ZNF469 is a profibrotic regulator of extracellular matrix in hepatic stellate cells.

Activation of quiescent hepatic stellate cells (HSCs) into proliferative myofibroblasts drives extracellular cellular matrix (ECM) accumulation and liver fibrosis; nevertheless, the transcriptional network that promotes such a process is not completely understood. ZNF469 is a putative C2H2 zinc finger protein that may bind to specific genome sequences. It is found to be upregulated upon HSC activation; however, the molecular function of ZNF469 is completely unknown. Here, we show that knockdown of ZNF469 in primary human HSCs impaired proliferation, migration, and collagen production. Conversely, overexpression of ZNF469 in HSCs yielded the opposite results. Transforming growth factor-ß 1 promoted expression of ZNF469 in a Smad3-dependent manner, where the binding of Smad3 was confirmed at the ZNF469 promoter. RNA sequencing data of ZNF469-knockdown HSCs revealed the ECM-receptor interaction, which provides structural and signaling support to cells, was the most affected pathway, and significant downregulation of various collagen and proteoglycan genes was observed. To investigate the function of ZNF469, we cloned a full-length open reading frame of ZNF469 with an epitope tag and identified a nuclear localization of the protein. Luciferase reporter and chromatin immunoprecipitation assays revealed the presence of ZNF469 at the promoter of ECM genes, supporting its function as a transcription factor. Analysis of human fibrotic and cirrhotic tissues showed increased expression of ZNF469 and a positive correlation between expression levels of ZNF469 and ECM genes. Moreover, this observation was similar in other fibrotic organs, including the heart, lung, and skin, suggesting that myofibroblasts from various origins generally require ZNF469 to promote ECM production. Together, this study is the first to reveal the role of ZNF469 as a profibrotic factor in HSCs and suggests ZNF469 as a novel target for antifibrotic therapy.

Regional Differences in Clinical Presentation and Prognosis of Patients With Post-Sustained Virologic Response Hepatocellular Carcinoma.

BACKGROUND & AIMS: Widespread use of direct-acting antivirals for hepatitis C virus infection has been paralleled with increased numbers of patients with hepatocellular carcinoma (HCC) after achieving sustained virologic response (post-SVR HCC) worldwide. Few data compare regional differences in the presentation and prognosis of patients with post-SVR HCC. METHODS: We identified patients with advanced fibrosis (F3/F4) who developed incident post-SVR HCC between March 2015 and October 2021 from 30 sites in Europe, North America, South America, the Middle East, South Asia, East Asia, and Southeast Asia. We compared patient demographics, liver dysfunction, and tumor burden by region. We compared overall survival by region using Kaplan-Meier analysis and identified factors associated with survival using multivariable Cox regression analysis. RESULTS: Among 8796 patients with advanced fibrosis or cirrhosis who achieved SVR, 583 (6.6%) developed incident HCC. There was marked regional variation in the proportion of patients detected by surveillance (range: 59.5%-100%), median maximum tumor diameter (range, 1.8-5.0 cm), and the proportion with multinodular HCC (range, 15.4%-60.8%). The prognosis of patients highly varied by region (hazard ratio range, 1.82-9.92), with the highest survival rates in East Asia, North America, and South America, and the lowest survival rates in the Middle East and South Asia. After adjusting for geographic region, HCC surveillance was associated with early stage detection (Barcelona Clinic Liver Cancer stage 0/A, 71.0% vs 21.3%; P < .0001) and lower mortality rates (adjusted hazard ratio, 0.29; 95% CI, 0.18-0.46). CONCLUSIONS: Clinical characteristics, including early stage detection, and prognosis of post-SVR HCC differed significantly across geographic regions. Surveillance utilization appears to be a high-yield intervention target to improve prognosis among patients with post-SVR HCC globally.

Distinct alterations of gut microbiota between viral- and non-viral-related hepatocellular carcinoma.

