Objective To systematically summarize the published literature on the genetic variants associated with nonalcoholic fatty liver disease(NAFLD). Methods Literature from Web of Science,PubMed,and Embase between January 1980 and September 2022 was systematically searched.Meta-analyses of the genetic variants were conducted using at least five data sources.The epidemiologic credibility of the significant associations was graded using the Venice criteria. Results Based on literature screening,399 eligible studies were included,comprising 381 candidate gene association,16 genome-wide association,and 2 whole-exome sequencing studies.We identified 465 genetic variants in 173 genes in candidate gene association studies,and 25 genetic variants in 17 genes were included in the meta-analysis.The meta-analysis identified 11 variants in 10 genes that were significantly associated with NAFLD,with cumulative epidemiological evidence of an association graded as strong for two variants in two genes(HFE,TNF),moderate for four variants in three genes(TM6SF2,GCKR,and ADIPOQ),and weak for five variants in five genes(MBOAT7,PEMT,PNPLA3,LEPR,and MTHFR). Conclusion This study identified six variants in five genes that had moderate to strong evidence of an association with NAFLD,which may help understand the genetic architecture of NAFLD risk.

Objective:Non-alcoholic fatty liver disease(NAFLD)is a common metabolic disorder in overweight and obese children,and its etiology and pathogenesis remain unclear,lacking effective preventive and therapeutic measures.This study aims to explore the association between whole blood copper,zinc,calcium,magnesium and iron levels and NAFLD in overweight and obese children aged 6 to 17 years,providing a scientific basis for the prevention and intervention of early NAFLD in overweight and obese children. Methods:A cross-sectional study design was used to collect relevant data from overweight and obese children who visited the Hunan Children's Hospital from January 2019 to December 2021 through questionnaire surveys.Fasting blood samples were collected from the subjects,and various indicators such as blood glucose,blood lipid,and mineral elements were detected.All children were divided into an overweight group(n=400)and a NAFLD group(n=202).The NAFLD group was divided into 2 subgroups according to the ALT level:A non-alcoholic fatty liver(NAFL)group and a non-alcoholic steatohepatitis(NASH)group.Logistic regression analysis was used to analyze the association between minerals(copper,zinc,calcium,magnesium,and iron)and NAFLD,NAFL and NASH. Results:A total of 602 subjects were included,of whom 73.6%were male,with a median age of 10(9,11)years,and a body mass index(BMI)of 24.9(22.7,27.4)kg/m2.The intergroup comparison results showed that compared with the overweight group,the NAFLD group had higher levels of age,BMI,diastolic blood pressure(DBP),systolic blood pressure(SBP),triglyceride(TG),low density lipoprotein(LDL),alanine transaminase(ALT)and aspartate aminotransferase(AST),and lower level of high density lipoprotein(HDL).The NAFL group had higher levels of age,BMI,DBP,SBP,ALT,and AST,and lower levels of HDL compared with the overweight group.The levels of age,BMI,DBP,SBP,TG,LDL,ALT,and AST of NASH were higher than those in the overweight group,while the level of HDL was lower than that in overweight group(all P<0.017).After adjusting for a variety of confounders,the OR of NAFLD for the highest quantile of iron was 1.79(95%CI 1.07 to 3.00)compared to the lowest quantile,and no significant association was observed between copper,zinc,calcium,and magnesium,and NAFLD.The subgroup analysis of NAFLD showed that the OR for the highest quantile of iron in children with NAFL was 2.21(95%CI 1.26 to 3.88),while no significant association was observed between iron level and NASH.In addition,no significant associations were observed between copper,zinc,calcium,and magnesium levels and NAFL or NASH. Conclusion:High iron level increases the risk of NAFLD(more likely NAFL)in overweight and obese children,while copper,zinc,calcium,magnesium,and other elements are not associated with the risk of NAFLD in overweight and obese children.

