulate ferroptosis.Methods C.elegans treated with 10 μg/mL BS were monitored for survival time and changes in body length,motility,and reproductive function.The effect of ETS-5 gene knockdown on survival time of C.elegans was observed,and the changes in fat accumulation and lipid redox homeostasis in the transfected C.elegans were assessed using Oil Red O staining and by detecting MDA levels and the GSH/GSSG ratio.The mRNA expression levels of ferroptosis-related genes(FTN-1,GPX-1 and AAT-9)were detected using qPCR.The effects of BS treatment and ETS-5 knockdown on AAT-9 enzyme activity in C.elegans were examined.The effect of BS on nuclear localization of FEV(the human homolog of ETS-5)was validated in cultured human umbilical venous endothelial cells(HUVECs).Results Both BS treatment and ETS-5 knockdown significantly prolonged the lifespan,promoted lipid accumulation and reduced lipid peroxidation in C.elegans.ETS-5 knockdown resulted in upregulated expressions of the ferroptosis repressors GPX-1,AAT-9 and FTN-1 and increased the GSH/GSSG ratio in C.elegans.Conclusion BS inhibits ferroptosis in C.elegans by suppressing the expression of ETS-5 transcription factor and hence the activity of AAT-9 enzyme,a key gene for ferroptosis,which in turn prolongs the lifespan of C.elegans.

Objective To explore the diagnosis of infertility and clinical analysis of tubal patency by three-dimensional ultrasonic hysterosalpingography.Methods A total of 102 infertility patients admitted to Lishui Hospital of Traditional Chinese Medicine Affiliated to Zhejiang Chinese Medicine University from December 2022 to December 2024 were selected as the study objects to analyze the diagnosis of infertility and the clinical application of three-dimensional ultrasound hysterosalpingography on tubal patency.Results Of 102 infertility patients,the detection rate was 97.06％,the misdiagnosis rate was 0,and the missed diagnosis rate was 2.94％.The sensitivity,specificity and area under the curve of three-dimensional ultrasound hysterosalpingography for infertility were 72.65％,84.83％and 0.819.A total of 204 fallopian tubes were examined in 102 patients.Hysteroscopic fallopian tube staining showed patency(30.39％),obstruction(31.37％),and three-dimensional ultrasonic hysterosalpingography showed patency(31.86％),obstruction(32.35％).Compared with inflammation,the injection pressure,flow time and reverse flow of contrast agent in tumor and cyst lesions and congenital dysplasia were decreased(P<0.05),but there was no significant difference between tumor and cyst lesions and congenital dysplasia(P<0.05).In 102 patients,there was no hypersensitivity or other adverse reactions in the examination of three-dimensional ultrasound hysterosalpingography,and the satisfaction rate was 96.08％.Conclusion Three-dimensional ultrasound hysterosalpingography has a high diagnostic value for infertility,and can effectively evaluate the patency of fallopian tube,with high safety and patient satisfaction.

An assay was established for detection of toxigenic Clostridioides difficile by combining microfluidic chip analysis with immunochromatography,and its performance was evaluated and compared with those of the Xpert C.difficile/Epi and VIDAS CD AB tests.Primer pairs were designed according to the tcdB and tpi genes in C.difficile.The specificity,limit of detection,reproducibility,and stability were evaluated.A total of 215 stool samples from patients with diarrhea were collected and tested in parallel with the Xpert C.difficile/Epi,VIDAS CDAB,and our assay.C.difficile was isolated from samples,and the tcdB gene was identified when discrepant results were obtained from the three above assays.Our assay showed no cross-reaction with other diarrhea-associated pathogens.Its reproducibility was 100％in testing of two standard plasmids containing tcdB and tpi genes at two concentrations(105 and 102 copies/μL).Two standard plasmids were detected after the PCR and immunochromatography reagents had been stored for 3,6,9,and 12 months,and all the results were posi-tive.The limit of detection was 10 copies/μL for toxigenic C.difficile.Testing of 33 samples positive for C.difficile with our assay(33/215,15.3％)yielded findings statistically coherent with those of the Xpert C.difficile/Epi test(kappa value=0.965).The sensitivity,specificity,positive predictive value,and negative predictive value of our assay,with respect to Xpert C.difficile/Epi as the standard,were 94.3％,100.0％,100.0％,and 98.9％;these values were significantly higher than those of VIDAS CDAB(60.0％,98.9％,91.3％,and 92.7％)(Kappa=0.714,OR=157.50,95％CI:62.03-847.28,P=0.013).In conclusion,our newly developed assay is specific,stable,and reproducible,and may be used for rapid and accu-rate detection of toxigenic C.difficile.The assay could be used for C.difficile infection screening in outpatient and emergen-cy,community medical service center,and epidemiological settings.

