Background/Aims: Liver transplantation (LT) following total hepatectomy is a life-saving treatment for hepatocellular carcinoma (HCC). The HCC recurrence after LT hinders the effectiveness of the procedure. The objective of this study is to develop a pre-operative risk stratification model based on a liquid biopsy. Methods: We conducted a comprehensive multi-omics study of 260 HCC patients from three centers, including clinical data, low-coverage whole-genome sequencing of cell-free DNA (cfDNA) from plasma, as well as whole-exome, single-nucleus RNA, and spatial transcriptomics from matched tumor and non-tumor tissues. Results: We identified cfDNA-derived copy number alteration (CNA) signatures associated with post-transplant recurrence. By integrating cfDNA-derived CNA profiles with single-cell transcriptomic data, we traced recurrence-associated cfDNA to a distinct subpopulation of malignant cells within the primary tumor. These cells were embedded in a pro-metastatic microenvironment of specialized endothelial subtypes and cancer-associated fibroblasts. Notably, most recurrence-associated lesions were detectable in cfDNA prior to liver transplantation (LT). Building on these insights, we developed the ZJU Criteria based on CNA fragments and tumor markers, a pre-LT risk prediction tool that integrates conventional clinical factors with cfDNA-derived CNA signatures, and validated it using internal and independent external cohorts. Conclusion Our findings suggest that post-transplant recurrence commonly originates from advanced subclones that emerge late during tumor evolution. The ZJU Criteria provides an accurate, non-invasive strategy that significantly improves pre-LT risk stratification and clinical decision-making for patients with HCC.

This study assessed the role and mechanism of the recombinant Bacillus Calmette-Gue?rin vaccine(rBCG)with c-di-AMP adjuvant in regulating metabolism and immunity in epididymal white adipose(eWAT)in mice.Male C57BL/6 mice were intravenously immunized with BCG and rBCG,and their body weights were monitored.eWAT was isolated from the mice,and the stromal vascular fractions(SVFs)cell number was counted with a hemocytometer.Sections of mouse adipose tissue were prepared,and the size,number,and morphology of eWAT adipocytes and crown-like structure(CLS)formation were compared under a microscope after HE staining.The transcription levels of lipid metabolism-associated factors,cytokines and aging-associated genes in each group were determined with qRT-PCR.The body weights of mice gradually increased after immunization with BCG and rBCG.The proportions of eWAT increased,and the SVFs cell number decreased,in rBCG immunized mice.HE staining indicated that BCG immunization promoted hyperplasia,whereas rBCG immunization promoted hypertrophy of eWAT adipocytes;moreover,both BCG and rBCG immunization induced CLS formation in eWAT.The qRT-PCR results indicated that rBCG immunization inhibited the expression of genes associated with lipolysis and energy expenditure in eWAT.BCG immunization had little effect on cytokine transcription,whereas rBCG significantly induced the transcription of IFN-γ and IL-1Ra,and inhibited that of IL-15 and IL-2,but did not induce the expression of aging-associated genes.Thus,rBCG immunization induced eWAT adipocyte hypertrophy,which was associated with the inhibition of eWAT lipolysis and the regulation of cytokine expression.

The objective of this study was to evaluate the toxicity and safety of novel Mycobacterium tuberculosis fusion strain protein derivatives,referred to as B/R strain active proteins.In cellular experiments,RAW264.7 cells were treated with each vaccine preparation,and apoptosis rates were measured.In subsequent animal experiments,C57BL/6 mice were immunized via subcutaneous injection,and their survival and body weight changes were monitored and recorded at 2,4,8,12,and 16 weeks.The lungs and spleens were harvested to calculate organ coefficients,and pathological examinations were conducted.At the eighth week of immunization,the mice were infected with high concentrations of BCG,and pathological changes in the lungs and spleens were observed 4 weeks post-infection.The apoptosis rate at 6 hours was significantly higher in the experimental group than the PBS group(P<0.05).At 12 and 24 hours,the apoptosis rate in the experimental group remained higher than that in the PBS group,although this difference was not statistically significant.After immunization,mice in all four groups exhibited normal growth patterns,as indicated by stable body weight changes.At 4 and 12 weeks post-immunization,the lung coefficients in the protein group were significantly higher than those in the PBS group at the same time points.Additionally,the lung coefficients in the BCG group were significantly elevated across all time periods(P<0.05).The spleen coefficients in the protein and BCG groups were significantly higher than those in the PBS group at 2,4,8,12,and 16 weeks,whereas the ICD B/R group showed higher spleen coefficients than the PBS group only at week 8(P<0.05).Pathological examination revealed normal lung and spleen tissues in the PBS group.However,during the 2-8 weeks immunization period,lung and spleen tissues in all experimental groups exhibited varying degrees of damage,which gradually diminished by 12-16 weeks.Notably,no tuberculosis nodules were observed in any experimental group.After infection with high concentrations of BCG,no overt pathological changes were observed on the surfaces of the lungs and spleens in any group.Microscopic examination revealed less severe pathological changes in the lungs and spleens of mice in the experimental groups than the PBS group.Furthermore,no statistically significant differences were observed between the protein group and the BCG group.Our findings suggested that the B/R strain active proteins'toxicity and safety profiles were comparable to those of BCG,and showed immunoprotective effects.This study provides an experimental foundation for the development of a novel tuberculosis vaccine.

