Background/Aims: Dysregulated cholesterol metabolism is a hallmark of hepatocellular carcinoma (HCC) that drives tumor initiation and progression. However, clinical targeting of cholesterol metabolism has yielded limited benefits due to stringent feedback in tumor cells. Identifying a central mediator capable of restoring cholesterol homeostasis within the cell’s intrinsically fine-tuned regulatory framework is urgently needed. Methods: We integrated a proteomic dataset from patients with cholesterol-dysregulated HCC into a global cholesterol metabolic regulatory network to identify potential therapeutic targets for disrupted cholesterol homeostasis. The prognostic significance of the candidate targets was further validated in an independent cohort through immunohistochemistry. Functional and mechanistic studies were conducted in vitro using HCC cell lines and in vivo using mouse models. The pharmacological efficacy of the candidate agent was evaluated in both subcutaneous and orthotopic HCC mouse models. Results: ER lipid raft-associated 1 (ERLIN1), a pivotal regulator of cholesterol metabolism reprogramming, was identified as an independent favorable prognostic indicator in HCC. ERLIN1 constrains HCC progression both in vitro and in vivo by stabilizing the INSIG1–SCAP–SREBP2 axis and maintaining the metabolic balance of intracellular cholesterol. Under hypoxia, impaired factor-inhibiting hypoxia-1-dependent hydroxylation of ASB11 at asparagine residues 90 and 92 enhances ASB11-mediated ERLIN1 degradation. Pharmacological targeting of this axis using zoledronic acid (ZoA) attenuated HCC progression by weakening the ASB11–ERLIN1 interaction and restoring cholesterol homeostasis. Conclusions ERLIN1 represents a druggable metabolic vulnerability in cholesterol-dysregulated HCC. Targeting the ASB11–ERLIN1 axis with the clinically approved ZoA reestablishes cholesterol homeostasis and offers a promising therapeutic strategy to overcome the current limitations of cholesterol-targeted HCC therapies.

Objective To investigate mutations in OTU domain-containing protein 3(OTUD3)and their functional mechanisms in the initiation and progression of colorectal cancer(CRC).Methods Gene expression profiling interactive analysis(GEPIA2)and the human protein atlas database(THPA)were used to analyze gene transcription and protein expressions.Samples from 32 patients with CRC were collected to identify OTUD3 mutants.Based on the information about mutation sitesof OTUD3 in an existing database,a plasmid vector containing the OTUD3 gene mutant was constructed.Plasmid vectors containing the phosphatase and tensin homolog deleted on chromosome 10(PTEN)and the OTUD3 gene mutant were co-transfected into HCT116 cells.Western blotting,half-life,immunoprecipitation,ubiquitination,and hybrid algorithm molecular docking(H-DOCK)assays were employed to find out whether and why the OTUD3 mutants affected PTEN protein levels.Functional alterations in CRC cells after OTUD3 mutation were verified by CCK-8 cell proliferation,transwell cell invasion,scratch,and clonal formation assays.Results OTUD3 mutations were highly frequent in CRC.OTUD3 mutants R178W and N321S resulted in the loss of function of the stable PTEN protein,leading to enhanced proliferation,invasion,migration,and survival of CRC.Conclusion In CRC,OTUD3 mutation reduces the ability to stabilize PTEN and promotes the occurrence and development of CRC.

Objective To profile ubiquitination in cysteinyl aspartate-specific proteinase-2(CASP2)deficient cells under heat shock and investigate the role of CASP2 in stress response.Methods Ubiquitination levels in subcellular fractions of control and C ASP2 knockout(KO)cells were detected via Western blotting.After 2 hours of heat shock treatment,Soluble Ⅱ and Pellet fractions were collected from both control and CASP2 KO cells for ubiquitinome analysis.Anti-di-glycine remnant(K-ε-GG)antibody-based proteomic analysis was performed to identify differentially ubiquitinated proteins and associated key signaling pathways.Proteins that displayed significantly upregulated ubiquitination in CASP2 KO cells under heat shock were subjected to His-tag pull-down assays to find out whether CASP2 regulated the ubiquitination of these proteins.Results Under heat shock,CASP2 KO cells displayed significantly higher accumulation of overloaded ubiquitinated conjugates in the Pellet fraction compared to controls.Ubiquitinomics analysis revealed substantial alterations in protein ubiquitination patterns following CASP2 KO.One hundred proteins exhibited significantly elevated ubiquitination levels while 36 proteins had their ubiquitination reduced relative to controls.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis indicated that hyper-ubiquitinated proteins were primarily associated with Huntington disease,Alzheimer disease,bile secretion,carbon metabolism and autophagy.His-tag pull-down assays combined with Western blotting revealed increased ubiquitination of nicotinamide adenine dinucleotide reduced-ubiquinone oxidoreductase 1 beta subcomplex subunit 3(NDUFB3)and autophagy-related protein 9A(ATG9A)in CASP2 KO cells under heat shock.Conclusion Overloaded ubiquitinated conjugates are accumulated due to CASP2 deficiency during heat shock.CASP2 modulates ubiquitination levels through multiple signaling pathways.

