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 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 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.

Liquid biopsy is a new noninvasive detection method and is also the main molecular detection method to guide the application of precision medicine. Liquid biopsy mainly includes the measurement of circulating free DNA (cf-DNA), circulating tumor cells, and exosomes, and in particular, cf-DNA is becoming a valuable molecular detection tool, especially in the early detection and diagnosis of liver cancer and parasitic infections involving the liver. This article reviews the clinical application of cf-DNA in liver cancer and liver-related parasitic diseases.

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.

Objective:To investigate the effect of hepatitis B virus (HBV) infection in patients with hepatic alveolar echinococcoisis after surgical resection and determine the differences of liver function between patients with different HBV-DNA levels.Methods:Patients were selected from January 2014 to July 2018 in the Affiliated Hospital of Qinghai University. Twenty-eight patients with hepatitis B and hepatic alveolar echinococcoisis were included in the experimental group, and 20 patients with hepatic alveolar echinococcoisis but without hepatitis B virus were included in the control group. Based on HBV-DNA level, the experimental group was divided into low-level group (HBV-DNA level<200 IU/ml, n=6), intermediate-level group (HBV-DNA level 200-20 000 IU/ml, n=15) and high level group (HBV-DNA level>20 000 IU/ml, n=7). Comparison of complications and liver function after liver resection in two groups.Univariate and multivariate logistic regression were used to analyze the influential factors of postoperative complications in patients.Comparison of postoperative liver function indexes in patients with different HBV-DNA levels. Results:In the control group, postoperative total bilirubin 10.6(8.3, 16.9) μmol/L, direct bilirubin 5.3(3.4, 10.0) μmol/L, prothrombin time 13.6(13.0, 15.8)s, and the incidence of complications 25.0%(5/20), were better than the experimental group 12.6(8.4, 46.9) μmol/L, 6.7(3.1, 26.4) μmol/L, 15.4(13.5, 18.1)s, 78.6% (22/28), the differences were statistically significant significance (all P<0.05). Multivariate logistic analysis showed that patients with HBV infection ( OR=4.593, 95% CI: 1.128-18.708) and intraoperative blood loss ≥1 000 ml ( OR=2.200, 95% CI: 1.106-4.378) were the hepatic alveolar echinococcoisis independent risk factors for postoperative complications. There were no significant differences in total bilirubin and albumin between the three groups of patients with different HBV-DNA levels ( P>0.05). Conclusion:Patients with HBV and hepatic alveolar echinococcoisis have worse liver function and are more prone to complications after surgical resection, but there is no significant difference in liver function among patients with different HBV-DNA levels.

Objective To preliminarily study the use of indocyanine green in hepatic alveolar echinococcosis surgery.Methods The data of 13 patients with hepatic alveolar ecbinococcosis treated at the Affiliated Hospital of Qinghai University from May 2017 to May 2018 with laparotomy and intraoperative indocyanine green injection were retrospectively studied.Images were collected by the fluorescence acquisition system.Results There were 7 females and 6 males,with an average age of (37.9±14.7) years.The mean weight was (57.4±11.3) kg.Except for one patient of Han nationality,the rest were Tibetans.The lesions were not visualized but normal liver tissues emitted green fluorescence.One patient had a slightly enhanced fluorescence circle around the lesion.The fluorescence intensity of some areas were between normal liver tissues and the lesion,which were suspected to be the marginal zone (which needed to be confirmed by pathology).Three patients (23.1％,3/13) had small lesions on the liver surface,2 were not found by imaging examination,and 1 showed strip calcification on CT.No residual lesions in the liver (except for the microwave ablated lesions) were found after resection.No postoperative complications occurred in all the patients.Seven patients were followed up and no recurrence was found.Conclusions Fluorescence imaging of indocyanine green has the advantages of detecting small and residual lesions in surgery carried out for hepatic alveolar echinococcosis.More studies are needed to confirm the findings.