Objective:To establish a stable rat model of sequelae of pelvic inflammatory disease(SPID)with clinical characteristics,and to provide a reliable experimental model for the study of the pharmcological effect and mechanism of SPID.Methods:Twenty-four 7-week-old SD rats were divided into sham operation group,model-A(108 cfu/mL mixed bacterial solution,0.2 mL),model-B(109 cfu/mL mixed bacterial solution 0.2 mL),and model-C(108 cfu/mL E.coli 0.2 mL).The weight of the rat's uterine was weighed and the uterine index was calculated.The automatic hematology analyzer was used to detect the blood routine;hematoxylin-eosin staining(HE)and masson staining were used to detect uterine pathlogical changes in rats.Enzyme-linked immunosorbent assay(ELISA)was used to detect interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in rat uterine tissue homogenates.Western blot was used to detect the expression of proteins related to NF-κB signaling pathway.Results:Compared with the sham operation group,the uterine index of model-A,model-B,and model-C were significantly increased(P＜0.05,P＜0.01).The levels of WBC and NE in the model-A increased significantly(P＜0.01).The level of LY in model-B decreased significantly(P＜0.01).The levels of IL-1β,TNF-α in model-A,model-B,and model-C were significantly increased(P＜0.01).The levels of IL-6 in model-A and model-B were significantly increased(P＜0.05,P＜0.01).The collagen volume fraction of model-A and model-B were significantly increased(P＜0.01).Mechanism study indicates that the expression levels of p-IKKβ/IKKβ,p-IκBα/IκBα and p-p65/p65 in model-A were significantly increased(P＜0.01),and the expression levels of IκBα/β-actin were significantly decreased(P＜0.01).The expression level of p-IKKβ/IKKβ in model-B was significantly increased(P＜0.01).Conclusions:A stable rat model of SPID that conforms to clinical characteristics can be successfully constructed by combining 0.2 mL of mixed bacterial solution with a concentration of 108 cfu/mL and mechanical injury.This modeling method intervened in the expression of the NF-κB inflammatory signaling pathway.

Aim To establish and evaluate an alternative meth-od for detecting skin sensitization of cosmetics based on local lymph node assay using bromodeoxyuridine(BrdU)with flow cytometry(FCM).Methods(1)25％hexyl cinnamic alde-hyde(HCA)was chosen as a positive control with an acetone:olive oil(4∶1,V/V,AOO)mixture as a vehicle control for the experiment.The dorsal sides of both ears of mice were treated with test solutions on day 1,day 2,and day 3.Brdu solution was injected inter-peritoneally on day 5.On day 6,the bilateral ears and mandibular lymph nodes were excised,and the number of Brdu positive cells was measured by flow cytometry.The stim-ulation index(SI)was calculated to identify whether it was ≥3,in order to establish the method of LLNA:Brdu-FCM.(2)BrdU-FCM test was conducted using a blind method with the fif-teen reference substances listed in OECD TG429 whose skin sensitization potentials were known.The test substances were dissolved in AOO,N,N-dimethylformamide(DMF)or dimeth-yl sulfoxide(DMSO)at three different concentrations.Tests were performed the same as above.SI and EC2.7 were calculat-ed to evaluate whether the test substance was categorized as a skin sensitizer.The reliability and accuracy of the method were validated by comparing the classification of test substances with that in OECD TG429.Results The SI for 25％HCA was 3.9,showing positive in the skin sensitization test.It demonstrated that the LLNA:Brdu-FCM test method was properly implemen-ted.Nine test substances(2,4-dinitrochlorobenzene,4-pheny-lenediamine,cobalt chloride,2-mercaptobenzothiazole,hexyl-cinnamaldehyde,eugenol,phenyl benzoate,cinnamic alcohol,imidazolidinyl urea)were positive,and six test substances(methyl methacrylate,chlorobenzene,isopropanol,lactic acid,methyl salicylate,salicylic acid)were negative.The method was evaluated with sensitivity of 90％,specificity of 100％,positive prediction rate of 100％,negative prediction rate of 83％,false positive rate of 0％,false negative rate of 17％and accuracy of 93％.The LLNA:BrdU-FCM assay could correctly categorize the test substances that were skin sensitizers or non-sensitizers.Conclusion The LLNA:BrdU-FCM assay appears to be a relia-ble predictor of skin sensitization protential of chemicals,and it is expected to an alternative method for identifying skin sensitization as a supplementary in safety evaluation of cosmetic ingredient.