Objective To investigate how deubiquitinase OTU domain-containing protein 3(OTUD3)suppresses the progression of hepatocellular carcinoma via gut-liver axis metabolic remodeling and microbiome dynamics.Methods A total of 24 male 2-week-old littermate C57BL/6J mice(12 wild-type and 12 Otud3-/-)were divided into two differential genotype groups before 6 mice from each group were randomly chosen to receive intraperitoneal injections of N-nitrosodieth-ylamine(DEN)for hepatocellular carcinoma(HCC)induction.The mice were divided into four groups(n=6/group):Otud3+/+control(WT CON),Otud3-/-control(KO CON),Otud3+/+DEN-induced HCC(WT DEN),and Otud3-/-DEN-induced HCC(KO DEN).At 40 weeks of age,liver tissues were collected for metabolomic profiling,and fecal samples were obtained for 16S rRNA sequencing.Results Multivariate analyses,including principal component analysis(PCA),partial least squares-discriminant analysis(PLS-DA),sparse partial least squares-discriminant analysis(sPLS-DA),and orthogonal partial least squares-discriminant analysis(OrthoPLS-DA),demonstrated complete intergroup separability.Fifty-four differential metabolites were identified between the WT DEN and KO DEN groups through metabolomic profiling,with gut-liver axis-associated pathways such as cholesterol metabolism and fatty acid biosynthesis revealed by KEGG pathway analysis.Microbiome analysis indicated an upregulation of Bacteroides at the genus level in the KO DEN group compared to WT DEN.Pearson correlation analysis highlighted amino acids and derivatives as predominant metabolite classes and revealed Bacteroidetes and Firmicutesas the dominant gut microbial phyla.Conclusion OTUD3 suppresses HCC progression by modulating gut-liver axis metabolism,potentially mediated by elevated betaine and increased abundance of Odoribacter,Alistipes,and Lachnoclostridium.

Objective To construct a mouse model with conditional knockout of deubiquitinase Otulin gene in the endocardium and to analyze the phenotype ofthe model.Methods A model ofmice with endocardium-specific conditional Otulin gene knockout was constructed with the Cre-loxP technique.The genotypes of Otulin gene knockout mice were determined by using polymerase chain reaction(PCR).Data on the reproduction of the knockout mice was statistically analyzed and the growth and development of the offspring were observed.The Cardiac function of mice was monitored by ultrasound and the changes of cellular components in blood were measured by a fully automatic hematology analyzer.Hematoxylin-eosin(HE)staining was used to analyze the pathological changes in all the tissues and organs ofthe offspring.Results An endocardium-specific Otulin gene knockout mouse model was established.The conditional knockout mice could grow and reproduce normally and the percentage of genotypes of their offspring conformed to Mende's law.Compared with littermate control mice,the conditional knockout mice had smaller body size,lighter weight,significantly enlarged spleens and spiking inflammatory cells in blood.In addition,the tricuspid valve defect and cardiac function were attenuated.Conclusion A mouse model of endocardium-specific Otulin gene knockout has been constructed.These mice show impaired cardiac function and obvious inflammatory response.