Altered gut microbiota has been connected to hepatocellular carcinoma (HCC) occurrence and advancement. This study was conducted to identify a gut microbiota signature in differentiating between viral-related HCC (Viral-HCC) and non-hepatitis B-, non-hepatitis C-related HCC (NBNC-HCC). Fecal specimens were obtained from 16 healthy controls, 33 patients with viral-HCC (17 and 16 cases with hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, respectively), and 18 patients with NBNC-HCC. Compositions of fecal microbiota were assessed by 16S rRNA sequencing. Bioinformatic analysis was performed by the DADA2 pipeline in the R program. Significantly different genera from the top 50 relative abundance were used to classify between subgroups of HCC by the Random Forest algorithm. Our data demonstrated that the HCC group had a significantly decreased alpha-diversity and changed microbial composition in comparison with healthy controls. Within the top 50 relative abundance, there were 11 genera including Faecalibacterium, Agathobacter, and Coprococcus that were significantly enhanced in Viral-HCC, while 5 genera such as Bacteroides, Streptococcus, Ruminococcus gnavus group, Parabacteroides, and Erysipelatoclostridium were enhanced in NBNC-HCC. Compared to Viral-HCC, the NBNC-HCC subgroup significantly reduced various short-chain fatty acid-producing bacteria, as well as declined fecal butyrate but elevated plasma surrogate markers of microbial translocation. Based on the machine learning algorithm, a high diagnostic accuracy to classify HCC subgroups was achieved with an area under the receiver-operating characteristic (ROC) curve (AUC) of 0.94. Collectively, these data revealed that gut dysbiosis was distinct according to etiological factors of HCC, which might play an essential role in hepatocarcinogenesis. These findings underscore the possible use of a gut microbiota signature for the diagnosis and therapeutic approaches regarding different subgroups of HCC. KEY POINTS: • Gut dysbiosis is connected to hepatocarcinogenesis and can be used as a novel biomarker. • Gut microbiota composition is significantly altered in different etiological factors of HCC. • Microbiota-based signature can accurately distinguish between Viral-HCC and NBNC-HCC.

Size-Dependent Electrochemistry of Laser-Induced Graphene Electrodes.

Laser-induced graphene (LIG) electrodes have become popular for electrochemical sensor fabrication due to their simplicity for batch production without the use of reagents. The high surface area and favorable electrocatalytic properties also enable the design of small electrochemical devices while retaining the desired electrochemical performance. In this work, we systematically investigated the effect of LIG working electrode size, from 0.8 mm to 4.0 mm diameter, on their electrochemical properties, since it has been widely assumed that the electrochemistry of LIG electrodes is independent of size above the microelectrode size regime. The background and faradaic current from cyclic voltammetry (CV) of an outer-sphere redox probe [Ru(NH3)6]3+ showed that smaller LIG electrodes had a higher electrode roughness factor and electroactive surface ratio than those of the larger electrodes. Moreover, CV of the surface-sensitive redox probes [Fe(CN)6]3- and dopamine revealed that smaller electrodes exhibited better electrocatalytic properties, with enhanced electron transfer kinetics. Scanning electron microscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy showed that the physical and chemical surface structure were different at the electrode center versus the edges, so the electrochemical properties of the smaller electrodes were improved by having rougher surface and more density of the graphitic edge planes, and more oxide-containing groups, leading to better electrochemistry. The difference could be explained by the different photothermal reaction time from the laser scribing process that causes different stable carbon morphology to form on the polymer surface. Our results give a new insight on relationships between surface structure and electrochemistry of LIG electrodes and are useful for designing miniaturized electrochemical devices.

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease without Type 2 Diabetes: Stratified by Body Mass Index.

The relationship between gut dysbiosis and body mass index (BMI) in non-diabetic patients with non-alcoholic fatty liver disease (NAFLD) is not adequately characterized. This study aimed to assess gut microbiota's signature in non-diabetic individuals with NAFLD stratified by BMI. The 16S ribosomal RNA sequencing was performed for gut microbiota composition in 100 patients with NAFLD and 16 healthy individuals. The differential abundance of bacterial composition between groups was analyzed using the DESeq2 method. The alpha diversity (Chao1, Shannon, and observed feature) and beta diversity of gut microbiota significantly differed between patients with NAFLD and healthy controls. However, significant differences in their diversities were not observed among subgroups of NAFLD. At the phylum level, there was no trend of an elevated Firmicutes/Bacteroidetes ratio according to BMI. At the genus level, patients with lean NAFLD displayed a significant enrichment of Escherichia-Shigella and the depletion of Lachnospira and Subdoligranulum compared to the non-lean subgroups. Combining these bacterial genera could discriminate lean from non-lean NAFLD with high diagnostic accuracy (AUC of 0.82). Non-diabetic patients with lean NAFLD had a significant difference in bacterial composition compared to non-lean individuals. Our results might provide evidence of gut microbiota signatures associated with the pathophysiology and potential targeting therapy in patients with lean NAFLD.

Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis With Liver Fibrosis as Predictors of New-Onset Diabetes Mellitus in People With HIV: A Longitudinal Cohort Study.

