The class III phosphatidylinositol 3-kinase VPS34 supports EV71 replication by promoting viral replication organelle formation.

Enterovirus 71 (EV71) belongs to the family of Picornaviridae; it could cause a variety of illnesses and pose a great threat to public health worldwide. Currently, there is no specific drug treatment for this virus, and a better understanding of virus-host interaction is crucial for novel antiviral development. Here, we find that the class III phosphatidylinositol 3-kinase, VPS34, is an essential host factor for EV71 infection. VPS34 inhibition with either shRNA or specific chemical inhibitor significantly reduces EV71 infection. Meanwhile, EV71 infection upregulates phosphatidylinositol 3-phosphate (PI3P) production in viral replication organelles (ROs), while the depletion of PI3P by phosphatase overexpression inhibits EV71 infection. In addition, the PI3P-binding protein, double FYVE-containing protein 1 (DFCP1), is also required for an efficient replication of EV71. DFCP1 could interact with viral 2C protein and facilitate viral association with lipid droplets (LDs), which are important lipid sources for viral RO biogenesis. Taken together, these results indicate that EV71 virus exploits the VPS34-PI3P-DFCP1-LDs pathway to promote viral RO formation and viral infection, and they also illuminate novel targets for antiviral development.IMPORTANCEEnterovirus 71 (EV71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD) and other serious complications, which are big threats to children under 5 years old. Unravelling the interactions between virus and the host cells will open new avenues in antiviral research. Here, we found the class III phosphatidylinositol 3-kinase, VPS34, and its effector, double FYVE-containing protein 1 (DFCP1), were essential for EV71 infection, both of which could support EV71 viral replication by enhancing the biogenesis of viral replication organelles (ROs). As DFCP1 localizes to lipid droplets, hijacking of these host factors will enable viral utilization of lipids from LDs for the generation of membrane structures during RO biogenesis. In addition, the VPS34 kinase inhibitor was found to be potent against EV71 infection; therefore, this study also brings up a novel target for future anti-EV71 drug development.

Association of HBeAg decline rate from mid-pregnancy to delivery with HBeAg seroconversion after delivery in hepatitis B virus-infected mothers.

There is still controversy about whether to continue antiviral therapy (AVT) after delivery, especially for pregnant women in the immune tolerance (IT) phase. In this study, a retrospective cohort study was conducted to explore the relationship between hepatitis B e antigen (HBeAg) decline rate (%) from mid-pregnancy to delivery and HBeAg seroconversion postpartum among patients using nucleos(t)ide analogs (NAs) to prevent mother-to-child transmission (MTCT), with the goal of identifying the ideal candidates for postpartum AVT continuation. This retrospective cohort study included 151 postpartum women. Univariate and multivariable logistic regression analyses were conducted to assess the association between the HBeAg decline rate (%) from mid-pregnancy to delivery and HBeAg seroconversion postpartum. Receiver operating characteristic (ROC) analysis was utilized to evaluate the predictive capacity of the HBeAg decline rate (%) and determine the optimal cut-off point. The univariate analysis revealed a significant association between the HBeAg decline rate (%) and HBeAg seroconversion postpartum (OR 1.068, 95% CI: 1.034-1.103, p < .001). In the multivariate regression analysis, adjusting for age, hepatitis B surface antigen (HBsAg) titre (log10 IU/mL) at mid-pregnancy, HBeAg titre (log10 S/CO) at mid-pregnancy, and hepatitis B virus (HBV) DNA load decline rate (%) from mid-pregnancy to delivery, the HBeAg decline rate(%) remained significantly associated with HBeAg seroconversion postpartum (OR 1.050, 95% CI: 1.015-1.093, p = .009). Then HBeAg decline rate (%) was treated as a categorical variable (tertiles) for sensitivity analysis. In the three distinct models, taking Tertile1 as a reference, women in Tertile3 still had a 4.201-fold (OR 4.201, 95% CI: 1.382-12.773, p = .011) higher risk of developing HBeAg seroconversion (p for trend <.05) after adjusting above covariates. The area under the curve (AUC) was 0.723 (95% CI: 0.627-0.819). The optimal cut-off value was 5.43%, with a sensitivity of 0.561, specificity of 0.791, and Youden's index of 0.352.A higher HBeAg decline rate (%) from mid-pregnancy to delivery independently correlated with an increased risk of HBeAg seroconversion postpartum. This decline rate can serve as a valuable clinical indicator for predicting HBeAg seroconversion.