Objective This study aimed to investigate the antimicrobial resistance profiles of bacterial strains isolated from pediatric intensive care units(PICU)in China for better antimicrobial therapy.Methods Clinical isolates were collected from 17 institutions,including tertiary care children's hospitals and pediatric department of tertiary general hospitals in China from January 1,2020 to December 31,2022.Antimicrobial susceptibility testing was carried out according to a unified protocol using Kirby-Bauer method or automated systems.Results were interpreted according to the breakpoints released by the Clinical and Laboratory Standards Institute(CLSI)in 2020.Results A total of 10 688 isolates were collected,including gram-positive organisms(39.2％)and gram-negative organisms(60.8％).The top three organisms were S.aureus(13.6％,1 453/10 688),A.baumannii(10.0％,1 067/10 688),and coagulase-negative Staphylococcus(9.9％,1 058/10 688).Multi-drug resistant organisms(MDROs)were very common in children.The prevalence of methicillin-resistant Staphylococcus aureus(MRSA),carbapenem-resistant Enterobacterales(CRE),carbapenem-resistant E.coli,carbapenem-resistant K.pneumoniae(CRKP),carbapenem-resistant A.baumannii(CRAB),and carbapenem-resistant P.aeruginosa(CRPA)was 41.1％,19.4％,8.8％,30.9％,67.4％,and 28.8％,respectively.Overall,more than 50％of Enterobacteriales isolates were resistant to cephalosporins,while nearly 25％of Enterobacteriales isolates were resistant to carbapenems.MDROs were highly resistant to commonly used antibiotics.More than 80％of CRE and CRAB strains were resistant to all beta-lactam antibiotics.CRE and CRAB showed low resistance rates to tigecycline and polymyxin.CRPA showed lower resistance rates to piperacillin,beta-lactamase inhibitor combinations than the resistance rates to third and fourth generation cephalosporins.All of the Staphylococcus and Enterococcus isolates were susceptible to vancomycin and tigecycline.None of PRSP strains isolated from meningitis and nonmeningitis samples were resistant to rifampicin,vancomycin,or linezolid.The prevalence of β-lactamase-negative ampicillin-resistant(BLNAR)strains was 43.3％in Haemophilus influenzae.Conclusions MDROs were prevalent in PICU.It is necessary to establish an effective multidisciplinary team(MDT)to control the antimicrobial resistance.