Objective To compare the pain relief and long-term clinical success rate of vital pulp therapy and root canal treatment in mature permanent teeth with carious irreversible pulpitis.Methods A total of 90 patients diagnosed with carious irreversible pulpitis in mature permanent teeth were collected at Shanghai Stomatological Hospital from Jan 2021 to Jun 2022.They were randomly divided into two groups:test group(n=45)undergoing vital pulp therapy(VPT)and control group(n=45)undergoing root canal treatment(RCT).Pain scores were recorded before treatment,24 hours after operation and 7 days after operation.We conducted clinical evaluation and imaging analysis at 1,6,and 12 months after the surgery,then compared the pain scores and treatment success rates between the two groups.Results Eighty-one patients,including 39 patients in group VPT aged(31.00±1.43)years old and 42 patients in group RCT aged(30.60±1.54)years old,received follow-up for more than 1 year,and the success rate of the test group and control was 97.44%and 95.24%.The pain degree of the two groups was reduced at 24 hours and 7 days after operation(P<0.05),and the pain score of the test group was reduced compared with that in the control group 7 days after operation(P<0.01).Conclusion Compared with root canal treatment,vital pulp therapy for mature permanent teeth with carious irreversible pulpitis can achieve good results in short-term pain evolution and long-term clinical success.

Objective To retrospectively analyze the association between metabolic factors and high-risk colorectal adenoma(CRA).Methods The medical records of patients aged 18-75 years who underwent their initial colonoscopy at Karamay Central Hospital of Xinjiang Uygur Autonomous Region from Jul 2000 to Mar 2017 were collected.The comparison between normal colonoscopy(NC)and high-risk CRA patients was conducted using an unpaired t-test,while chi-square test was used for categorical variables.Least absolute shrinkage and selection operator(LASSO)regression and Logistic regression were utilized to analyze the association between metabolic factors and high-risk CRA.Results A total of 1 798 patients meeting the inclusion and exclusion criteria were enrolled and divided into normal colonoscopy(NC)findings group(n=972)and high-risk CRA group(n=826).The high-risk CRA group exhibited significantly lower levels of high-density lipoprotein cholesterol(HDL-C)in comparison to the NC group,while uric acid and fibrosis 4(FIB-4)index levels were significantly higher than those observed in the NC group(all P<0.05).Based on LASSO regression analysis,we identified 12 variables that potentially influence the occurrence of high-risk CRA,including age,gender,smoking history,alcohol consumption history,non-alcoholic fatty liver disease(NAFLD),hypertension,coronary artery disease,hyperglycemia,hypercholesterolemia,low levels of HDL-C,elevated alanine aminotransferase,and elevated gamma-glutamyl transferase.Multivariate analysis revealed that individuals aged over 50 years,male gender,cigarette and alcohol consumption,low HDL-C levels,history of NAFLD and hypertension were identified as independent risk factors associated with high-risk CRA(P<0.05).In addition,without or with adjusting for age,sex,smoking,and drinking history,patients with a high TG/HDL-C ratio(the ratio≥2.68)had a significantly higher risk of high-risk CRA than those with a low TG/HDL-C ratio(the ratio<2.68)[odds ratios(ORs)were1.430 and 1.235 respectively,all P<0.05)].Without or with adjusting variables,the ORs for NAFLD patients with FIB-4 index>2.67 were 1.849(P=0.466)and 1.435(P=0.707),respectively.Conclusion A significant association exists between metabolic factors and high-risk CRA.Independent risk factors for high-risk CRA include older age(≥50 years),male,smoking history,alcohol consumption history,low levels of HDL-C,and a history of NAFLD and hypertension.Individuals exhibiting a TG/HDL-C ratio exceeding 2.68 manifest a significantly heightened susceptibility to the development of high-risk CRA.Therefore,elderly males with one or more aforementioned metabolic abnormalities should be considered a priority population for colorectal screening.

