ObjectiveTo investigate the impact of anticentromere antibody （ACA） on the clinical features and prognosis of patients with primary biliary cholangitis （PBC） by comparing clinical classification， ursodeoxycholic acid （UDCA） response， GLOBE score， and UK-PBC score between ACA-positive PBC patients and ACA-negative PBC patients. MethodsA total of 749 patients who were admitted to Beijing Ditan Hospital， Capital Medical University， from August 2013 to December 2022 and were diagnosed with PBC were enrolled and divided into ACA-positive group with 147 patients and ACA-negative group with 602 patients. According to their conditions on admission， the two groups were compared in terms of the distribution of clinical types， i.e.， chronic progression-type PBC， portal hypertension-type PBC， and standard jaundice/liver failure-type PBC. There were 261 patients with complete data after 1-year follow-up， among whom there were 53 patients with positive ACA and 208 with negative ACA. A statistical analysis was performed， and propensity score matching was performed based on sex and age at a ratio of 1∶2. The two groups were compared in terms of 1-year UDCA response rate， GLOBE score， and UK-PBC score before and after matching. The independent-samples t test or the Mann-Whitney U test was used for comparison of continuous data between two groups， and the chi-square test was used for comparison of categorical data between two groups. ResultsCompared with the ACA-negative group， the ACA-positive group had a significantly higher age （61.28±10.35 years vs 56.74±12.17 years， t=4.164， P<0.001）， a significantly higher proportion of female patients （93.9% vs 77.6%， χ²=20.221， P<0.001）， a significantly higher proportion of patients with portal hypertension （48.3% vs 27.6%， χ²=23.289， P<0.001）， and a significantly lower proportion of patients with jaundice/liver failure （24.5% vs 38.5%， χ²=10.205， P<0.001）. After 1-year follow-up， for the 261 PBC patients with complete data， there was no significant difference in UDCA response rate before propensity score matching between the ACA-positive group and the ACA-negative group （41.5% vs 41.8%， P>0.05）， and there was a significant difference in the proportion of patients with a GLOBE score of >0.3 between the ACA-positive group and the ACA-negative group （92.5% vs 80.3%， χ²=3.935， P=0.047）. There were 53 patients in the ACA-positive group and 106 patients in the ACA-negative group after propensity score matching， and there were no significant differences between the two groups in UDCA response rate， GLOBE score， and UK-PBC score （all P>0.05）. ConclusionACA-positive patients tend to have an older age， with a higher proportion of female patients or patients with portal hypertension， while there is a relatively low proportion of patients with jaundice/liver failure. Positive ACA has no significant impact on UDCA response rate， GLOBE score， and UK-PBC score.

Objective To develop and evaluate a nucleic acid amplification test for spectinomycin-resistant Neisseria gonorrhoeae(N.gonorrhoeae).Methods N.gonorrhoeae-specific primers NG1/NG2 and primers specific to the N.gonorrhoeae rpsE gene mutation(80_82 delTTA)were designed.Genomic nucleic acids of spectinomycin-sensitive and resistant N.gonorrhoeae,Escherichia coli,Pseudomonas aeruginosa,and Salmonella typhi were used as templates to be amplified by PCR and quantitative real-time PCR(qPCR).The sensitivity and specificity of the method were evaluated accordingly.Results The NG1/NG2 primers could effectively amplify specific fragments of N.gonorrhoeae,yielding negative results for the nucleic acid amplification test of the other types of bacteria tested.E64/E175R and E-87/E95R could effectively differentiate the wild type and mutant(80_82 delTTA)rpsE genes.In PCR reactions,the minimum limits of NG1/NG2,E64/E175R,and E87/E95R for the target genes were 414.8 copies,414.8 copies,and 4.1 copies/μL,respectively,while those for qPCR reactions were 41.5,41.5,and 4.1×10-2 copies/μL,respectively.Conclusion A nucleic acid amplification test for spectinomycin-resistant N.gonorrhoeae with high specificity and sensitivity was successfully established in this study,which is expected to provide support for the rapid diagnosis of N.gonorrhoeae infection and treatment decision-making in clinical settings.