Objective:Non-alcoholic fatty liver disease(NAFLD)has significant genetic susceptibility.Adipocytokines play a crucial role in NAFLD development by participating in insulin resistance and hepatic steatosis.However,the association between adipocytokine pathway genes and NAFLD remains unclear.This study aims to explore the association of gene polymorphisms in the adipocytokine pathway and their interactions with NAFLD in obese children. Methods:A case-control study was conducted,dividing obese children into NAFLD and control groups.Peripheral venous blood(2 mL)was collected from each participant for DNA extraction.A total of 14 single nucleotide polymorphisms(SNP)in the adipocytokine pathway were genotyped using multiplex PCR and high-throughput sequencing.Univariate and multivariate Logistic regression analyses were used to assess the association between SNP and NAFLD in obese children.Dominant models were used to analyze additive and multiplicative interactions via crossover analysis and Logistic regression.Generalized multifactor dimensionality reduction(GMDR)was used to detect gene-gene interactions among the 14 SNPs and their association with NAFLD in obese children. Results:A total of 1 022 children were included,with 511 in the NAFLD group and 511 in the control group.After adjusting for age,gender,and BMI,multivariate Logistic regression showed that PPARG rs1801282 was associated with NAFLD in the obese children in 3 genetic models:heterozygote model(CG vs CC,OR=0.58,95%CI 0.36 to 0.95,P=0.029),dominant model(GG+CG vs CC,OR=0.62,95%CI 0.38 to 1.00,P=0.049),and overdominant model(CC+GG vs CG,OR=1.72,95%CI 1.06 to 2.80,P=0.028).PRKAG2 rs12703159 was associated with NAFLD in 4 genetic models:heterozygous model(CT vs CC,OR=1.51,95%CI 1.10 to 2.07,P=0.011),dominant model(CT+TT vs CC,OR=1.50,95%CI 1.10 to 2.03,P=0.010),overdominant model(CC+TT vs CT,OR=0.67,95%CI 0.49 to 0.92,P=0.012),and additive model(CC vs CT vs TT,OR=1.40,95%CI 1.07 to 1.83,P=0.015).No significant multiplicative or additive interaction between PPARG rs1801282 and PRKAG2 rs12703159 was found in association with NAFLD.GMDR analysis,adjusted for age,gender,and BMI,revealed no statistically significant interactions among the 14 SNPs(all P>0.05). Conclusion:Mutations in PPARG rs1801282 and PRKAG2 rs12703159 are associated with NAFLD in obese children.However,no gene-gene interactions among the SNP are found to be associated with NAFLD in obese children.

Objective:To prepare a recombinant hemagglutinin trimer (HA-Tri) vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods:A stable CHO cell line that could express HA-Tri was constructed. Western blot, single radial immunodiffusion, protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein. According to the different treatment conditions such as dosage and adjuvant, BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures. Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results:The constructed CHO cells could secret and express HA-Tri proteins. The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion. The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected, including high mannose, complex glycoforms and heterozygous glycoforms. After prime-boost immunization, there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75 μg of HA-Tri in combination with RFH01 adjuvant and 15 μg of monovalent vaccine stock solution ( P=0.431 2, U=36). Serum antibody titers in the HA-Tri+ RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant, and the highest titer was induced in the 15 μg HA-Tri+ RFH01 group, which was 1 280. Conclusions:The recombinant HA-Tri protein was successfully prepared. HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice, which would provide reference for the development of influenza virus recombinant subunit vaccines.

Objective:To purify H5N1 influenza virus concentrate prepared by MDCK cells with a new mixed-mode chromatography medium Capto Core700 and the traditional medium Sepharose 4FF, and to compare the separation and purification efficacy of the two media.Methods:Capto Core700 and Sepharose 4FF were used to purify inactivated H5N1 influenza virus concentrate. The morphology of virus particles in different samples was then observed under a transmission electron microscope. Single radial immunodiffusion (SRID), Folin-Phenol (Lowry) method, double-antibody sandwich ELISA and qPCR were used to detect hemagglutinin, total protein, host cell protein (HCP) and host cell DNA (HCD) before and after purification. The recovery rate of virus antigen and the removal rate of impurities were calculated. The immunogenicity of the viruses purified with different media was analyzed using animal experiments. Difference in the purification efficacy of the two chromatography media was analyzed by t-test. Results:H5N1 influenza viruses purified by Capto Core700 or Sepharose 4FF showed the typical influenza virus morphology under transmission electron microscope. There was no significant difference in the recovery rate of hemagglutinin between the two chromatography media ( P>0.05), but compared with Sepharose 4FF, Capto Core700 had a higher removal rate of impurities (total protein, HCP, HCD) and the difference was statistically significant ( P<0.05). Animal experiments showed that the viruses purified by the two chromatography media had good immunogenicity. Conclusions:Compared with Sepharose 4FF chromatography medium, Capto Core700 could more effectively remove process-related impurities such as HCP, HCD and total protein without affecting the recovery rate of viral antigen. This study provided reference for the development of purification technology in the production of H5N1 influenza virus vaccine in MDCK cells.

