Hepatitis B virus (HBV) infection can cause acute or chronic liver injury. Chronic hepatitis B (CHB)-related liver cirrhosis and hepatocellular carcinoma usually lead to an increase in mortality, and their pathogenesis may be associated with immune response. Intestinal flora plays an important role in host metabolism and immune regulation, and the studies on “gut-liver axis” and intestinal flora have shown that the structural change of intestinal flora, bacterial translocation, and related immunologic injury may affect the development and progression of liver inflammation on the basis of CHB. This article summarizes and discusses the immunological role of intestinal flora in CHB and explores the potential treatment methods for HBV infection based on intestinal flora.

ObjectiveTo investigate the expression of TM6SF2 in hepatocellular carcinoma (HCC) tissue and its biological functions by data mining in tumor databases. MethodsThe GEPIA database was applied to measure the change in the mRNA expression level of TM6SF2 in HCC tissue, and OncoLnc was used to analyze the association of TM6SF2 expression with the survival time of HCC patients. The cBioPortal and LinkedOmics databases were used to analyze the genes associated with the expression of TM6SF2 in HCC tissue, and the DAVID6.8 and STRING databases were used to perform a bioinformatics analysis of TM6SF2 and the genes associated with its expression. The t-test was used to investigate the difference in the mRNA expression of TM6SF2 between HCC tissue and adjacent tissue. The Spearman correlation coefficient was used to analyze the correlation of gene expression. The Kaplan-Meier method was used to calculate survival percentage, and the log-rank test was used to analyze the difference in survival percentage. ResultsCompared with the normal liver tissue, the HCC tissue had low mRNA expression of TM6SF2 (｜log2FC｜cut-off = 0.5, P＜0.01). Compared with those with high expression of TM6SF2, the patients with low expression had a significant reduction in overall survival time (χ2=9.897，P＜0.01). Data analysis showed that a total of 49 genes were associated with the expression of TM6SF2 in HCC tissue, and the gene ontology analysis showed that these genes were enriched in the biological processes and functions including fatty acid synthesis, fatty acid ligase activation, and thrombin regulation (P＜0.05). The Kyoto Encyclopedia of Genes and Genome pathway analysis showed that these genes were mainly involved in the signaling pathways of alanine metabolism, peroxisome proliferator-activated receptor signaling pathway, and bile secretion (P＜0.05). The protein-protein interaction network analysis showed that the genes of SERPINC1, NR1I2, SERPINA10, and SLC10A1 had marked or potential interaction with TM6SF2 (P＜0.01). ConclusionTumor data mining can quickly obtain the information on the expression of TM6SF2 in HCC tissue and provide a bioinformatics basis for exploring the role of TM6SF2 in the development and progression of HCC.

ObjectiveTo investigate the association of KCNJ11 rs5210 single nucleotide polymorphism with nonalcoholic fatty liver disease (NAFLD) and coronary artery disease (CAD) in the Chinese Han population in Qingdao, China. MethodsA total of 246 patients with NAFLD who attended Qingdao Municipal Hospital from December 2018 to September 2019 were enrolled as NAFLD group, 201 patients with CAD were enrolled as CAD group, and 116 patients with NAFLD and CAD were enrolled as NAFLD+CAD group; 342 healthy individuals were enrolled as control group. Fasting venous blood samples were collected for biochemical analysis. Whole blood genomic DNA was extracted, and PCR was used to determine KCNJ11 rs5210 genotype. The chi-square test was used to analyze whether the distribution of KCNJ11 rs5210 gene frequencies met the Hardy-Weinberg equilibrium, in order to determine whether the tested samples could represent the population. The chi-square test was used to analyze the differences in sex and genotype/allele frequency between groups. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups; the Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups, and the Bonferroni method was used for further comparison between two groups. The unconditional logistic regression model was used to calculate odds ratio (OR) and 95% confidence interval. ResultsThree genotypes (AA, GA, and GG) of KCNJ11 rs5210 were found by gene sequencing. There were no significant differences in rs5210 allele frequency and genotype distribution between the control group, the NAFLD group, the CAD group, and the NAFLD+CAD group (all P＞0.05), and there were still no significant differences after adjustment for sex, age, and body mass index (BMI) (all P＞0.05). For all subjects, the subjects with AA genotype had a higher level of alkaline phosphatase than those with GA genotype (P=0.048); in the NAFLD group, the patients with GA genotype had significantly higher BMI and total bilirubin than those with AA genotype (P=0.042 and 0.002). The unconditional logistic regression analysis showed that elevated BMI was associated with the risk of NAFLD (OR=1.35, P＜0.01), while decreased high-density lipoprotein (HDL) might indicate an increase in the risk of NAFLD (OR=0.33, P＜0.01); elevated fasting plasma glucose and decreased HDL might indicate an increase in the risk of CAD (OR=1.51 and 0.11, both P＜0.01) and NAFLD with CAD (OR=1.46 and 0.06, both P＜0.01). ConclusionThere is no significant association between KCNJ11 rs5210 polymorphism and the risk of NAFLD and CAD in the Chinese Han population in Qingdao.