BACKGROUND: We investigated the association between nonalcoholic fatty liver disease (NAFLD) plus or minus a concurrent diagnosis of nonalcoholic steatohepatitis (NASH) and incident diabetes mellitus (DM) and the risk factors associated with NAFLD or NASH development. METHODS: In this prospective study, we analyzed people with human immunodeficiency virus (HIV; PWH) aged ≥18 years without excessive alcohol consumption or hepatitis coinfections. NAFLD was defined as controlled attenuation parameter ≥248 dB/m, whereas NASH with significant disease activity and liver fibrosis was defined as a FibroScan-AST score ≥0.67. Cox proportional hazard regression was used to investigate the association between NAFLD with or without NASH and new-onset DM. RESULTS: Of 847 PWH, the median age at baseline was 45 years (interquartile range, 38-51; 43% female). Baseline NAFLD was associated with 2.8-fold higher risk of new-onset DM after adjusting for age, sex, family history of DM, antiretroviral therapy duration, smoking, statin use, stavudine/didanosine/zidovudine exposure, time-updated body mass index, hypertension, and dyslipidemia. Combined NAFLD and NASH at baseline had 3.1-fold higher new-onset DM risk. In separate analyses, baseline DM did not predict progression to NAFLD or NASH, but tenofovir alafenamide use was associated with an increased risk of NAFLD (hazard ratio [HR], 2.01; 95% confidence interval [CI], 1.02-4.02) or NASH development (2.31; 95% CI, 1.12-5.11). CONCLUSIONS: NAFLD alone or combined with NASH strongly predicts new-onset DM. This highlights the need for systematic risk assessments and management of NAFLD/NASH, as it may contribute to metabolic complications such as DM and subsequent cardiovascular diseases in PWH.

First-in-human randomized study of RNAi therapeutic RG6346 for chronic hepatitis B virus infection.

BACKGROUND & AIMS: RG6346 is an N-acetyl-D-galactosamine (GalNAc)-conjugated, double-stranded RNA interference agent targeting the HBV genome S-region. We investigated the safety, tolerability, pharmacokinetics, and pharmacodynamics of RG6346 in healthy volunteers and patients with chronic HBV infection (CHB). METHODS: This first-in-human, adaptive, randomized, double-blinded, phase I study recruited three groups of participants: Group A, 30 healthy volunteers received single-dose RG6346 at 0.1, 1.5, 3.0, 6.0, or 12.0 mg/kg, or placebo; Group B, nucleos(t)ide analogue-naïve participants with CHB received single-dose RG6346 at 3.0 mg/kg (n = 6) or placebo (n = 3); Group C, participants with nucleos(t)ide-suppressed CHB received four doses (every 28 days) of RG6346 at 1.5, 3.0, or 6.0 mg/kg (n = 4 in each cohort) or placebo (n = 6). RESULTS: RG6346 treatment for up to 4 months was safe and well tolerated. The most common adverse event was a mild injection site reaction. Several nucleos(t)ide-naïve participants exhibited self-resolving transaminase elevations with preserved liver function. By the end of RG6346 treatment in Group C (Day 112), the mean reduction from baseline in hepatitis B surface antigen (HBsAg) was 1.39, 1.80, and 1.64 log10 IU/ml in the 1.5, 3.0, and 6.0 mg/kg cohorts, respectively. Of the 12 participants in Group C, 11 (91.7%) achieved a ≥1 log10 IU/ml reduction in HBsAg (3 of 11 [27.3%] had the response sustained at conditional follow-up Day 448). No dose-response relationship was apparent between RG6346 and serum HBsAg levels. The RG6346-induced HBsAg response was independent of hepatitis B e antigen status. Moderate-to-marked sustained reductions of hepatitis B core-related antigen, HBV RNA, HBV DNA (in nucleos[t]ide analogue-naïve participants), and hepatitis B e antigen levels were observed. CONCLUSIONS: These favorable safety and pharmacodynamic data support the clinical development of RG6346 as the backbone of a finite antiviral treatment regimen, with the goal of sustained HBsAg loss (functional cure) in patients with CHB. CLINICAL TRIAL NUMBER: ClinicalTrials.gov NCT03772249. IMPACT AND IMPLICATIONS: Currently available therapies for chronic HBV infection are associated with low rates of functional cure and new, more efficacious treatments are needed. This first-in-human study of RG6346, an RNA interference therapy, showed a favorable safety profile as well as marked and durable reductions in hepatitis B surface antigen levels. These results support the continued development of RG6346 as the backbone of a finite treatment regimen targeting high functional cure rates and are important for HBV researchers and physicians.

Metabolic-Associated Fatty Liver Disease (MAFLD) is associated with immune activation, increased epicardial fat volume, and steatohepatitis among people with HIV in a Thai cohort.