Objective:To evaluate the immunogenicity of a quadrivalent subunit vaccine combined with RFH01 adjuvant in a mouse model.Methods:Identification tests were performed on four monovalent influenza virus subunit vaccine stock solutions according to the methods described in Part 3 of the Chinese Pharmacopoeia 2020 Edition. In the study of the quadrivalent subunit vaccine combined with RFH01 adjuvant, 460 female BALB/c mice (6-8 weeks old) were randomly divided into 46 groups including experimental groups, vaccine control group, negative control group and blank group with 10 mice in each group. In the study of the quadrivalent subunit vaccine in old and young mice, 80 female 10-month-old and 80 female 10-week-old BALB/c mice were randomly divided into 16 groups ( n=10) including monovalent influenza virus vaccine group, quadrivalent subunit vaccine group, quadrivalent subunit vaccine+ RFH01 adjuvant group, chicken embryo quadrivalent split vaccine control group and PBS group. All mice were immunized by intramuscular injection. At 21 d after the primary immunization, a booster immunization was conducted using the same strategy. Blood samples were collected at 21 d and 42 d after the primary immunization for serum separation. Haemagglutination inhibition (HI) test was performed to detect the antibody levels in mouse serum samples. Results:After the booster immunization, the positive conversion rates in all vaccine+ RFH01 adjuvant groups reached 100%, and the geometric mean titers (GMTs) of serum antibodies were significantly higher than those of the vaccine groups without RFH01 adjuvant. There were significant differences in serum antibody titers between the monovalent/quadrivalent subunit vaccine groups with and without RFH01 adjuvant. After the booster immunization, the titers of serum antibodies against H1N1, H3N2, B/Victoria and B/Yamagata in the 10-week-old mice were significantly higher than those in the 10-month-old mice.Conclusions:The monovalent and quadrivalent influenza virus vaccines in combination with RFH01 adjuvant could elicit higher antibody titers in young (6-10 weeks old) and old (10 months old) mice, showing good immunogenicity.

Influenza is a worldwide infectious disease caused by influenza virus. It has posed great challenges on public health and social stability since 1918. At present, vaccination is the most effective way to prevent and control influenza epidemics. Broad-spectrum antiviral drugs and neutralizing antibodies against influenza virus have been widely studied in recent years. Hemagglutinin (HA), which is on the surface of influenza virus, plays an important role in the stage of viral invasion into host cells. It is the main effective antigenic component of current influenza vaccines, as well as the main target of broad-spectrum neutralizing antibodies and broad-spectrum antiviral drugs. This review summarized the progress in the development of novel influenza vaccines, neutralizing antibodies, and antiviral drugs based on influenza virus HA, as well as other prevention and control measures, hoping to present new ideas for future influenza prevention and control.

Transglutaminase 2 (TGM2) is a ubiquitous multifunctional protein, which is related to the adhesion of different cells and tumor formation. Previous studies found that TGM2 is involved in the interaction between host cells and viruses, but the effect of TGM2 on the proliferation of influenza virus in cells has not been reported. To explore the effect of TGM2 during H1N1 subtype influenza virus infection, a stable MDCK cell line with TGM2 overexpression and a knockout cell line were constructed. The mRNA and protein expression levels of NP and NS1 as well as the virus titer were measured at 48 hours after pot-infection with H1N1 subtype influenza virus. The results showed that overexpression of TGM2 effectively inhibited the expression of NP and NS1 genes of H1N1 subtype influenza virus, while knockout of TGM2 up-regulated the expression of the NP and NS1 genes, and the expression of the NP at protein level was consistent with that at mRNA level. Virus proliferation curve showed that the titer of H1N1 subtype influenza virus decreased significantly upon TGM2 overexpression. On the contrary, the virus titer in TGM2 knockout cells reached the peak at 48 h, which further proved that TGM2 was involved in the inhibition of H1N1 subtype influenza virus proliferation in MDCK cells. By analyzing the expression of genes downstream of influenza virus response signaling pathway, we found that TGM2 may inhibit the proliferation of H1N1 subtype influenza virus by promoting the activation of JAK-STAT molecular pathway and inhibiting RIG-1 signaling pathway. The above findings are of great significance for revealing the mechanism underlying the interactions between host cells and virus and establishing a genetically engineering cell line for high-yield influenza vaccine production of influenza virus.
Animals ; Cell Proliferation ; Dogs ; Humans ; Influenza A Virus, H1N1 Subtype/genetics* ; Influenza, Human ; Madin Darby Canine Kidney Cells ; Protein Glutamine gamma Glutamyltransferase 2