ObjectiveTo investigate the association of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) rs8192678 single nucleotide polymorphism (SNP) with the risk of nonalcoholic fatty liver disease (NAFLD) and the influence of PPARGC1A rs8192678 SNP on NAFLD-related biochemical parameters. MethodsA total of 119 NAFLD patients who attended Qingdao Municipal Hospital Affiliated to Qingdao University from December 2017 to December 2018 were enrolled as NAFLD group, and 213 individuals who underwent physical examination during the same period of time were enrolled as control group. Clinical data and blood samples were collected from all subjects to measure related biochemical parameters and detect PPARGC1A rs8192678 SNP. The chi-square test was used to determine whether the genotype distribution of samples was in accordance with the Hardy-Weinberg equilibrium. The t-test or the Wilcoxon rank-sum 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. A binary logistic regression analysis was used to investigate the risk factors for NAFLD. ResultsThere were no significant differences in the genotype and allele frequencies of PPARGC1A rs8192678 between the NAFLD group and the control group (χ2=0.011 and 0.015, P=0.918 and 0.904). The binary logistic regression analysis showed that CT genotype of PPARGC1A rs8192678 was not a risk factor for NAFLD (odds ratio=0.951, 95% confidence interval: 0.368-2.457, P=0.918). In the NAFLD group, the patients carrying CT genotype had a significantly higher level of gamma-glutamyl transpeptidase (GGT) than those carrying CC genotype (Z=-2.331, P=0.020). ConclusionPPARGC1A rs8192678 SNP does not increase the risk of NAFLD, while NAFLD patients carrying CT genotype tend to have a higher serum level of GGT.

Objective To investigate assess the bidirectional causal relationship between ulcerative colitis(UC)and hypothyroidism using a two-sample Mendelian randomization(TSMR).Methods Single nucleotide polymorphism(SNP)data relevant to UC and hypothyroidism were retrieved from the Finnish Biobank and the IEU database,respectively.Independent SNPs strongly associated with UC were selected as instrumental variables.Causal associations between UC and hypothyroidism were evaluated using the inverse variance weighted(IVW)method,MR-Egger regression,and weighted median estimator.Additionally,MR-PRESSO was employed to assess the hori-zontal pleiotropy and outlier SNPs.Cochran's Q test and funnel plots were performed to evaluate the heterogeneity among the SNPs.A leave-one-out analysis was conducted to examine the influence of individual SNPs on causal assessments.Results Four instrumental variables strongly associated with UC were identified.The IVW method indicated a causal relationship between UC and hypothyroidism(OR = 0.975,95%CI:0.924～0.990,P = 0.011).Cochran's Q test yielded a Q statistic of 2.566 with a p-value of 0.463,suggesting no heterogeneity among the SNPs.Both MR-Egger(P = 0.523)and MR-PRESSO(P = 0.548)tests suggested the absence of horizontal pleiotropy.However,the results of the reverse TSMR did not support a reverse causal relationship.Conclusion The findings from the TSMR analysis reveal a negative causal relationship between UC and hypothyroidism.