Protein folding dependence on selenoprotein M contributes to steady cartilage extracellular matrix repressing ferroptosis via PERK/ATF4/CHAC1 axis.

OBJECTIVE: Initiation of endoplasmic reticulum (ER) stress is pivotal to the advancement of osteoarthritis (OA). We aimed to explore the function of ER-resident selenoprotein M (SELM) in cartilage-forming chondrocytes, investigating how SELM participates in cartilage extracellular matrix (ECM) metabolism and ER stress modulation. METHODS: Articular cartilage samples with knee OA undergoing total knee arthroplasty were categorised into OA-smooth and OA-damaged groups, with primary chondrocytes extracted from smooth areas. Destabilization of the medial meniscus was induced in male C57BL6/J mice, with sham operations on the left knee as controls. After 8 weeks, knee joint tissues were collected for analysis. Histology and immunohistochemistry examined cartilage damage. Molecular biology techniques investigated how SELM affects ECM metabolism and ER stress regulation. RNA sequencing revealed the pathway changes after SELM intervention. AlphaFold demonstrated how SELM interacts with other molecules. Cultured cartilage explants helped determine the effects of SELM supplementation. RESULTS: SELM expression was reduced in the damaged cartilage. Increasing SELM levels positively impacted ECM equilibrium. Decreasing SELM expression activated genes linked to degenerative ailments and impaired the cellular response to misfolded proteins, initiating the PERK/P-EIF2A/ATF4 pathway and exacerbating GSH/GSSG imbalance via the ATF4/CHAC1 axis. SELM likely participated in protein folding and modification by leveraging its thioredoxin domains. In vitro SELM supplementation mitigated IL-1ß effects on damaged cartilage explants and suppressed beneficial chondrocyte phenotypes. CONCLUSIONS: Our results confirm the involvement of SELM in ER stress-induced cartilage damage as well as protein folding, pointing to new directions in molecular therapy for degenerative diseases.

Effects of sputum bacillary load and age on GeneXpert and traditional methods in pulmonary tuberculosis: a 4-year retrospective comparative study.

BACKGROUND: The purpose of this study was to evaluate the diagnostic value of the GeneXpert® MTB/RIF (Xpert®), Auramine O staining method, and Lowenstein-Jensen medium for bacteriologically confirmed pulmonary tuberculosis and explore the effects of the sputum bacillary load (SBL) and qRT‒PCR threshold cycle (Ct) value on the detection methods. METHODS: We retrospectively analysed the results in the Department of Infectious Disease for 49 months. The χ2 test was used to compare the performances of each method, receiver operating characteristic curve analysis was used to determine the optimal cut-off values, and the factors associated with a false-negative result from Xpert® were analysed by logistic regression. RESULTS: Simultaneous analysis of 980 sputum specimens showed that the positive detection rate of Xpert® did not increase with increasing SBL, and there were differences between the three when SBL ≤ 1 + (all P < 0.05). There was a good negative correlation between the Ct value and the SBL (P < 0.0001). Age was an independent risk factor for false-negative Xpert® results (P = 0.029), and when Ct < 16, the diagnostic sensitivity and specificity were both 100.00%. The optimal cut-off Ct values for resegmentation based on the drug resistance classification were < 18.6, 18.6-34.1, and > 34.1 cycles. CONCLUSIONS: Xpert® was not affected by SBL but it was by age, and it is more advantageous when SBL ≤ 1 + . The results regarding rifampicin resistance were reliable, and the novel Ct segmentation was a practical and more clinically meaningful classification method for diagnosing rifampicin resistance. These findings will help improve physicians' ability to accurately diagnose TB.

MAPK p38/Ulk1 pathway inhibits autophagy and induces IL-1ß expression in hepatic stellate cells.