Objective To explore the characteristics and mechanism of phase separation between TAR DNA binding protein-43(TDP-43)and ubiquitin.Methods The TARDBP gene and its truncated genes were inserted into vectors to construct recombinant plasmids for expression and protein purification.The phase separation system of ubiquitin and TDP-43 was constructed in vitro.The characteristics of the droplets formed via liquid-liquid phase separation were observed by fluorescence microscopy.The plasmids of ubiquitin and TDP-43 were co-transfected into HEK293T cells to observe aggregates containing TDP-43 and ubiquitin and find out whether TDP-43 could be ubiquitinated.Results The GFP-8Ub,TDP-43 full-length(FL)and truncated proteins were purified.TDP-43 FL and C-terminal domain(CTD)proteins were able to form droplets via phase separation with ubiquitin.The droplets changed into solid-like aggregates after prolonged incubation.Insolvable aggregates containing TDP-43 and ubiquitin were formed.TDP-43 was ubiquitinated under stress conditions in HEK293T cells after being co-transfected with ubiquitin and TDP-43 recombinant plasmids.Conclusion TDP-43 undergoes co-phase separation with ubiquitin,mainly driven by the multivalent interaction between TDP-43′s CTD structural domain and ubiquitin.The droplets finally form aggregates with solid-like properties.Under stress conditions,especially when the protein homeostasis is disrupted,TDP-43 and ubiquitin form aggregates while TDP-43 is ubiquitinated.This study reveals the basic mechanism of TDP-43 co-phase separation with ubiquitin and liquid-solid transformation.

Objective This study aimed to identify PAX9 variants in non-syndromic tooth agenesis families of Chi-na,as well as to analyze the genotype-phenotype of non-syndromic tooth agenesis caused by PAX9 variants,which can provide a basis for the genetic diagnosis of tooth agenesis.Methods We collected the data of 44 patients with non-syn-dromic oligodontia who underwent treatment at Stomatological Hospital of Hebei Medical University between 2018 and 2023.Whole-exome sequencing was performed on the peripheral blood of the proband and its core family members,and the variants were verified by Sanger sequencing.Pathogenicity analysis and function prediction of the variants were per-formed using bioinformatics tools.The correlation between the genotype of PAX9 variant and its corresponding pheno-type was examined by reviewing 55 publications retrieved from PubMed.The studies involved 232 tooth agenesis pa-tients with PAX9 variants.Results A novel PAX9 c.447delG(p.Pro150Argfs*62)and a reported PAX9 c.406C>T(p.Gln136*)were identified in two Chinese families.Through bioinformatics analysis and three-dimensional structural mod-eling,we postulated that the frameshift variant was pathogenic.The outcome was the premature cessation of PAX9 pro-tein,which caused severe structural and functional deficiencies.Summarizing the PAX9 genotype-phenotype relationship revealed that patients carrying the PAX9 variant commonly led to loss of the second molars.Conclusion We identified the novel PAX9 c.447delG(p.Pro150Argfs*62)in a Chinese family of non-syndromic oligodontia,expanding the known variant spectrum of PAX9.The most susceptible tooth position for PAX9 variants of tooth agenesis was the second mo-lars and the deciduous molars during the deciduous dentition.

Objective To investigate the association between serum macrophage polarization-related factors and liver fibrosis in patients with alveolar echinococcosis (AE). Methods A total of 120 patients with AE who attended Department of Hepatobiliary and Pancreatic Surgery in The Affiliated Hospital of Qinghai University from September 2018 to October 2020 were enrolled as AE group, and 33 healthy controls were enrolled as normal control group. The two groups and the patients with varying degrees of liver fibrosis were compared in terms of the levels of interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and transforming growth factor-β1 (TGF-β1). Comparison of normally distributed continuous data between two groups was made by the independent samples t -test, while comparison of non-normally distributed continuous data was made by the Mann-Whitney U test or Kruskal-Wallis H test. Comparison of categorical data between groups was made by the chi-square test. Univariate and multivariate logistic regression analyses were used to investigate the association between serum macrophage polarization-related factors and liver fibrosis in patients with AE, and the receiver operating characteristic (ROC) curve was used to analyze the value of serological examination in the diagnosis of liver fibrosis in patients with AE. A Spearman correlation analysis was used to analyze the correlation of each index with HAI score and Metavir score. Results Compared with the normal control group, the AE group had significant increases in the serum levels of IL-6 [13.97 (9.64-23.62) pg/mL vs 1.30 (0.35-2.71) pg/mL, Z =-5.980, P < 0.001], TNF-α [2.26 (1.65-4.13) pg/mL vs 1.40 (1.04-2.10) pg/mL, Z =-3.114, P < 0.01], and TGF-β1 [3.64(2.71-5.72) pg/mL vs 2.91(2.20-3.35) pg/mL, Z =-2.594, P < 0.05], and increases in the serum levels of IL-6 (hazard ratio [ HR ]=2.721, 95% confidence interval [ CI ]: 1.730-4.280, P < 0.05) and TNF-α( HR =3.527, 95% CI : 1.158-10.747, P < 0.05) were independent risk factors for the onset of liver fibrosis in AE patients. The ROC curve analysis showed that hydatid IgG combined with the serum levels of IL-6 and TNF-α had a sensitivity of 88.4%, a specificity of 95.8%, and an area under the ROC curve of 0.951(95% CI : 0.937-0.964) in the diagnosis of liver fibrosis, which were significantly higher than those of IL-6, TNF-α, or hydatid IgG alone ( Z =-3.458, -4.011, and 2.379, all P < 0.05). The Spearman analysis showed that the serum levels of IL-6, TNF-α, and TGF-β1 were positively correlated with HAI score ( r =0.560, 0.644, and 0.465, all P < 0.001) and Metavir fibrosis score ( r =0.530, 0.758, and 0.567, all P < 0.001), and the serum level of IL-10 was negatively correlated with HAI score ( r =-0.232, P =0.011) and Metavir fibrosis score ( r =-0.288, P =0.001). Conclusion Macrophage polarization is often observed in patients with hepatic AE, and the levels of the macrophage polarization-related factors IL-6, TNF-α, and TGF-β1 are associated with the development and progression of liver fibrosis, which can provide certain reference information for predicting the onset of liver fibrosis.