INTRODUCTION: A change in terminology from fatty liver disease to metabolic-associated fatty liver disease (MAFLD), along with modified diagnostic criteria, was proposed in 2020, and data regarding MAFLD burden in people living with HIV are limited. We investigated associations between MAFLD and immune activation, cardiovascular disease (CVD) risks including epicardial fat volume, and steatohepatitis in an Asian cohort. METHODS: We evaluated CVD risk (epicardial fat tissue, coronary artery calcium [CAC] score, and 10-year atherosclerotic CVD [ASCVD] score) in people living with HIV aged >50 years. Individuals with excessive alcohol consumption and viral hepatitis infections were excluded. MAFLD diagnosis was based on 2020 International Consensus criteria. Non-alcoholic steatohepatitis (NASH) with significant activity and liver fibrosis was defined as FibroScan-aspartate aminotransferase (FAST) score ≥0.67 and >0.35. Multivariate logistic regression models were used to investigate factors associated with MAFLD and NASH with significant activity and liver fibrosis. RESULTS: The median age was 54 years (interquartile range [IQR] 52-60) and current CD4 count was 613 (IQR 467-804) cells/mm3 . A total of 37% were female, and most (98%) people living with HIV were virally suppressed. The prevalence of MAFLD and non-alcoholic fatty liver disease was 35% and 38%, respectively. In multivariate analyses, higher body mass index, albumin, epicardial fat volume, and liver stiffness were significantly associated with MAFLD. A higher CD4/CD8 ratio was associated with a lower risk of MAFLD. People with HIV with MAFLD had higher odds of having NASH with significant activity and liver fibrosis (adjusted odds ratio 3.3; 95% confidence interval 1.6-6.6), and similar associations were also observed among different MAFLD categories. CONCLUSIONS: The complex relationship between MAFLD and immune activation, steatohepatitis, and epicardial fat tissue suggests an increased risk of advanced liver disease and CVDs beyond the traditional risk factors in people living with HIV with fatty liver disease.

Circulating Extracellular Vesicle-Derived microRNAs as Novel Diagnostic and Prognostic Biomarkers for Non-Viral-Related Hepatocellular Carcinoma.

Extracellular vesicle-derived microRNAs (EV-miRNAs) are promising circulating biomarkers for chronic liver disease. In this study, we explored the potential significance of plasma EV-miRNAs in non-hepatitis B-, non-hepatitis C-related HCC (NBNC-HCC). We compared, using the NanoString method, plasma EV-miRNA profiles between NBNC-HCC and control groups including patients with non-alcoholic fatty liver disease (NAFLD) and healthy controls. The differentially expressed EV-miRNAs were validated in another set of plasma samples by qRT-PCR. A total of 66 significantly differentially expressed EV-miRNAs between the HCC and the control groups were identified in the discovery set. In the validation cohort, including plasma samples of 70 NBNC-HCC patients, 70 NAFLD patients, and 35 healthy controls, 5 plasma EV-miRNAs were significantly elevated in HCC, which included miR-19-3p, miR-16-5p, miR-223-3p, miR-30d-5p, and miR-451a. These miRNAs were found to participate in several cancer-related signaling pathways based on bioinformatic analysis. Among them, EV-miR-19-3p exhibited the best diagnostic performance and displayed a high sensitivity for detecting alpha-fetoprotein-negative HCC and early-stage HCC. In multivariate analysis, a high EV-miR-19-3p level was demonstrated as an independently unfavorable predictor of overall survival in patients with NBNC-HCC. In conclusion, our data have indicated, for the first time, that EV-miR-19-3p could serve as a novel circulating biomarker for the diagnosis and prognosis of NBNC-HCC.

Safety, antiviral activity and pharmacokinetics of JNJ-64530440, a novel capsid assembly modulator, as 4 week monotherapy in treatment-naive patients with chronic hepatitis B virus infection.