The purpose of this study was to establish a luciferase immunosorbent assay(LISA)using the Crimean-Congo hemorrhagic fever virus(CCHFV)glycoprotein C(Gc),a specific antigen,for the detection of CCHFV IgG antibodies.Three antigenic fragments based on CCHFV glycoprotein C were designed,and three recombinant plasmids were constructed by liga-tion with the NanoLuc luciferase(NLuc)expression vector pNLF1-N through molecular cloning.The accuracy of the sequences in the recombinant plasmids was confirmed through sequencing.The recombinant plasmids were transfected into eukaryotic cells to obtain fusion proteins containing specific antigens and luciferase,and the expression of the fusion proteins was verified by western blotting,thereby facilitating the establishment of the CCHFV-LISA detection technique.The assay's sensitivity,specificity,and stability were evaluated and compared with those of a commercial CCHFV IgG antibody test kit.Three recom-binant antigen fragments of CCHFV Gc—NLuc-Gc-Full,NLuc-Gc-C1,and NLuc-Gc-C2—were expressed,with molecular weights of 80.1 kDa,62.8 kDa,and 53.9 kDa,respectively.The optimal fragment for CCHFV detection was NLuc-Gc-C2.The sensitivity of the CCHFV-LISA was 90.9％,and the specificity was 100％;the findings were highly concordant with those for the commercial CCHFV enzyme-linked immunosorbent assay kit.Repeatability tests indicated no statistically significant differ-ences in inter-and intra-assay variability within the same batch.The LISA was highly specific,sensitive,and user-friendly in detecting IgG antibodies against the CCHFV.Therefore,this method may facilitate serological diagnosis and epidemiological studies in endemic regions,and provide essential technical support for disease surveillance and early warning.

Objective:To investigate the impact of postoperative complications on adverse outcomes following curative-intent resection for gallbladder cancer (GBC).Methods:The multi-center real-world study was conducted. The clinicopathological data of 629 patients with GBC, who were admitted to 14 medical centers including The First Affiliated Hospital of Army Medical University from the national multicenter database of Biliary Surgery Group of Elite Group of Chinese Journal of Digestive Surgery, from April 2020 to April 2024 were collected. There were 225 males and 404 females, aged (64±10)years. Patients underwent open curative-intent resection for GBC. Observation indicators: (1)surgery, postoperative complica-tions and adverse outcomes; (2) analysis of risk factors affecting postoperative adverse outcomes in patients and population attributable fraction (PAF). Missing data in predictor variables were addressed using multiple imputation with chained equations, while cases with missing outcome variables were addressed using the "multiple imputation then deletion (MID)" strategy. The severity of multicollinearity among independent variables was assessed using the variance inflation factor (VIF) test. Multivariable possion regression models with log link and robust error variance were construc-ted incorporating restricted cubic splines (3 knots) to address nonlinear relationships in continuous variables, calculating adjusted relative risk ( RR) with corresponding 95% confidence interval ( CI). Adjusted PAF was calculated for each imputed dataset using the AF package of R software, with subsequent pooling performed according to Rubin's rules. Results:(1) Surgery, postoperative complications and adverse outcomes. All 629 patients underwent curative-intent resection for GBC, of which 143 cases had postoperative complications, including 68 cases of intra-abdominal ascites, 39 cases of pulmonary infection, 21 cases of bile leakage, 12 cases of intra-abdominal hemorrhage, 11 cases of liver failure, 10 cases of pan-creatic fistula, 10 cases of wound infection, 10 cases of gastroparesis, 7 cases of cholangitis, 7 cases of sepsis. The same patient could have more than one kind of complication. Of 629 patients, there were 19 cases of postoperative 90-day death and 11 cases of missing data, 42 cases with post-operative 90-day reoperation and 7 cases with missing data, 44 cases with postoperative 90-day readmission and 3 cases with missing data, 155 cases with prolonged postoperative hospital stay and 3 cases with missing data. (2) Analysis of risk factors affecting the postoperative adverse outcomes in patients and PAF. Results of multivariate analysis showed that pulmonary infection and liver failure were independent risk factors for postoperative 90-day mortality ( RR=3.74, 12.15, 95% CI as 1.18-11.83, 1.98-74.48, P<0.05). Pulmonary infection demons-trated the highest PAF as 4.61% (95% CI as 3.94%-5.28%, P<0.05). Intra-abdominal ascites, pulmonary infection, bile leakage, and intra-abdominal hemorrhage were independent risk factors for post-operative 90-day reoperation ( RR=4.80, 3.62, 3.46, 4.99, 95% CI as 2.49-9.26, 1.42-9.21, 1.34-8.92, 1.55-16.06, P<0.05). Intra-abdominal ascites demonstrated the highest PAF as 8.65% (95% CI as 8.22%-9.08%, P<0.05). Intra-abdominal ascites, bile leakage, and liver failure were independent risk factors for postoperative 90-day readmission ( RR=6.20, 3.33, 14.33, 95% CI as 3.21-11.95, 1.33-8.35, 3.72-55.28, P<0.05). Intra-abdominal ascites demonstrated the highest PAF as 9.11% (95% CI as 8.85%-9.37%, P<0.05). Intra-abdominal ascites, pulmonary infection, bile leakage, liver failure, and wound infection were independent risk factors for prolonged postoperative hospital stay ( RR=2.29, 2.21, 2.26, 2.14, 3.35, 95% CI as 1.63-3.23, 1.41-3.46, 1.32-3.86, 1.11-4.13, 1.70-6.60, P<0.05). Intra-abdominal ascites demonstrated the highest PAF as 6.03% (95% CI as 5.71%-6.35%, P<0.05). Conclusion:Pulmonary infection is the most significant risk factor for postoperative 90-day mortality after curative-intent resection for GBC, while intra-abdominal ascites is the most significant risk factor for postoperative 90-day reoperation, postoperative 90-day readmission, and prolonged postoperative hospital stay.