In the past, hepatic stellate cells (HSCs) were considered to be noninflammatory cells and to contribute to liver fibrosis by producing extracellular matrix. Recently, it was found that HSCs can also secrete cytokines and chemokines and therefore participate in hepatic inflammation. Autophagy participates in many immune response processes in immune cells. It is unclear whether autophagy is involved in inflammatory cytokine induction in HSCs. MAPK p38, Ulk1 phosphorylation, and the Ulk1-Atg13 complex were analyzed in HSC-T6 cells after LPS treatment. The relationship between autophagy inhibition and inflammation was investigated in primary rat HSCs. We discovered that LPS inhibited autophagy through MAPK p38. The activation of MAPK p38 induced Ulk1 phosphorylation, which disrupted the Ulk1-Atg13 complex and therefore inhibited autophagy. Furthermore, in primary rat HSCs, we demonstrated that autophagy inhibition regulated IL-1ß induction, which depended on the MAPK p38/Ulk1 pathway. Our results reveal a continuous signaling pathway, MAPK p38-Ulk1 phosphorylation-Ulk1-Atg13 disruption, which inhibits autophagy and induces IL-1ß expression in HSCs.NEW & NOTEWORTHY LPS inhibits autophagy in a concentration- and dose-dependent manner in HSC-T6 cells. MAPK p38 induces phosphorylation of Ulk1, which disrupts the Ulk1-Atg13 complex and is therefore required for the inhibition of autophagy by LPS. LPS induces IL-1ß expression via the MAPK p38/Ulk1 pathway in HSCs.

Inhibition of miR-188-5p alleviates hepatic fibrosis by significantly reducing the activation and proliferation of HSCs through PTEN/PI3K/AKT pathway.

Persistent hepatic damage and chronic inflammation in liver activate the quiescent hepatic stellate cells (HSCs) and cause hepatic fibrosis (HF). Several microRNAs regulate the activation and proliferation of HSCs, thereby playing a critical role in HF progression. Previous studies have reported that miR-188-5p is dysregulated during the process of HF. However, the role of miR-188-5p in HF remains unclear. This study investigated the potential role of miR-188-5p in HSCs and HF. Firstly, we validated the miR-188-5p expression in primary cells isolated from liver of carbon tetrachloride (CCl4 )-induced mice, TGF-ß1-induced LX-2 cells, livers from 6-month high-fat diet (HFD)-induced rat and 4-month HFD-induced mice NASH models, and human non-alcoholic fatty liver disease (NAFLD) patients. Furthermore, we used miR-188-5p inhibitors to investigate the therapeutic effects of miR-188-5p inhibition in the HFD + CCl4 induced in vivo model and the potential role of miR-188-5p in the activation and proliferation of HSCs. This present study reported that miR-188-5p expression is significantly increased in the human NAFLD, HSCs isolated from liver of CCl4 induced mice, and in vitro and in vivo models of HF. Mimicking the miR-188-5p resulted in the up-regulation of HSC activation and proliferation by directly targeting the phosphatase and tensin homolog (PTEN). Moreover, inhibition of miR-188-5p reduced the activation and proliferation markers of HSCs through PTEN/AKT pathway. Additionally, in vivo inhibition of miR-188-5p suppressed the HF parameters, pro-fibrotic and pro-inflammatory genes, and fibrosis. Collectively, our results uncover the pro-fibrotic role of miR-188-5p. Furthermore, we demonstrated that miR-188-5p inhibition decreases the severity of HF by reducing the activation and proliferation of HSCs through PTEN/AKT pathway.

Obesity interacts with hyperuricemia on the severity of non-alcoholic fatty liver disease.

BACKGROUND: A series of evidence revealed that body mass index was an important confounding factor in the research of uric acid and ischemic heart disease/hypertension. The objective of this study was to investigate whether obesity status can modify the association between serum uric acid and the severity of liver damage in NAFLD, and the possible interactive effect of hyperuricemia and obesity. METHODS: We conducted a cross-sectional study in a total of 557 ultrasound diagnosed-NAFLD. The hepatic steatosis and liver fibrosis were quantitatively evaluated by transient elastography. Hyperuricemia was defined as serum uric acid > 420 µmol/L in men, > 360 µmol/L in women and obesity was defined as body mass index ≥ 25 kg/m2. The adjusted OR values of hyperuricemia and obesity were analyzed by multivariate logistic regression analysis, and the additive model was used to investigate the possible interactive effect. RESULTS: Multivariate regression analysis showed that hyperuricemia was associated with serious hepatic steatosis (1.74[1.09-2.79]) and elevated ALT (2.17[1.38-3.41]), but not with advanced fibrosis (1.61[0.91-2.85]). The association was further investigated in different BMI group. Hyperuricemia was associated with higher odds of serious hepatic steatosis (2.02[1.14-3.57]) and elevated ALT (2.27[1.37-3.76]) only in obese NAFLD, not in non-obese subjects. Similarly, patients with hyperuricemia had higher odds of advanced fibrosis in obese subjects (2.17[1.13-4.18]), not in non-obese subjects (0.60[0.14-2.70]). Furthermore, there was an additive interaction between hyperuricemia and obesity on the odds of serious hepatic steatosis (AP: 0.39[0.01-0.77]) and advanced fibrosis. (AP: 0.60[0.26-0.95]). CONCLUSIONS: Hyperuricemia and obesity had a significantly synergistic effect on the hepatic steatosis and fibrosis. Thus, management of uric acid may need to be targeted in obese NAFLD.