Objective:To express the head domain of influenza A virus hemagglutinin (HA) in a prokaryotic expression system and to evaluate its immunogenicity.Methods:The genes encoding the HA head domains of H1N1 and H3N2 influenza viruses were cloned into pET-22b(+ ) prokaryotic expression plasmid. After the induction with IPTG, the fusion proteins rH1N1-HA and rH3N2-HA containing HA head domain and His-tag were expressed and obtained from E. coli BL21. SDS-PAGE and Western blot was used to verify the expression of the recombinant proteins. Rabbits were immunized with multiple doses of the purified recombinant proteins to obtain polyclonal antibodies against the HA head domains of H1N1 and H3N2. The immunogenicity of the recombinant proteins was evaluated in BALB/c mice. Results:rH1N1-HA and rH3N2-HA induced protective antibodies (geometric mean titer ≥40) in mice and could be used as protective antigens. Polyclonal antibodies against rH1N1-HA and rH3N2-HA could be used as important materials for Western blot, ELISA and other immunological assays.Conclusions:The HA head domains prepared in this study could be used as protective antigens to induce protective antibodies in mice. Polyclonal antibodies against the HA head domains could be used for immunological and serological studies of influenza A viruses.

OBJECTIVES: Cervical cancer is the most common malignant tumor in the female reproductive system worldwide. The recurrence rate for the treated cervical cancer patients is high, which seriously threatens women's lives and health. At present, the risk prediction study of cervical cancer has not been reported. Based on the influencing factors of cervical cancer recurrence, we aim to establish a risk prediction model of cervical cancer recurrence to provide a scientific basis for the prevention and treatment of cervical cancer recurrence. METHODS: A total of 4 358 cervical cancer patients admitted to the Hunan Cancer Hospital from January 1992 to December 2005 were selected as research subjects, and the recurrence of cervical cancer patients after treatment was followed up. Univariate analysis was used to analyze the possible influencing factors. Variables that were significant in univariate analysis or those that were not significant in univariate analysis but may be considered significant were included in multivariate Cox regression analysis to establish a cervical cancer recurrence risk prediction model. Line graphs was used to show the model and it was evaluated by using receiver operating characteristic (ROC) curves, calibration plots, and decision curve analysis. RESULTS: Univariate analysis showed that the recurrence rates of cervical cancer patients with different age, age of menarche, parity, miscarriage, clinical stage, and treatment method were significantly different (all P<0.05). Multivariate Cox regression analysis showed that RR=-0.489×(age≥55 years old)+0.481×(age at menarche >15 years old)+0.459×(number of miscarriages≥3)+0.416×(clinical stage II)+0.613×(clinical stage III/IV)+0.366×(the treatment method was surgery + chemotherapy) + 0.015×(the treatment method was chemotherapy alone). The area under the ROC curve (AUC) of the Cox risk prediction model for cervical cancer recurrence constructed was 0.736 (95% CI 0.684 to 0.789), the best prediction threshold was 0.857, the sensitivity was 0.576, and the specificity was 0.810. The accuracy of the Cox risk model constructed by this model was good. From the clinical decision curve, the net benefit value was high and the validity was good. CONCLUSIONS Patient age, age at menarche, miscarriages, clinical stages, and treatment methods are independent factors affecting cervical cancer recurrence. The Cox proportional hazards prediction model for cervical cancer recurrence constructed in this study can be better used for predicting the risk of cervical cancer recurrence.
Pregnancy ; Humans ; Female ; Middle Aged ; Adolescent ; Prognosis ; Uterine Cervical Neoplasms/epidemiology* ; Abortion, Spontaneous ; Neoplasm Recurrence, Local/pathology* ; Proportional Hazards Models ; Risk Factors ; Retrospective Studies