Objective To explore the biological mechanisms of isorhamnetin(ISO)inhibition of breast cancer(BC)progression and effects on paclitaxel sensitivity by in vivo and in vitro experiments.Methods The effects of ISO on MDA-MB-231 cell viability,migration,invasion and apoptosis were explored by CCK-8,scratch,Transwell invasion and apoptosis assays.ISO biological functions were analyzed and core target genes were identified by genome-wide microarrays,functional enrichment and protein-protein interactions(PPI).CYP1B1 expression characterization and prognosis in BC were assessed by quantitative Real-time polymerase chain reaction(qRT-PCR),Western blotting(WB),and Kaplan-Meier plotter database.The effects of CYP1B1 overexpression on MDA-MB-231 cell viability,migration,invasion and apoptosis were explored by CCK-8,scratch,Transwell invasion and apoptosis assays.The effects of ISO and CYP1B1 on BC tumor growth were analyzed by establishing a subcutaneous graft tumor animal model and verified by histological analysis with immunohistochemistry(IHC)and hematoxylin and eosin(HE)staining.The effects of ISO and CYP1B1 on NF-κB signaling were analyzed by qRT-PCR and WB.The effect of ISO combined with paclitaxel on BC progression was analyzed by in vitro and in vivo experiments.Results The results of CCK-8 assay showed that ISO inhibited the viability of MDA-MB-231 cells with an IC50 value of 11.93 μmol/L;the scratch,Transwell invasion and apoptosis assays confirmed that ISO inhibited the migration and invasion of MDA-MB-231 cells and promoted apoptosis(P＜0.05).Volcano plot showed a total of 80 differentially expressed genes(DEGs),of which ISO promoted the expression of 27 genes and inhibited the expression of 53 genes.Gene set enrichment analyses showed that DEGs were mainly enriched in the signaling of organic hydroxyl compound metabolic process,inflammatory response,sterol metabolic process,and nuclear factor-κB(NF-κB).qRT-PCR,WB,and Kaplan-Meier plotter results showed that CYP1B1 mRNA and protein expression was increased in BC and mediated worse prognosis(P＜0.05).CCK-8,scratch,Transwell invasion and apoptosis assays showed that CYP1B1 overexpression enhanced MDA-MB-231 cell viability,migration and invasion,and inhibited apoptosis,which was reversed by the addition of ISO(P＜0.05).The results of in vivo experiments showed that ISO downregulated CYP1B1 expression and attenuated NF-κB signaling(P＜0.05).The results of in vitro and in vivo experiments showed that ISO combined with paclitaxel significantly inhibited BC cell viability and tumor growth(P＜0.05).Conclusion ISO inhibits BC malignant biological behavior by modulating the CYP1B1/NF-κB signaling axis,and ISO enhances paclitaxel sensitivity.