OBJECTIVES: We investigated JNJ-64530440 (a hepatitis B virus capsid assembly modulator) safety, antiviral activity and pharmacokinetics in patients with chronic hepatitis B (CHB) (Phase 1b, NCT03439488). METHODS: Twenty treatment-naive, HBeAg-positive or -negative CHB patients were randomized 4|:|1 to JNJ-64530440 750 mg once or twice daily, or placebo for 28 days. RESULTS: All patients (mean age 43.8 years; 85% male; 70% White; 20% HBeAg positive) completed dosing/28 day follow-up. Mild-to-moderate treatment-emergent adverse events occurred in 3/4 (placebo), 6/8 (once-daily) and 4/8 (twice-daily) patients; mostly fatigue, increased alanine aminotransferase, decreased neutrophil count, and headache. Hepatitis B virus (HBV) DNA was substantially reduced; mean (range) changes from baseline at day 29 were: -3.2 (-2.4 to -3.9) (once-daily) and -3.3 (-2.6 to -4.1) (twice-daily) log10 IU/mL; placebo 0.1 (0.7 to -0.6) log10 IU/mL. HBV DNA levels were below the lower limit of quantification (LLOQ) in 5/8 (once-daily) and 3/8 (twice-daily) patients. For patients with detectable baseline HBV RNA, mean (SE) changes versus baseline in HBV RNA at day 29 were: -2.65 (0.81) (once-daily) and -2.94 (0.33) (twice-daily) log10 copies/mL. HBV RNA levels were 'target not detected' in 4/6 (once-daily) and 3/7 (twice-daily) patients. JNJ-64530440 pharmacokinetics in CHB patients were comparable with those in healthy volunteers. CONCLUSIONS: JNJ-64530440 750 mg once-daily or twice-daily for 28 days was well tolerated and achieved potent antiviral activity in CHB patients.

Prevalence and genotype distribution of hepatitis C virus within hemodialysis units in Thailand: role of HCV core antigen in the assessment of viremia.

BACKGROUND: Individuals with end-stage renal disease have a higher risk of hepatitis C virus (HCV) acquisition during long-term hemodialysis (HD). Our report was designed to investigate HCV prevalence and genotype, in addition to the clinical use of HCV core antigen (HCVcAg), within multiple HD facilities in Thailand. METHODS: This cross-sectional report was investigated between January and June 2019. HCV infection was assessed by anti-HCV and confirmed active infection by measuring HCV RNA and HCVcAg. HCV genotype was determined by phylogenetic analysis using nucleotide sequences of NS5B region. RESULTS: Overall, 140 of 3,305 (4.2%) patients in 15 dialysis centers had anti-HCV positive. Among them, HCV RNA was further assessed in 93 patients and was detectable in 59 (63.4%) persons. Considering HCV viremia, HCVcAg measurement exhibited high accuracy (96.8%), sensitivity (94.9%) and specificity (100%) in comparison with HCV RNA testing. Moreover, individuals infected with HCV received a longer duration of dialysis vintage when compared to anti-HCV negative controls. The major sub-genotypes were 1a, 1b, 3a, 3b, 6f and 6n. Regarding phylogenetic analysis, there were 7 clusters of isolates with high sequence homology affecting 17 individuals, indicating possible HCV transmission within the same HD centers. CONCLUSIONS: HCV frequency and common sub-genotypes in HD centers were different from those found in the Thai general population. HCVcAg might be an alternate testing for viremia within resource-limited countries. Enhanced preventive practices, dialyzer reuse policy and better access to antiviral therapy are crucial for HCV micro-elimination within HD facilities.

meta-Ureidophenoxy-1,2,3-triazole hybrid as a novel scaffold for promising HepG2 hepatocellular carcinoma inhibitors: Synthesis, biological evaluation and molecular docking studies.

Thirty-one meta-ureidophenoxymethyl-1,2,3-triazole derivatives were designed and synthesized via nucleophilic addition, nucleophilic substitution and copper-catalyzed azide-alkyne cycloaddition (CuAAC). The evaluation of their cytotoxicity using MTT assay indicated that almost all derivatives exhibited significantly superior inhibitory activity against hepatocellular carcinoma cell line HepG2 compared to the parental molecule sorafenib (1). Among the series, 5r was the most potent anti-HepG2 agent with IC50 = 1.04 µM, which was almost 5-fold more active than sorafenib (IC50 = 5.06 µM), while the cytotoxic activity against human embryonal lung fibroblast cell line MRC-5 remained comparable to sorafenib. The synthetic derivative 5r, thus, possessed 5.2-time higher selectivity index (SI) than that of sorafenib. Molecular docking studies revealed an efficient interaction of 5r at the same sorafenib's binding region in both B-Raf and VEGFR-2 with lower binding energies than those of sorafenib, consistent with its cytotoxic effect. Furthermore, 5r was proven to induce apoptosis in a dose-dependent manner similar to sorafenib. In addition, the prediction using SwissADME suggested that 5r possessed appropriate drug properties conforming to Veber's studies. These findings revealed that the newly designed meta-ureidophenoxy-1,2,3-triazole hybrid scaffold was a promising structural feature for an efficient inhibition of HepG2. Moreover, derivative 5r emerged as a promising candidate for further development as a targeted anti-cancer agent for hepatocellular carcinoma (HCC).