Objective To investigate the role of a novel rpsE gene mutation in mediating high-level spectinomycin resistance in Neisseria gonorrhoeae and to evaluate its effect on the biological fitness of the bacteria.Methods Spectinomycin-containing medium was used to screen for Neisseria gonorrhoeae strains with spontaneous mutations that conferred spectinomycin resistance.Minimum inhibitory concentrations(MIC)were determined,and the rpsE gene was sequenced.Changes in the growth rates of spectinomycin-resistant strain were assessed using the drop plate method and growth curves.Additionally,in vitro competition experiments were conducted with spectinomycin at different concentrations to assess changes in the biological fitness of the spectinomycin-resistant strain.Results A Neisseria gonorrhoeae strain with high-level spectinomycin resistance mediated by a novel rpsE gene mutation(88_90delGTT)was successfully identified and designated NG-SPTR.Compare with the wild-type strain,the NG-SPTR exhibited reduced growth rate(optical density[OD]comparison,P<0.05).In addition,in vitro competition experiments showed a competitive index(CI)<1 in gonococcal base liquid(GCBL)without or with low-concentration spectinomycin(≤16 μg/mL).In the GCBL with 32 μg/mL spectinomycin,the CI value gradually increased from<1 before 18 h to>1 after 18 h.The mutant strain showed CI>1 in GCBL with spectinomycin concentrations≥64 μg/mL.Conclusion The rpsE gene mutation(88_90delGTT)mediates high-level spectinomycin resistance in Neisseria gonorrhoeae,and imposes a fitness cost on the bacteria.The biological fitness of the mutant strain is influenced by the concentration of spectinomycin.

Objective To explore the effect and mechanism of quercetin on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).Methods BMSCs were divided into a blank control group(Control)and quercetin(Quercetin)low-dose group(4.8 mL/kg),medium-dose group(9.6 mL/kg),and high-dose group(19.2 mL/kg)intervened with drug-containing serum,while the positive control group was treated with osteogenic differentiation medium,respectively.The cell cycle was analysed by flow cytometry,cell proliferation was detected by MTT assay,cell activity was determined by Alkaline phosphatase(ALP)assay kit,and calcified nodule formation was observed by alizarin red staining.The expression of β-catenin and the key factors of osteogenic differentiation,runt-related transcription factor 2(RUNX2)and osteocalcin(OCN),were detected by qPCR and Western blot,respectively.Results Compared with the control group,quercetin-containing serum significantly promoted the proliferation of BMSCs(P=0.000205,P=0.000063)and enhanced the formation of calcium nodules,and increased osteogenic and ALP activity after osteogenic differentiation.The results of qPCR and Western blot showed that the quercetin group significantly up-regulated the mRNA and protein expression of β-catenin(P<0.0001),RUNX2(P<0.0001)and OCN(P<0.0001)during osteogenic differentiation.Conclusion Quercetin can effectively promote the osteogenic differentiation of BMSCs,and its mechanism is achieved by activating the Wnt/β-catenin signaling pathway and up-regulating the expression of the osteogenesis-related transcription factor RUNX2.

The clinical application of chemotherapeutic drugs has been limited due to the lack of tumor selectivity which causes different degrees of adverse reactions. Transferrin receptor(TfR) is one of the targets for tumor targeted drug delivery system because it is over-expressed in many kinds of tumor cells and tumor-associated blood vessels, but is low-expressed in normal cells. By modifying drug carriers or coupling antitumor drugs with ligands that have high specific affinity for TfR, it is expected to achieve targeted therapy of TfR over-expressed tumors. In this paper, the application progress of TfR functional ligands in tumor targeted drug delivery in recent years is summarized, in order to provide reference for the research of TfR targeted drug delivery systems.