Short-term and long-term safety and efficacy of tenofovir alafenamide, tenofovir disoproxil fumarate and entecavir treatment of acute-on-chronic liver failure associated with hepatitis B.

BACKGROUND & AIMS: There is limited evidence on the efficacy and safety of nucleos(t) ide analogues (NAs) in the treatment of HBV-ACLF. Our objective was to evaluate the outcomes among TAF, TDF and ETV, three first-line antivirals against chronic hepatitis B, in patients with HBV-ACLF. METHODS: Patients with HBV-related ACLF were recruited and received daily TAF (25 mg/d), TDF (300 mg/d) and ETV (0.5 mg/d). They were prospectively followed-up. The primary endpoint was overall survival at week 12 and week 48, the secondary endpoints were virological response and biochemical response. RESULTS: Forty gender and age matched eligible subjects were recruited and divided into three groups: TAF group, TDF group and ETV group. By week 48, 8 (80%) patients in TAF group, 6 (60%) patients in TDF group and 17 (85%) patients in ETV group survived without liver transplantation (P = 0.251). After 4 weeks of NAs treatment, all three groups showed paralleling reduction of HBV DNA levels. All three groups presented similar biochemical responses at week 4, patients treated with TAF showed a priority in total bilirubin reduction, albumin and cholesterol maintenance. Additionally, although there was no significant difference in changes of serum urea, serum creatinine, serum cystatin C and estimated GFR among the three groups by treatment week 4, TDF showed unfavorable renal safety even in short -term treatment. The treatment using NAs was well-tolerated and there was no serious drug-related adverse event reported. CONCLUSIONS: TAF, TDF and ETV are of similar efficacy and safety in short-term and long-term treatment of HBV-ACLF. TRIAL REGISTRATION: This study is ongoing and is registered with ClinicalTrials.gov , NCT03640728 (05/02/2019).

High Prevalence of Preexisting HBV Polymerase Mutations in Pregnant Women Does Not Limit the Antiviral Therapy Efficacy.

BACKGROUND: HBV-resistant mutants in treatment-naïve patients may lead to antiviral treatment failure. It is not clear if HBV mutants are present in pregnant women and about the influence of the preexisting mutants on the short-term antiviral therapy during pregnancy. METHOD: We enrolled 73 pregnant women with high HBV DNA load and telbivudine (TBV) treatment during pregnancy in this retrospective study. The UDPS was used to detect the HBV mutations before and after the TBV treatment. RESULTS: Before TBV treatment, the complexity of HBV quasispecies of all subjects was 0.40 ± 0.09; 41.1% (30/73) and 53.4% (39/73) subjects had rtM204I/V and rtN236 T/A detected, respectively; and 9.6% (7/73) patients had more than 20% frequency mutation of rtM204I/V, which was also similar with high frequency of rtN236 T/A mutation (41.1% vs. 53.4%, P=0.136; frequencies >20%: 9.6% vs. 5.5%, P=0.347). After TBV treatment, 71.2% (52/73) subjects had HBV DNA load ≥ 103 IU/mL at delivery. Among them, 75.0% of patients with rtM204I positive had HBV DNA load ≥103 IU/mL at delivery, which was comparable with the subjects without rtM204I (75.0% vs. 70.8%, P=0.710). No changes were found in the frequencies and the complexity of HBV quasispecies of rtM204I mutation after the TVB treatment. CONCLUSION: The prevalence of preexisting drug-resistant mutations among pregnant women was high using UPDS. However, the preexisting HBV mutation had limited influence on the efficacy of short-term TBV treatment, and TBV treatment during late pregnancy seemed not to increase the risk of emerging HBV-resistant mutants.