After sequencing, we amplified and cloned foot-and-mouth disease virus (FMDV) O/QYYS/s/06 whole genome by three fragments. These three fragments were cloned into vector P43 one by one to construct recombinant plasmid P43C, which carried the full-length cDNA of FMDV O/QYYS/s/06. Then, plasmid P43C and plasmid T7 expressing T7 RNA polymerase were co-transfected into BHK-21 cells. After 48 h, we harvested the culture broth from transfected BHK-21 cells and inoculated into 2-3 day-old sucking mice. After four generation passage, the virus harvested from sucking mice was confirmed to be type O FMDV by the indirect hemagglutination test, sucking mice's neutralization test and sequencing. The results showed that we have successfully constructed the full-length cDNA clone of FMDV O/QYYS/s/06 strain.
Animals ; Animals, Newborn ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Viral ; biosynthesis ; genetics ; Foot-and-Mouth Disease ; virology ; Foot-and-Mouth Disease Virus ; classification ; genetics ; pathogenicity ; Mice ; Transcription, Genetic ; Transfection

OBJECTIVETo investigate the degradation process of collagens and identify the key unit operation during manufacturing process of E'jiao.

METHODSamples in different unit operations were withdrawn, and their amino acid compositions and the molecular weight ranges were determined. The peptide composition was analyzed by high-performance liquid chromatography/mass spectrometry.

RESULTThe content of sample during atmospheric condensation unit increased by 16.8% compared to the thermal extraction unit. Gel filtration chromatographic analysis indicated that the degradation process of collagen primarily occurred during the atmospheric condensation unit. The peptides in samples mainly resulted from the degradation of collagens and cytoskeleton proteins such as tubulin, actinin, and so on. The relative abundance of degraded collagens increased with the decrease of no-collagen proteins.

CONCLUSIONCollagen degradation mainly occurred during the atmospheric condensation unit, which was the key process affecting the composition of E-jiao.

Chromatography, Gel ; Chromatography, High Pressure Liquid ; methods ; Collagen ; analysis ; metabolism ; Mass Spectrometry ; methods ; Molecular Weight ; Peptides ; analysis

Objective To investigate whether there was a correlation between serum liver enzyme levels and blood pressure in the Chinese Han population with nonalcoholic fatty liver disease (NAFLD) in Shandong coastal regions in China. Methods A total of 269 NAFLD patients who lived in Shandong coastal regions and attended or underwent physical examination in Qingdao Municipal Hospital from December 2019 to June 2020 were enrolled, among whom 105 had hypertension and 164 did not have hypertension. Morning blood pressure was measured to calculate mean arterial pressure (MAP), and laboratory tests were performed to measure the serum levels of liver enzymes [alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), and alkaline phosphatase (ALP)] and fasting blood glucose (FBG). The independent samples t -test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U 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. A Pearson correlation analysis was used to investigate the correlation of four liver enzymes with the indices including MAP, and a binary logistic regression model was used to analyze the impact of serum liver enzymes on hypertension. Results Compared with the non-hypertension group, the hypertension group had significantly higher body mass index (BMI), MAP, and GGT (all P < 0.05). For all NAFLD patients and the NAFLD patients without hypertension, male patients had significantly higher BMI, MAP, ALT, AST, and GGT than female patients (all P < 0.05), and for the NAFLD patients with hypertension, male patients had a significantly higher level of GGT than female patients ( P < 0.05). There was a significant difference in the distribution of GGT between the hypertension group and the non-hypertension group, and compared with the non-hypertension group, the hypertension group had a significantly higher proportion of patients with GGT exceeding the normal range ( χ 2 =4.781, P =0.029). Serum GGT level was correlated with MAP within the normal range (70-105 mm Hg) ( r =0.178, P =0.011), while there was no significant correlation when MAP exceeded the normal range ( P =0.415). After adjustment for age and sex, the binary logistic regression model showed that AST level was positively associated with hypertension in the population with NAFLD (odds ratio [ OR ]=1.011, 95% confidence interval [ CI ]: 1.000-1.022, P =0.040), and after further adjustment for BMI and FBG, the results showed that AST level was still positively associated with hypertension ( OR =1.011, 95% CI : 1.000-1.022, P =0.044). Conclusion In Chinese Han population with NAFLD in Shandong coastal regions, higher levels of AST may predict an increased risk of hypertension.