Objective:To analyze the risk factors for bile leakage in patients undergoing cystic echinococcosis surgery to establish a nomogram model to predict the risk of bile leakage.Methods:The clinical data were retrospectively analyzed that 203 patients with cystic echinococcosis treated in Qinghai University Affiliated Hospital from January 2015 to October 2018. Logistic regression was used to screen out independent risk factors of biliary leakage in patients undergoing cystic hydatid surgery. Then the R software was used to establish a predictive nomogram model. Bootstrap method was used to validate the nomogram model and ROC curve was used to explore the predictive efficacy of the model in predicting the bile leakage.Results:There were 54 cases of biliary leakage in 203 patients with cystic echinococcosis, the incidence was 26.1%. ALP ( OR=11.193, 95% CI 5.066-24.731), GGT ( OR=2.728, 95% CI 1.246-5.975), cyst diameter ( OR=3.491, 95% CI 1.550-7.861), hilar cyst ( OR=2.503, 95% CI 1.176-5.329) were all independent risk factors for biliary leakage of cystic echinococcosis. The consistency index was 0.835 for predicting PPC risk, and the area was 0.823 (95% CI 0.754-0.892) under the curve for predicting the risk of cystic echinococcal bile leakage. Conclusions:ALP, GGT, cyst diameter and hepatic hilar cyst are independent risk factors for cystic echinococcal bile leakage. Nomogram model can evaluate the risk of cystic echinococcal bile leakage more intuitively, with potentially high clinical application value.

Objective:To investigate the effect of 125I particles in alveolar echinococcosis with the animal model (nude mice and Sprague Dawley rats). Methods:Twenty 10 weeks nude mice with body weight ranged from 20 to 24 g were divided into three groups. Sixteen nude mice were divided into experimental group ( n=8), puncture group ( n=4) and model group ( n=4). There was no intervention in the model group and only particle puncture needle was used in the puncture group. 125I particles were implanted in the experimental group. 14 male Sprague Dawley rats without specific pathogen, with body weight 280-320 g, 12 weeks old, were used to construct the model of hepatic alveolar echinococcosis. Then the rats were divided into intervention group ( n=10) and control group ( n=4). In the intervention group, 125I particles were pushed into the lesions. The abdomen was only open and closed in the control group. All the mice were sacrificed 45 days after intervention. The tumor size was measured. The activity of protoscolex and pathological changes of Echinococcus multilocularis in each group were observed. Results:At the timepoint of 22nd, 30th and 40th day of intervention, the largest diameter of tumor in nude mice experimental group was (10.7±5.2) mm, (10.9±5.0) mm, (8.5±4.3) mm, smaller than that in puncture group (24.5±4.4) mm, (25.4±4.1) mm, (31.4±2.8) mm and model group (22.5±7.3) mm, (25.0±5.4) mm, (26.7±6.3) mm, with statistically significant difference ( P<0.05). The number and activity of protoscoleces in experimental group were lower than those in puncture group and model group. Under the light microscope, the structure of echinococcus vesiculae and its body in the experimental group was obviously destroyed, and the cuticle and germinal layer of echinococcus vesiculae in the puncture group and the model group were normal, with multiple intact protoscoleces. The pathological changes of Sprague Dawley rats in the intervention group and the control group were basically the same as those in the nude mice model. Conclusion:The 125I particle radiation effect can kill Echinococcus multilocularis protoscoleces and inhibit the growth of alveolar echinococcosis.