Nitric oxide inhibits autophagy and promotes apoptosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related mortality worldwide. The expression of nitric oxide synthase (NOS) and the inhibition of autophagy have been linked to cancer cell death. However, the involvement of serum nitric oxide (NO), the expression of NOS and autophagy have not been investigated in HCC. In the present study, we first established that the NO level was significantly higher in hepatitis B virus-related HCC than in the liver cirrhosis control (53.60 ± 19.74 vs 8.09 ± 4.17 µmol/L, t = 15.13, P < 0.0001). Using immunohistochemistry, we found that the source of NO was at least partially attributed to the expression of inducible NOS and endothelial NOS but not neuronal NOS in the liver tissue. Furthermore, in human liver cancer cells, NO-induced apoptosis and inhibited autophagy. Pharmacological inhibition of autophagy also induced apoptosis, whereas the induction of autophagy could ameliorate NO-induced apoptosis. We also found that NO regulates the switch between apoptosis and autophagy by disrupting the Beclin 1/Vps34 association and by increasing the Bcl-2/Beclin 1 interaction. Overall, the present findings suggest that increased NOS/NO promotes apoptosis through the inhibition of autophagy in liver cancer cells, which may provide a novel strategy for the treatment of HCC.

Efficacy and safety of telbivudine and tenofovir disoproxil fumarate in preventing hepatitis B vertical transmission: A real-life practice.

Mother-to-child transmission (MTCT) is a major obstacle in the elimination of hepatitis B virus (HBV) infection. Telbivudine (LdT) and tenofovir disoproxil fumarate (TDF) are the two most common antiviral medicines for preventing MTCT. However, the efficacy and safety of LdT and TDF in preventing HBV vertical transmission during the second to third trimester have not been compared rigorously. Therefore, we carried out a prospective multicentre cohort study of chronic hepatitis B in mothers with HBV DNA > 106  IU/mL, receiving LdT or TDF during the second to third trimester. Among the 893 mothers enrolled, 857 (LdT/TDF/untreated group (NTx) = 396/325/136) completed consecutive follow-up with 854 infants (LdT/TDF/NTx = 395/323/136). LdT and TDF treatment resulted in a similar decrease of HBV DNA in mothers at delivery. Multivariate analysis indicated that only HBsAg titre at the baseline correlated with viral DNA decrease (P = 0.015). With intention-to-treat analysis, MTCT rates in the LdT, TDF and NTx group were 4.41%, 2.42% and 22.08%, respectively. An increasing vertical transmission rate was found to be closely associated with higher HBsAg titre, 5.32% and 17.65% infection rate was estimated in infants born to mothers with HBsAg > 4 and >5 log10 IU/mL, respectively. No serious side effects were reported in either mothers or infants. LdT and TDF treatments were well tolerated and showed comparable efficacy in reducing MTCT. Higher risk of MTCT was shown in pregnant women with HBsAg > 4 log10 IU/mL.

Expression of autophagy-modulating genes in peripheral blood mononuclear cells from familial clustering patients with chronic hepatitis B virus infection.

We previously found that genetic factors are associated with a familial predisposition for developing liver cirrhosis and hepatocellular carcinoma during chronic hepatitis B virus (HBV) infection. Autophagy has been shown to play a role in HBV replication and the course of disease. More than 190 host genes have been identified that modify the process of autophagy, but which of these genes are involved in chronicity of HBV infection and how this occurs remains unclear. Chronic hepatitis B (CHB) patients were recruited to investigate the expression of autophagy-modulating genes in peripheral blood mononuclear cells (PBMCs). mRNA prepared from PBMCs from members of two families with clustering HBV infection, including 11 CHB patients and nine healthy spouses, was hybridized to high-density oligonucleotide arrays. Immunoblot analysis was used to determine the level of autophagy. Of the 192 autophagy-modulating genes, 18 were found to be differently expressed. Of these, 11 displayed decreased expression in CHB patients, while seven displayed increased expression compared to those in healthy controls. Functional analysis showed that these genes are closely involved in initiation, nucleation, elongation of phagophores, formation of autophagosomes, transportation to lysosomes, and the process of degradation. Western blot analysis revealed inhibited autophagy in PBMCs based on decreased lipidation of LC3II. A differential expression profile of autophagy-modulating genes was observed, and decreased autophagy in PBMCs could be closely associated with chronicity of HBV infection, suggesting a novel strategy for the treatment of patients with chronic HBV infection.

Recurrent hyperkalemia in patients with chronic kidney disease and hepatitis C treated with direct antiviral agents.