Objective To investigate the differences in clinical features between the primary biliary cholangitis(PBC)patients with negative or positive anti-mitochondrial antibody(AMA)by analyzing related immune and biochemical parameters.Methods This study was conducted among the patients who attended Beijing Ditan Hospital,Capital Medical University,from January 2013 to December 2022 and were diagnosed with PBC,and they were divided into AMA negative group with 139 patients(24.5%)and AMA positive group with 428 patients(75.5%).Propensity score matching at a ratio of 1∶1 was performed with age and sex as matching factors and a matching tolerance of 0.06.Liver function,coagulation,and immune parameters on admission were analyzed,as well as the changes in liver function and other indicators after 6 months of treatment and the response to ursodeoxycholic acid(UDCA)at 6 and 12 months of treatment.The independent-samples t test was used for comparison of normally distributed continuous data between two groups,and the Mann-Whitney U rank sum test was used for comparison of non-normally distributed continuous data between two groups;the chi-square test was used for comparison of categorical data between two groups.Results There were 139 AMA-negative PBC patients and 139 AMA-positive PBC patients after propensity score matching.Compared with the AMA positive group on admission,the AMA negative group had significantly lower levels of direct bilirubin and globulin(Glo)and significantly higher levels of albumin,albumin/globulin ratio,prealbumin,and fibrinogen(all P<0.05).After 6 months of UDCA treatment,there were significant differences in Glo and prealbumin between the AMA negative group and the AMA positive group(P<0.05).Both the AMA negative group and the AMA positive group had an increase in prealbumin after 6 months of treatment,and the AMA negative group had a significantly greater increase than the AMA positive group(U=41.00,P=0.015).After UDCA treatment for 6 and 12 months,there was no significant difference in treatment response to UDCA between the AMA negative group and the AMA positive group(all P>0.05).Conclusion After matching for age and sex,compared with the AMA-positive PBC patients,the AMA-negative PBC patients tend to have a milder degree of liver inflammation and damage,significantly greater improvements in inflammation and liver synthesis ability after UDCA treatment,and better response to UDCA.

Objective To evaluate the clinical effect of Tension-free laparoscopic lateral suspension with mesh for pelvic organ prolapse.Methods A total of 85 patients who underwent pelvic organ prolapse were selected as the study group,and 40 patients underwent Laparoscopic sacral fixation surgery(LSC)as the control group in Henan Provincial People's Hospital from March 1,2021 to October 31,2023.The patients were divided into two subgroups:uterine preservation group and uterine resection group and followed up until April 30,2024.The intra-operative conditions and postoperative complications were recorded and analyzed.POP quantitative staging(POP-Q)scores were used for evaluation.Preoperative and postoperative quality of life and therapeutic effect were evaluated using the pelvic floor distress inventory short form-20(PFDI-20),urinary distress inventory-6(UDI-6),colorectal-anal distress inventory-8(CRADI-8),pelvic organ prolapse distress inventory-6(POPDI-6),pelvic floor impact questionnaire-7(PFIQ-7),prolapse and Incontinence sexual function questionnaire short form(PISQ-12).Results The median follow-up time for patients in the study group is 13.13 months,with an objective cure rate of 96.47%and a reoperation rate of 1.18%.The perioperative complication rates are 6.45%for uterine resection and 4.35%for uterine preservation,while the mesh exposure rate is 1.61%for uterine resection.In comparison,the median follow-up time for patients in the control group is slightly longer at 13.76 months,with an objective cure rate of 92.5%and a reoperation rate of 2.5%.The perioperative complication rates are higher at 14.71%for uterine resection and as high as 33.33%for uterine preservation,while the mesh exposure rate is also elevated at 8.82%for uterine resection.Despite these differences,there was no significant disparity in objective cure rates or reoperation rates between the study group and the control group.Furthermore,it was observed that the study group experienced shorter operation times,less bleeding,faster postoperative recovery,shorter hospitalization periods,lower perioperative complications,and reduced mesh exposure rates-especially among patients with uterine preservation.Additionally,intra-group comparisons revealed significant improvements in all POP-Q indicators one year after surgery(P<0.05),along with significantly lower scores on PFDI-20,UDI-6,CRADI-8,POPDI-6,PFIQ-7,and PISQ-12 scales com-pared to pre-surgery levels(P<0.05).However,no significant inter-group differences were noted.Conclusions Tension-free laparoscopic lateral suspension with mesh proves to be an effective surgical approach for treating ante-riorand middle pelvic organ prolapse.It demonstrates few perioperative complications while significantly improving prolapse symptoms and enhancing patient qualityof life.It stands as a viable alternative to sacrocolpopexy,particu-larly beneficial for patients with a preserved uterus.