Objective To investigate the independent predictive factors for functional cure after long-term nucleos(t)ide analogue (NUC) antiviral therapy followed by pegylated interferon α-2b therapy in chronic hepatitis B (CHB) patients. Methods A total of 162 CHB patients who were admitted to several hospitals in Qingdao, China, from 2018 to 2021 were enrolled as subjects, and all patients received pegylated interferon α-2b for at least 48 weeks after NUC therapy for one year or longer. According to whether HBsAg clearance was achieved at week 48 of pegylated interferon α-2b treatment, the patients were divided into functional cure group with 79 patients and non-cure group with 83 patients, and related clinical indices were compared between the two groups. The two-independent-samples t test and the Mann-Whitney U rank sum test were used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between two groups. The Spearman correlation analysis was performed, and the univariate and multivariate logistic regression analyses were used to investigate the independent predictive factors for functional cure. The receiver operating characteristic (ROC) curve was plotted for related variables, and the area under the ROC curve (AUC) was used to evaluate the prediction accuracy of the variables. Results Compared with the non-cure group, the functional cure group had a significantly lower HBsAg level at baseline [21.63 (3.33-157.60) IU/mL vs 794.70 (336.10-1 185.34) IU/mL, Z =-8.869, P < 0.001], at week 12 of pegylated interferon α-2b treatment [1.34 (0.04-16.59) IU/mL vs 567.11 (226.09-1 047.86) IU/mL, Z =-9.847, P < 0.001), and at week 24 of pegylated interferon α-2b treatment [0.01 (0.00-0.34) IU/mL vs 304.79 (89.24-772.23) IU/mL, Z =-10.474, P < 0.001) and a significantly greater reduction in HBsAg at weeks 12 and 24 of pegylated interferon α-2b treatment [week 12: 89.6% (57.5%-99.4%) vs 21.8% (2.0%-40.9%), Z =-7.926, P < 0.001; week 24: 99.9% (99.0%-100.0%) vs 44.1% (20.6%-73.8%), Z =-9.593, P < 0.001]. Compared with the non-cure group, the functional cure group had a significantly lower HBeAg positive rate at baseline (8.9% vs 25.3%, χ 2 =7.652, P =0.006), a significantly lower proportion of patients with baseline HBV DNA > 1000 IU/mL (0 vs 8.4%, χ 2 =5.073, P =0.024), a significantly lower level of total bilirubin at baseline [12.60 (10.12-15.93) μmol/L vs 15.50 (11.80-24.10) μmol/L, Z =-3.611, P < 0.001], a significantly higher level of aspartate aminotransferase (AST) at week 12 of treatment [47.00 (34.00-68.00) U/L vs 41.00 (30.00-56.50) U/L, Z =-2.031, P =0.042], and a significantly higher proportion of patients with AST > 2×upper limit of normal (16.5% vs 4.8%, χ 2 =5.835, P =0.016). The multivariate logistic regression analysis showed that baseline HBsAg (odds ratio [ OR ]=0.996, 95% confidence interval [ CI ]: 0.995-0.997, P < 0.001), HBsAg at week 12 of pegylated interferon α-2b treatment ( OR =0.990, 95% CI : 0.986-0.994, P < 0.001), HBsAg at week 24 of pegylated interferon α-2b treatment ( OR =0.983, 95% CI : 0.975-0.991, P < 0.001), and baseline total bilirubin ( OR =0.885, 95% CI : 0.826-0.949, P =0.001) were independent predictive factors for functional cure. The ROC curve of baseline HBsAg showed an AUC of 0.904 and the optimal cut-off value of 118.24 IU/mL; the ROC curve of HBsAg at week 12 of pegylated interferon α-2b treatment showed an AUC of 0.948 and the optimal cut-off value of 73.74 IU/mL; the ROC curve of HBsAg at week 24 of pegylated interferon α-2b treatment showed an AUC of 0.975 and the optimal cut-off value of 11.01 IU/mL; the ROC curve of baseline total bilirubin showed an AUC of 0.664 and the optimal cut-off value of 19.9 μmol/L. Conclusion Baseline HBsAg, HBsAg at week 12 of pegylated interferon α-2b treatment, HBsAg at week 24 of pegylated interferon α-2b, and baseline total bilirubin are independent predictive factors for functional cure at week 48 of pegylated interferon α-2b treatment in CHB patients receiving sequential therapy with NUC and pegylated interferon α-2b.