Identification of microRNA-96-5p as a postoperative, prognostic microRNA predictor in nonviral hepatocellular carcinoma.

AIM: The microRNA (miR) clusters miR-183/96/182 and miR-217/216a/216b are significantly upregulated in nonviral hepatocellular carcinoma (NBNC-HCC). Here, we investigate the impact of each member of these clusters on the clinical outcome of NBNC-HCC and analyze the antitumor effects of miR-96-5p. METHODS: The association between recurrence-free survival of 111 NBNC-HCC patients and the levels of miR-183-5p, miR-96-5p, miR-182-5p, miR-217-5p, miR-216a-5p, and miR-216b-5p in tumor and adjacent tissues was investigated. The impact of miR-96-5p on apoptosis and invasion of a hepatoma cell line, HepG2, was investigated by cell counting, Transwell assay, and flow cytometry, respectively. RESULTS: MicroRNA-183-5p, miR-96-5p, miR-182-5p, miR-217-5p, and miR-216b-5p were significantly upregulated in tumor tissues compared to the adjacent tissues (p = 0.0005, p = 0.0030, p = 0.0002, p = 0.0011, and p = 0.0288, respectively). By multivariate Cox regression analysis, high tumor/adjacent ratios of miR-182-5p (p = 0.007) and miR-217-5p (p = 0.008) were associated with poor recurrence-free survival. In contrast, a low tumor/adjacent ratio of miR-96-5p (p < 0.001) was associated with poor recurrence-free survival. It suggested that further upregulation of miR-96-5p in tumors might have an inhibitory effect on recurrence. Transfection of miR-96-5p mimic significantly induced apoptosis of HepG2 cells, in association with downregulation of Nucleophosmin 1 (NPM1) and a decrease of phosphorylated AKT protein. Interestingly, simultaneous knockdown of the NPM1 and AKT genes induced apoptosis. MicroRNA-96-5p also suppressed proliferation and invasion, which inhibited epithelial-to-mesenchymal transition of HCC cells. CONCLUSION: MicroRNA-96-5p as a tumor suppressor would be valuable to stratify NBNC-HCC patients at high risk of recurrence.

Electrochemical immunoassay for detection of hepatitis C virus core antigen using electrode modified with Pt-decorated single-walled carbon nanotubes.

Pt nanoparticles deposited on single-walled carbon nanotubes (PtSWCNTs), synthesized via the deposition precipitation (DP) method, were introduced as a substrate for immobilizing antibodies on an electrode surface and then enhancing the electrochemical sensitivity. A PtSWCNT-modified paper-based screen-printed graphene electrode was successfully developed to diagnose hepatitis C virus (HCV) infection. The hepatitis C virus core antigen (HCV-cAg) level was determined by differential pulse voltammetry (DPV) using [Fe(CN)6]3-/4- as a redox solution. In the presence of HCV-cAg, the DPV current response decreased with increasing HCV-cAg concentration. Under the optimal conditions, the change in current response provides a good linear correlation with the logarithm of HCV-cAg concentration in the range 0.05 to 1000 pg mL-1 (RSD < 5%), and the limit of detection was 0.015 pg mL-1 (or 0.71 fmol L-1). Furthermore, the proposed immunosensor has been utilized to quantify HCV-cAg in human serum samples with reliable results compared with standard immunoassays (% relative error < 10%). This sensor offers a simple, sensitive, selective, disposable, and inexpensive means for determination of HCV-cAg in human serum samples. The paper-based label-free immunosensor is versatile and feasible for clinical diagnosis.

Transcriptomic Analyses Reveal Long Non-Coding RNA in Peripheral Blood Mononuclear Cells as a Novel Biomarker for Diagnosis and Prognosis of Hepatocellular Carcinoma.

Novel biomarkers are highly required for the diagnosis and predicting prognosis of hepatocellular carcinoma (HCC). In this study, we investigated the profiles of long non-coding RNAs (lncRNAs) obtained from the peripheral blood mononuclear cells (PBMCs) of patients with HCC and PBMCs from a co-culture model using transcriptomic analysis. The differentially expressed lncRNAs (DElncRNAs) were then characterized and integrated as cancer-induced lncRNAs. Among them, three up-regulating DElncRNAs including MIR4435-2HG, SNHG9 and lnc-LCP2-1 and one down-regulating, lnc-POLD3-2, were identified. The functional analysis showed that these enriched lncRNAs were mainly associated with carcinogenesis and immune responses. Following further validation in PBMCs samples (100 HBV-related HCC, 100 chronic hepatitis B and 100 healthy controls), MIR4435-2HG, lnc-POLD3-2 and their combination were revealed to be sensitive biomarkers in discriminating HCC from non-HCC (AUROC = 0.78, 0.80, and 0.87, respectively), particularly among individuals with normal serum alpha-fetoprotein levels. Additionally, high circulating SNHG9 expression was shown to be an independent prognostic factor of overall survival in patients with HCC. These results indicate that determining these lncRNAs in PBMCs could serve as novel diagnostic and prognostic biomarkers for HBV-related HCC.