BACKGROUND: Sofosbuvir is the keystone of direct antiviral agents for the chronic hepatitis C (CHC). The safety of sofosbuvir in patients with stage 4-5 chronic kidney disease (CKD) needs further observation in real world. CASE PRESENTATION: Thirty-three patients with stage 5 CKD and hepatitis C virus (HCV) infection from 2 hemodialysis centers accepted sofosbuvir based treatment as we reported previously. Serum potassium concentrations were tested every 4 weeks or on demand. Ten of 33 patients showed recurrence of hyperkalemia. We summarized the characteristics of hyperkalemia occurrence in these 10 patients. Overall, 24 episodes of hyperkalemia were observed in these 10 patients, 21 were under treatment and 3 were after treatment. Patients with or without hyperkalemia before sofosbuvir treatment didn't show significantly differences in the median frequencies of hyperkalemia episodes during the observation period (3.5 vs. 2, p = 0.264). CONCLUSIONS: Patients with stage 5 CKD and HCV infection treated with sofosbuvir based regimens, even halved sofosbuvir, should be taken caution and closely monitoring serum potassium and renal function is necessary.

Role of mitophagy regulation by ROS in hepatic stellate cells during acute liver failure.

Liver sinusoids serve as the first line of defense against extrahepatic stimuli from the intestinal tract. Hepatic stellate cells (HSCs) are pericytes residing in the perisinusoidal space that integrate cytokine-mediated inflammatory responses in the sinusoids and relay these signals to the liver parenchyma. Oxidative stress has been shown to promote inflammation during acute liver failure (ALF). Whether and how oxidative stress is involved in HSC inflammation during ALF remains unclear. Level of systemic oxidative stress is reflected by superoxide dismutase (SOD). Thus, ALF patients were recruited to investigate the correlation between plasma SOD levels and clinical features. Liver tissues were collected from chronic hepatitis patients by biopsy and from ALF patients who had undergone liver transplantation. SOD2 expression and HSCs activation were investigated by immunohistochemistry. Inflammation, mitophagy, and apoptosis were investigated by immunoblot analysis and flow cytometry in HSCs treated with lipopolysaccharide (LPS) and reactive oxygen species (ROS) donors. The plasma SOD level was significantly increased in patients with ALF compared with those with cirrhosis (444.4 ± 23.58 vs. 170.07 ± 3.52 U/ml, P < 0.01) and was positively correlated with the Model for End-Stage Liver Disease-Na score ( R2 = 0.4720, P < 0.01). In vivo observations revealed that SOD2 immunostaining was increased in ALF patients and mice models, and in vitro experiments demonstrated that LPS/ROS promoted inflammation via inhibiting mitophagy. Moreover, the regulation of inflammation was apoptosis independent in HSCs. LPS-induced increases in oxidative stress promote inflammation through inhibiting mitophagy in HSCs during the process of ALF, providing a novel strategy for the treatment of patients with ALF. NEW & NOTEWORTHY Here we demonstrate that the serum superoxide dismutase (SOD) level is significantly increased in patients with acute liver failure (ALF), and, correlated with the Model for End-Stage Liver Disease-Na score, SOD level dropped in the remission stage of ALF. We identify that, in liver tissue from ALF patients and mice models, manganese-dependent SOD was overexpressed, and show lipopolysaccharide/H2O2 inhibits mitophagy via reactive oxygen species in hepatic stellate cells (HSCs). We show that inhibited mitophagy promotes inflammation in HSCs, whereas mitophagy inducer rescues HSCs from lipopolysaccharide-induced inflammation.

A molecular epidemiology study investigating familial clustering of hepatitis B virus infection in families with unfavorable prognoses in Northwest China.

Hepatitis B virus (HBV) infections and adverse outcome have been demonstrated to show characteristics of familial clustering. The aim of this study was to investigate the prevalence of different HBV genotypes, HBV sub-genotypes, and Pre-S mutations associated with familial HBV infection clusters with unfavorable prognoses. Families presenting with clustered HBV infections and unfavorable prognoses were enrolled in this study. Non-clustered HBV-infected individuals were used as the control group. DNA extracted from patient serum samples was used to facilitate characterization of the HBV genotypes, HBV sub-genotypes, and Pre-S mutations by phylogenetic analysis. The Pre-S/S gene was successfully amplified in 83 patients from the clustering group and 105 patients from the sporadic group. The prevalence of genotype C in the clustering group (71/83, 85.54%) was significantly higher than in the sporadic group (77/105, 73.33%) (P = 0.042). The prevalence of sub-genotype C2 in the clustering group (33/83, 39.76%) was also higher than in the sporadic group (21/105, 20%) (P = 0.003). Analyses of functional mapping of pre-S sequences showed that the prevalence of the mutation in the S promoter site (nt 3045-3189 of pre-S1 domain) was significantly increased in the clustering group compared with the sporadic group (15.7% vs. 3.8%) (P = 0.009). This study suggests that genotype C, especially sub-genotype C2, may be associated with the progression of HBV infection in familial clustering infection cohorts with unfavorable prognoses. We also observed that the natural occurrence of S promoter mutations in the clustering group was significantly prevalent.