Lung cancer ranks the top of malignancies that cause cancer-related deaths worldwide.The leaves of Morus alba L are traditional Chinese medicine widely applied in respiratory diseases.Our previous work has demonstrated the anti-lung cancer effect of secondary metabolites of mulberry leaf,but their mechanism of action has still not fully elucidated.We synthesized Moracin N(MAN)-Probe conjugated with alkyne to label lung cancer cells and identified protein targets by chemical proteomic analysis.MAN and its probe exerted similar growth-inhibitory effect on human lung cancer cells.Chemical proteomic results showed that MAN targeted the programmed death ligand 1(PD-L1)checkpoint pathway and T cell receptor(TCR)signaling pathway,indicating its immune-regulatory function.Cell-free surface plasmon resonance(SPR)results showed the direct interaction of MAN with PD-L1 protein.Molecular docking analysis demonstrated that MAN bound to E158 residue of PD-L1 protein.MAN downregulated the expression levels of PD-L1 in a time-and dose-dependent manner and disrupted the PD-L1/programmed death 1(PD-1)binding,including other secondary metabolites of mulberry leaves Guangsangon E(GSE)and Chalcomoracin(CMR).Human peripheral blood mononuclear cells(PBMCs)co-cultured with MAN-treated A549 cells,resulting in the increase of CD8+GZMB+T cells and the decrease of CD8+PD-1+T cells.It suggested that MAN exerts anti-cancer effect through blocking the PD-L1/PD-1 signaling.In vivo,MAN combined with anti-PD-1 antibody significantly inhibited lung cancer development and metastasis,indicating their synergistic effect.Taken together,secondary metabolites of mulberry leaves target the PD-L1/PD-1 signaling,enhance T cell-mediated immunity and inhibit the tumorigenesis of lung cancer.Their modulatory effect on tumor microenvironment makes them able to enhance the therapeutic efficacy of immune checkpoint inhibitors in lung cancer.