MicroRNA-223 Suppresses Human Hepatic Stellate Cell Activation Partly via Regulating the Actin Cytoskeleton and Alleviates Fibrosis in Organoid Models of Liver Injury.

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate target mRNA expression, and altered expression of miRNAs is associated with liver pathological conditions. Recent studies in animal models have shown neutrophil/myeloid-specific microRNA-223 (miR-223) as a key regulator in the development of various liver diseases including fibrosis, where hepatic stellate cells (HSCs) are the key player in pathogenesis. However, the precise roles of miR-223 in human HSCs and its therapeutic potential to control fibrosis remain largely unexplored. Using primary human HSCs, we demonstrated that miR-223 suppressed the fibrogenic program and cellular proliferation while promoting features of quiescent HSCs including lipid re-accumulation and retinol storage. Furthermore, induction of miR-223 in HSCs decreased cellular motility and contraction. Mechanistically, miR-223 negatively regulated expression of smooth muscle α-actin (α-SMA) and thus reduced cytoskeletal activity, which is known to promote amplification of fibrogenic signals. Restoration of α-SMA in miR-223-overexpressing HSCs alleviated the antifibrotic effects of miR-223. Finally, to explore the therapeutic potential of miR-233 in liver fibrosis, we generated co-cultured organoids of HSCs with Huh7 hepatoma cells and challenged them with acetaminophen (APAP) or palmitic acid (PA) to induce hepatotoxicity. We showed that ectopic expression of miR-223 in HSCs attenuated fibrogenesis in the two human organoid models of liver injury, suggesting its potential application in antifibrotic therapy.

Genetic variation in STAT4 is associated with treatment response to pegylated interferon in patients with chronic hepatitis B.

BACKGROUND: Signaling pathways in the STAT4 gene play an essential role in interferon-mediated antiviral effects. OBJECTIVE: This study was aimed at investigating the role of rs7574865, a single nucleotide polymorphism (SNP) in STAT4, in patients with chronic hepatitis B (CHB) treated with pegylated interferon (PEG-IFN). METHODS: A total 261 Thai patients (115 HBeAg-positive and 146 HBeAg-negative CHB) treated with 48-week PEG-IFN were recruited. Virological response (VR) at 48 weeks post treatment was defined as HBeAg seroconversion plus HBV DNA < 2,000 IU/mL for HBeAg-positive CHB and HBV DNA < 2,000 IU/mL for HBeAg-negative CHB. The SNP was analyzed by TaqMan PCR assay. RESULTS: The distribution of GG, GT and TT genotypes of rs7574865 was 41.8%, 42.9% and 15.3%, respectively. There was no different in its distribution according to HBeAg status. Overall, patients with TT genotype, compared with non-TT genotype, achieved higher VR (64.3% vs. 30.5%; P < 0.001) and HBsAg clearance (23.8% vs. 5.0%; P < 0.001). There was the same trend in the HBeAg-positive group (VR, 52.4% vs. 30.9%; P = 0.077; HBsAg clearance, 23.8% vs. 6.4%; P = 0.028) and in the HBeAg-negative group (VR, 68.4% vs. 32.3%; P = 0.004; HBsAg clearance, 21.1% vs. 4.7%; P = 0.026). Multiple regression analysis demonstrated that low baseline HBsAg level and TT genotype were factors independently associated with VR and HBsAg clearance. CONCLUSIONS: Our data support that SNP rs7574865 is associated with response to PEG-IFN therapy in Thai patients with CHB, regardless of baseline HBeAg status. Thus, the determination of this SNP could maximize cost-effectiveness of PEGIFN in patients with CHB.

Improvement of Gut Diversity and Composition After Direct-Acting Antivirals in Hepatitis C Virus-Infected Patients With or Without Human Immunodeficiency Virus Coinfection.