Role and regulation of autophagy and apoptosis by nitric oxide in hepatic stellate cells during acute liver failure.

BACKGROUND & AIMS: We previously found that hepatic stellate cell activation induced by autophagy maintains the liver architecture to prevent collapse during acute liver failure. Nitric oxide has shown to induce hepatic stellate cell apoptosis. Whether and how nitric oxide is involved in acute liver failure and autophagy remains unclear. METHODS: Acute liver failure patients were recruited to investigate the correlation between plasma nitric oxide levels and clinical features. Liver tissues were collected from chronic hepatitis patients by biopsy and from acute liver failure patients who had undergone liver transplantation. The expression of nitric oxide synthases and hepatic stellate cell activation (alpha-SMA), and autophagic activity (LC3) were investigated by immunohistochemistry. Autophagy and apoptosis were investigated by immunoblot analysis, confocal microscopy, and flow cytometry in hepatic stellate cells treated with nitric oxide donors. RESULTS: Plasma nitric oxide level was significantly increased in patients with acute liver failure compared to those with cirrhosis (53.60±19.74 µM vs 19.40±9.03 µM, Z=-7.384, P<.001) and positively correlated with MELD-Na score (r=.539, P<.001), implicating nitric oxide in acute liver failure. At least some Nitric oxide was produced by overexpression of inducible nitric oxide synthases and endothelial nitric oxide synthases, but not neuronal nitric oxide synthases in the liver tissue. In vivo observation revealed that autophagy was inhibited in hepatic stellate cells based on decreased LC3 immunostaining, and in vitro experiments demonstrated that Nitric oxide can inhibit autophagy. Moreover, nitric oxide promoted hepatic stellate cell apoptosis, which was rescued by an autophagy inducer. CONCLUSIONS: Increased nitric oxide synthases/ nitric oxide promotes apoptosis through autophagy inhibition in hepatic stellate cells during acute liver failure, providing a novel strategy for the treatment of patients with acute liver failure.

Effect of extrusion processing on immune activation properties of hazelnut protein in a mouse model.

Although food processing can alter food allergenicity, the impact of extrusion processing on in vivo hazelnut allergenicity is unknown. Here, we tested the hypothesis that extrusion processing will alter the immune activation properties of hazelnut protein (HNP) in mice. Soluble extrusion-processed HNP (EHNP) was prepared and evaluated for immune response using an established transdermal sensitization mouse model. Mice were sensitized with identical amounts of EHNP versus raw HNP. After confirming systemic IgE, IgG1 and IgG2a antibody responses, oral hypersensitivity reaction was quantified by hypothermia shock response (HSR). Mechanism was studied by measuring mucosal mast cell (MMC) degranulation. Compared to raw HNP, the EHNP elicited slower but similar IgE antibody (Ab) response, lower IgG1 but higher IgG2a Ab response. The EHNP exhibited significantly lower oral HSR as well as MMC degranulation capacity. These results demonstrate that the extrusion technology can be used to produce soluble HNP with altered immune activation properties.

Mechanisms of fibrosis in acute liver failure.