Background and purpose:Colorectal cancer(CRC)is one of the major malignant tumors threatening human health worldwide,with long-term high incidence and mortality rate.Potassium channel modulatory factor 1(KCMF1)is a member of the E3 ubiquitin ligase family.It binds to target proteins through the RING domain and participates in the regulation of a variety of biological processes in vivo.However,the function of KCMF1 in CRC remains unclear.This study aimed to investigate the expression level of E3 ubiquitin ligase KCMF1 in colorectal tumor,and to explore the effects of KCMF1 on the proliferation of CRC cells and its underlying molecular mechanism.Methods:The The Cancer Genome Atlas(TCGA)and Genotype-Tissue Expression(GTEx)databases were used to analyze the expression level of KCMF1 in CRC tissues and adjacent tissues and the association between the KCMF1 expression and the prognosis of CRC patients.Furthermore,immunohistochemical staining was performed to detect the protein level of KCMF1 in 90 paired human CRC tissues and adjacent non-tumor tissues.Lentiviral shRNA delivery system was employed to specifically target the KCMF1 gene(shKCMF1)in HCT116 and HCT15 CRC cell lines.The effects of KCMF1 knockdown on cell proliferation,apoptosis and cell cycle distribution were assessed by methyl thiazoyl terazolium(MTT)assay,colony formation assay,Western blot and flow cytometry.Changes in the transcriptional profile in HCT116 cells upon KCMF1 knockdown were identified by RNA sequencing(RNA-Seq),and the affected signaling pathways were evaluated by bioinformatics analysis.Real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR),Western blot,luciferase reporter assay and cell immunofluorescence assay were utilized to validate the alteration of the affected signaling pathway.Results:The TCGA and GTEx databases and IHC results showed that the mRNA and protein expression levels of KCMF1 in CRC tissues were significantly upregulated compared with adjacent tissues(P＜0.01).KCMF1 expression level was negatively correlated with the survival time of patients with CRC(P＜0.01),and was positively associated with CRC clinical stage(P＜0.05).Compared with control cells,KCMF1 knockdown significantly inhibited the proliferation of HCT116 and HCT15 cells(P＜0.001),induced cell apoptosis(P＜0.001),and led to cell cycle arrest in G1 phase(P＜0.01).RNA-Seq analysis showed that KCMF1 was involved in the regulation of several signaling pathways,including nuclear factor-κB(NF-κB)signaling pathway.KCMF1 knockdown reduced the transcription levels of the target genes of NF-κB signaling pathway,including BCL-XL,XIAP and CIAP(P＜0.05),and suppressed the expression of phosphorylated p65 and nuclear translocation of p65(P＜0.01).Meanwhile,the activity of NF-κB reporter was reduced in tumor cells upon KCMF1 knockdown(P＜0.01).Conclusion:The expression of KCMF1 is significantly upregulated in human CRC tissues and positively associated with advanced clinical stage and poor prognosis.KCMF1 may promote the proliferation of CRC cells by activating the NF-κB signaling pathway.KCMF1 may be a potential new therapeutic target for CRC.

Objective To establish and evaluate a microbial sensitivity test method for Neisseria gonorrhoeae based on resazurin coloration.Methods Based on the broth microdilution method,resazurin was added as a live bacteria indicator.WHO G,a WHO gonococcal reference strain,was used to optimize the incubation time for resazurin-stained bacteria and the color change was visually observed to obtain the results.Agar dilution method(the gold standard)and resazurin-based microdilution assay were used to determine the minimum inhibitory concentration(MIC)of azithromycin,ceftriaxone,and spectinomycin for 3 reference strains and 32 isolates of Neisseria gonorrhoeae.The results were analyzed based on essential agreement(EA),which reflected the consistency of the MIC values,category agreement(CA),which reflected the consistency in the determination of drug resistance,intermediary,and sensitivity,very major error(VME),which reflected false sensitivity,and major error(ME),which reflected pseudo drug resistance,to evaluate the accuracy of resazurin-based microdilution assay as a microbial sensitivity test of of Neisseria gonorrhoeae.CA and EA rates≥90%and VME and ME rates≤3%were found to be the acceptable performance rates.Results The results obtained 6 hours after resazurin was added were consistent with those of the agar dilution method and the resazurin-based microdilution assay was established accordingly based on this parameter.The EA of resazurin-based microdilution assay for measuring the MIC results of azithromycin,ceftriaxone,and spectinomycin was 97.1%,91.5%,and 94.3%,respectively,and the CA was 88.6%,94.3%,and 94.3%,respectively.The VME was 0%for all three antibiotics,while the ME was 11.4%,5.7%,and 5.7%,respectively.Conclusion The resazurin-based microdilution assay established in this study showed good agreement with agar dilution method for measuring the MIC of antibiotics against Neisseria gonorrhoeae.Moreover,the sensitivity results of this method were highly reliable and could be easily obtained through naked eye observation.Nonetheless,the results of drug resistance should be treated with caution and the optimization of parameters should be continued.