BACKGROUND: The influence of direct-acting antivirals (DAAs) on the composition of gut microbiota in hepatitis C virus (HCV)-infected patients with or without human immunodeficiency virus (HIV) is unclear. METHODS: We enrolled 62 patients with HCV monoinfection and 24 patients with HCV/HIV coinfection receiving elbasvir-grazoprevir from a clinical trial. Fecal specimens collected before treatment and 12 weeks after treatment were analyzed using amplicon-based 16S ribosomal RNA sequencing. RESULTS: Sustained virological response rates in the monoinfection and coinfection groups were similar (98.4% vs 95.8%). Pretreatment bacterial communities in the patient groups were less diverse and distinct from those of healthy controls. Compared with HCV-monoinfected patients, HCV/HIV-coinfected individuals showed comparable microbial alpha diversity but decreased Firmicutes-Bacteroidetes ratios. The improvement of microbial dysbiosis was observed in responders achieving sustained virological response across fibrosis stages but was not found in nonresponders. Responders with a low degree of fibrosis exhibited a recovery in alpha diversity to levels comparable to those in healthy controls. Reciprocal alterations of increased beneficial bacteria and reduced pathogenic bacteria were also observed in responders. CONCLUSIONS: This study indicates a short-term effect of direct-acting antivirals in restoration of microbial dysbiosis. The favorable changes in gut microbiota profiles after viral eradication might contribute toward the reduction of HCV-related complications among infected individuals.

Hepatitis B Core-Related Antigen to Indicate High Viral Load: Systematic Review and Meta-Analysis of 10,397 Individual Participants.

BACKGROUND & AIMS: To eliminate hepatitis B virus (HBV) infection, scale-up of testing and treatment in resource-limited countries is crucial. However, access to nucleic acid testing to quantify HBV DNA, an essential test to examine treatment eligibility, remains severely limited. We assessed the performance of a novel immunoassay, HBV core-related antigen (HBcrAg), as a low-cost (less than US $15/assay) alternative to nucleic acid testing to indicate clinically important high viremia in chronic HBV patients infected with different genotypes. METHODS: We searched Medline, Embase, Scopus, and Web of Science databases through June 27, 2018. Three reviewers independently selected studies measuring HBV DNA and HBcrAg in the same blood samples. We contacted authors to provide individual participant data (IPD). We randomly allocated each IPD to a derivation or validation cohort. We applied optimal HBcrAg cut-off values derived from the derivation set to the validation set to estimate sensitivity/specificity. RESULTS: Of 74 eligible studies, IPD were obtained successfully for 60 studies (81%). Meta-analysis included 5591 IPD without antiviral therapy and 4806 treated with antivirals. In untreated patients, the pooled area under the receiver operating characteristic curve and optimal cut-off values were as follows: 0.88 (95% CI, 0.83-0.94) and 3.6 log U/mL to diagnose HBV DNA level of 2000 IU/mL or greater; and 0.96 (95% CI, 0.94-0.98) and 5.3 log U/mL for 200,000 IU/mL or greater, respectively. In the validation set, the sensitivity and specificity were 85.2% and 84.7% to diagnose HBV DNA level of 2000 IU/mL or greater, and 91.8% and 90.5% for 200,000 IU/mL or greater, respectively. The performance did not vary by HBV genotypes. In patients treated with anti-HBV therapy the correlation between HBcrAg and HBV DNA was poor. CONCLUSIONS: HBcrAg might be a useful serologic marker to indicate clinically important high viremia in treatment-naïve, HBV-infected patients.

Amplification-free DNA Sensor for the One-Step Detection of the Hepatitis B Virus Using an Automated Paper-Based Lateral Flow Electrochemical Device.

Until now, an electrochemical lateral flow assay (eLFA) capable of detecting nucleic acids has remained a challenge and has been scarcely explored because of its complicated multistep nature. Here, we report an automated paper-based eLFA device for the quantitative detection of the hepatitis B virus (HBV)-the major cause of liver cirrhosis and hepatocellular carcinoma (HCC). Using a time-delayed microfluidic strategy fabricated on paper, an automated and precisely sequenced solution transfer was enabled by single sample loading. A gold metallization strategy was employed for the signal-on electrochemical detection of the target DNA. Furthermore, a pyrrolidinyl peptide nucleic acid (so-called "acpcPNA") was used as a probe in this study because it offers higher specificity and yields lower background currents than those of traditional probes. Under optimal conditions, a broad dynamic range (10 pM to 2 µM) with an excellent detection limit (down to 7.23 pM) was achieved. The overall operation can be completed within 7 min of sample loading. The proposed sensor was successfully applied in HBV DNA detection in sera from patients without any amplification step (e.g., PCR) required, thus simplifying the operation further. Additionally, the results obtained from this present device are in accordance with the standard real-time PCR, thus supporting the accuracy of the method.