BACKGROUND & AIMS: Acute liver failure (ALF) is a condition with high mortality and morbidity. Fibrosis in chronic liver disease was extensively researched, whereas fibrosis and underlying mechanism in acute liver failure remains unclear. METHODS: Hepatitis B virus related ALF patients were recruited to investigate if there was ongoing fibrosis by liver histology and liver stiffness measurement(LSM) analysis as well as fibrosis markers assay. Sera HMGB1 were kinetically detected in progression and remission stage of ALF. Hepatic stellate cell(HSC) activation by HMGB1 was explored by testing mRNA and protein level of α-SMA and collagen 1a1 by using qPCR and western blot. Autophagy induction by HMGB1 was explored by LC3-II conversion, autophagy flux and fluorescence. RESULTS: Firstly, ongoing fibrosis in progression stage of ALF was confirmed by histological analysis, LS measurement as well as fibrosis markers detection. HSC activation and autophagy induction in explanted liver tissue also revealed. Next, kinetic monitoring sera HMGB1 revealed elevated HMGB1 in progression stage of ALF vs HBsAg carrier, and drop back to base level in remission stage. Thirdly, rHMGB1 dose dependently activated HSCs, as indicated by increased mRNA and proteins level in α-SMA and collagen 1a1. Moreover, autophagy was induced in HSC treated with rHMGB1, as illustrated by increased LC3 lipidation, elevated autophagy flux and GFP-LC3 puncta. CONCLUSIONS: Acute liver failure is accompanied by ongoing fibrosis, HSC activation and autophagy induction. Increased HMGB1 activates HSC via autophagy induction. Those findings integrate HMGB1, HSCs activation, autophagy into a common framework that underlies the fibrosis in ALF.

Comparative study of the different activities of hepatitis B virus whole-X protein and HBx in hepatocarcinogenesis by proteomics and bioinformatics analysis.

The hepatitis B virus (HBV) whole-X gene comprises the HBV X gene and the 168-bp region immediately upstream. Although the functions of HBx in hepatocarcinogenesis are well known, the activity of the HBV whole-X protein (HBwx), with 56 additional amino acids, has not yet been explored. In this study, proteomic and bioinformatic analysis was done to determine the protein interaction profiles of HBwx and HBx and to describe their functions in carcinogenesis. A total of 203 proteins were identified that interacted with HBwx, of which 149 were unique, the rest interacting also with HBx, and 73% (148/203) of these proteins are involved in carcinogenesis. Gene ontology (GO) analysis showed that HBwx- and HBx-interacting proteins are involved in different processes, the former mainly in biosynthetic processes (glycolysis, cell-cycle functions, and protein folding), and the latter mainly in localization, viral transcription, biological adhesion and angiogenesis. Pathway networks analysis revealed that proteins interacting with HBx participate mainly in oxidative phosphorylation, localization, the cytoskeleton, and cell adhesion. In contrast, more-specific functional analysis showed that proteins interacting with HBwx are involved in apoptosis and survival, cell-cycle functions, glycolysis, and gluconeogenesis (Pathway Maps); to cellular macromolecular complex assembly, protein folding and mRNA metabolic process (GO Processes); and to regulation of protein folding in the endoplasmic reticulum and cytoplasm, transcription, cell cycle G2-M and cytoskeleton rearrangement (Process Networks). In conclusion, this study shows that HBwx functions in carcinogenesis in a way that is different from that of HBx.

A comparison of the physical activity levels of 3-to-6-year-old children with autism spectrum disorder and children with typical development.

Background: Physical activity during early development is closely related to health. Differences in physical activity between young children with autism spectrum disorder and those with typical development are unclear. The purpose of this study was to compare the physical activity levels in children with autism spectrum disorder and typically developing children from the same area, including their sedentary physical activity, light physical activity, moderate-to-vigorous physical activity, and number of days in which the moderate-to-vigorous physical activity guideline recommendation of 60 min per day was met. Methods: A total of 77 participants aged 3-6 years were included: 41 children with autism spectrum disorder (mean age = 61.41 ± 10.69 months) and 36 children with typical development (mean age = 60.36 ± 10.16 months). The physical activity of the children was measured using an ActiGraph GT3x accelerometer. Results: There were no significant differences in daily sedentary physical activity (439.70 ± 54.98 vs. 450.42 ± 53.67) or moderate-to-vigorous physical activity (46.62 ± 18.93 vs. 47.47 ± 18.26) between the two groups. The average daily moderate-to-vigorous physical activity of the two groups did not reach 60 min, and they had similar proportions of participants who reached 60 min a given number of times (24.4% vs. 25%). Daily light physical activity was significantly higher in the autism spectrum disorder group (263.96 ± 43.17 vs. 242.32 ± 37.91, p < 0.05). The moderate-to-vigorous physical activity of both groups was similar and lower than the recommended minimum physical activity. Conclusion: Targeted interventions should be considered in early intervention programs for children with autism spectrum disorder to increase their moderate-to-vigorous physical activity.