Objective:To establish a model of reactive Th17 cells proliferation induced by collagen type Ⅱ(C Ⅱ)in vitro and investigate its influencing factors.Methods:The splenic lymphocytes of normal and CIA mice were isolated and divided into groups.They were given inactivated or non-inactivated C Ⅱ or different concentrations of IL-23(2,10,50 ng/mL),or IL-23p19 antibody.Culturing for 60 hours,the ratio of CD4+RORγt+Th17 cells was detected by flow cytometry.Then,the results obtained are ana lyzed,and the corresponding conclusions are drawn.Results:After 60 hours of culture in vitro,the ratio of Th 17 cells stimulated by inactivated or non-inactivated C Ⅱ in normal mouse spleen lymphocytes was significantly lower than that before culture,and the ratio of Th17 cells not stimulated by C Ⅱ in CIA mouse spleen lymphocytes was also significantly lower than that before culture,while the ratio of Th17 cells stimulated by inactivated C Ⅱ or non-inactivated C Ⅱ in CIA mouse spleen lymphocytes was significantly higher than that before culture,and there was a significant difference compared with the CIA control group(P＜0.05).However,there was no statistical difference in the ratio of Th17 cells between the two groups without inactivated C Ⅱ and inactivated C Ⅱ(P=0.44).After the analysis of the data obtained from the study,it was further concluded that different concentrations of IL-23 did not affect the Th17 cell ratio of spleen lymphocytes of CIA mice in vitro,but after adding IL-23p19 antibody neutralization reagent,the Th17 cell ratio of spleen lymphocytes of CIA mice in vitro decreased significantly,with a statistical difference compared with the blank control group(P＜0.01).Conclusions:This study established an in vitro Th17 cell proliferation model induced by type Ⅱ collagen,exploring the effects of IL-23 and collagen activity on Th17 cell proliferation.The results showed that CⅡ stimulation significantly promoted Th17 cell proliferation in CIA mice,with both active and inactivated CⅡ inducing proliferation.IL-23 was found to be essential for the maintenance of Th17 cells,although its direct proliferative effect was limited.These findings provide new experimental evidence and theoretical support for the mechanism research of rheumatic diseases and IL-23/IL-17 pathway-targeted therapies,with important implications for immune regulation and drug development.

Objective:To establish a stable rat model of sequelae of pelvic inflammatory disease(SPID)with clinical characteristics,and to provide a reliable experimental model for the study of the pharmcological effect and mechanism of SPID.Methods:Twenty-four 7-week-old SD rats were divided into sham operation group,model-A(108 cfu/mL mixed bacterial solution,0.2 mL),model-B(109 cfu/mL mixed bacterial solution 0.2 mL),and model-C(108 cfu/mL E.coli 0.2 mL).The weight of the rat's uterine was weighed and the uterine index was calculated.The automatic hematology analyzer was used to detect the blood routine;hematoxylin-eosin staining(HE)and masson staining were used to detect uterine pathlogical changes in rats.Enzyme-linked immunosorbent assay(ELISA)was used to detect interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in rat uterine tissue homogenates.Western blot was used to detect the expression of proteins related to NF-κB signaling pathway.Results:Compared with the sham operation group,the uterine index of model-A,model-B,and model-C were significantly increased(P＜0.05,P＜0.01).The levels of WBC and NE in the model-A increased significantly(P＜0.01).The level of LY in model-B decreased significantly(P＜0.01).The levels of IL-1β,TNF-α in model-A,model-B,and model-C were significantly increased(P＜0.01).The levels of IL-6 in model-A and model-B were significantly increased(P＜0.05,P＜0.01).The collagen volume fraction of model-A and model-B were significantly increased(P＜0.01).Mechanism study indicates that the expression levels of p-IKKβ/IKKβ,p-IκBα/IκBα and p-p65/p65 in model-A were significantly increased(P＜0.01),and the expression levels of IκBα/β-actin were significantly decreased(P＜0.01).The expression level of p-IKKβ/IKKβ in model-B was significantly increased(P＜0.01).Conclusions:A stable rat model of SPID that conforms to clinical characteristics can be successfully constructed by combining 0.2 mL of mixed bacterial solution with a concentration of 108 cfu/mL and mechanical injury.This modeling method intervened in the expression of the NF-κB inflammatory signaling pathway.

Objective To compare the pain relief and long-term clinical success rate of vital pulp therapy and root canal treatment in mature permanent teeth with carious irreversible pulpitis.Methods A total of 90 patients diagnosed with carious irreversible pulpitis in mature permanent teeth were collected at Shanghai Stomatological Hospital from Jan 2021 to Jun 2022.They were randomly divided into two groups:test group(n=45)undergoing vital pulp therapy(VPT)and control group(n=45)undergoing root canal treatment(RCT).Pain scores were recorded before treatment,24 hours after operation and 7 days after operation.We conducted clinical evaluation and imaging analysis at 1,6,and 12 months after the surgery,then compared the pain scores and treatment success rates between the two groups.Results Eighty-one patients,including 39 patients in group VPT aged(31.00±1.43)years old and 42 patients in group RCT aged(30.60±1.54)years old,received follow-up for more than 1 year,and the success rate of the test group and control was 97.44%and 95.24%.The pain degree of the two groups was reduced at 24 hours and 7 days after operation(P<0.05),and the pain score of the test group was reduced compared with that in the control group 7 days after operation(P<0.01).Conclusion Compared with root canal treatment,vital pulp therapy for mature permanent teeth with carious irreversible pulpitis can achieve good results in short-term pain evolution and long-term clinical success.