JHEP Reports

Background: Metabolic adaptation is a recognized feature of therapeutic resistance in hepatocellular carcinoma (HCC), but it is unclear whether transcriptional states exposed during acquired resistance are restricted to drug adaptation or reflect broader aggressive tumor biology. We tested whether metabolic programs derived from a lenvatinib-resistance model identify a clinically adverse transcriptional state in an independent HCC patient cohort. Methods: The discovery framework was based on GSE186191, comprising parental and acquired lenvatinib-resistant Hep3B and Huh7 cells. A pre-specified 33-gene lipid-source ledger served as a biological anchor, and three discovery-derived programs, MYC Targets V2, mTORC1 Signaling, and Fatty Acid Metabolism, were frozen before patient-level evaluation. In TCGA-LIHC, single-sample enrichment scores for the three programs were population-standardized and summed to generate an integrated metabolic score. Overall survival was assessed by Kaplan-Meier and Cox analyses. Whole-transcriptome differences between high- and low-score tumors were characterized by preranked gene set enrichment analysis (GSEA). Results: The survival cohort comprised 282 patients (118 deaths), with 141 patients in each median-defined score group. High-score patients had shorter overall survival (log-rank P=0.000419). The continuous score was associated with mortality in univariable analysis (HR 1.86, 95% CI 1.33-2.61; P=0.000293) and in the frozen model adjusted for age, sex, and stage indicators (HR 1.93, 95% CI 1.35-2.76; P=0.000350; n=277). In 327 primary tumors, Fatty Acid Metabolism was strongly depleted in high-score tumors (NES -2.06; FDR<0.001). MYC Targets V2 (NES 1.18; FDR=0.232) and mTORC1 Signaling (NES 1.11; FDR=0.229) showed positive directional enrichment without FDR significance. Conclusions: A lenvatinib-resistance-derived transcriptional program is associated with an adverse-survival state in HCC. The strongest patient-level pathway feature is depletion of canonical fatty-acid metabolism, accompanied by directional MYC/mTORC1 features rather than statistically established pathway activation. These findings support a testable model of metabolic identity remodeling but do not establish causality or clinical prediction of lenvatinib response.

Background & Aims: Immune-mediated liver injury from immune checkpoint inhibitors (ILICI) is a major immune-related adverse event that limits cancer immunotherapy, yet its tissue-level immunobiology is poorly defined and its management is largely extrapolated from autoimmune hepatitis (AIH). We previously identified a tri-cellular CD8+ T cell-macrophage-hepatocyte injury niche in a murine model of ILICI; here, we tested whether this niche is recapitulated in human disease. Methods: We applied imaging mass cytometry with a 32-marker panel to liver biopsies from patients with ILICI (n = 12), AIH as a disease comparator (n = 14), and healthy controls (n = 2), profiling approximately 297,000 single cells across 144 regions of interest with spatially resolved detection of apoptosis (cleaved caspase-3, cC3) and pyroptosis (cleaved gasdermin D, cGSDMD). Results: We detected histiocyte-rich granulomas in ILICI consisting of macrophages and CD8+ T cells, including activated memory-effector subsets. Permutation-based spatial analysis identified CD8+ T cell-macrophage co-localization as the most frequent significant interaction in ILICI, organizing into integrated innate-adaptive cellular neighborhoods that concentrated cC3- and cGSDMD-positive cells. Descriptively, this contrasted with AIH, in which immune cells and stroma were more spatially compartmentalized. CD8+ T-cell and macrophage densities correlated with Ishak necroinflammation scores, jaundice, and granuloma formation. Conclusions: These findings provide the first single-cell spatial proteomic characterization of human ILICI in situ; they recapitulate the tri-cellular CD8-macrophage-hepatocyte niche we previously defined in a murine model and characterize ILICI as a spatially organized innate-adaptive inflammatory process, nominating myeloid signaling and CD8-macrophage interactions as candidate liver-directed targets to uncouple hepatotoxicity from anti-tumor immunity.

Background: Focal liver lesions (FLLs) encompass a wide spectrum of benign and malignant pathologies, and accurate diagnosis is essential for appropriate management. Although advances in imaging have improved lesion characterization, histopathologic assessment remains the diagnostic gold standard for indeterminate lesions. Data on the histopathologic spectrum and diagnostic utility of ultrasound-guided percutaneous liver biopsy (US-PLB) in sub-Saharan Africa (SSA) are limited. This study aimed to characterize the histopathologic findings of US-PLB performed for FLLs at a tertiary referral center in SSA and to identify factors associated with hepatocellular carcinoma (HCC). Methods: We conducted a retrospective observational study of adult patients ([&ge;]18 years) who underwent US-PLB for FLL between January 2021 and December 2024 at Adera Medical and Surgical Center. Patients with indeterminate pathology results, incomplete records, biopsies performed for diffuse liver disease, or lesions classified as LI-RADS 1, 2, or 5 were excluded. Demographic, clinical, laboratory, imaging, histopathologic, and outcome data were extracted from medical records. Descriptive statistics were used to summarize patient characteristics and histopathologic diagnoses. Logistic regression analysis was performed to identify factors associated with HCC. Results: A total of 119 were included in the final analysis. The median age was 56 years (IQR 45-65), and 59.7% were male. No major biopsy-related complications were reported. HCC was the most common histopathologic diagnosis, accounting for 42.9% of cases, followed by secondary metastatic tumors (15.9%) and regenerative nodules (15.9%). Other diagnoses included chronic hepatitis (8.4%), cholangiocarcinoma (5.9%), and hepatic abscess (3.4%). Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections were present in 14.3% and 12.4% of patients, respectively. On multivariate analysis, HBV infection (AOR 7.85, 95% CI 1.45-42.60; p=0.017), HCV infection (AOR 9.03, 95% CI 1.41-57.76; p=0.020), and larger tumor size (AOR 1.27, 95% CI 1.11-1.46; p<0.01) were significantly associated with HCC. Conclusion: Ultrasound-guided percutaneous liver biopsy demonstrated a favorable safety profile for the evaluation of FLL. HCC was the predominant histopathologic diagnosis, reflecting the substantial burden of primary liver cancer in this setting. Chronic viral hepatitis and larger tumor size were significantly associated with HCC. These findings support the continued role of US-PLB in the diagnostic evaluation of indeterminate focal liver lesions and underscores the importance of viral hepatitis prevention, surveillance, and early detection strategies in sub-Saharan Africa.

Background & Aims: Fatigue is a central symptom of chronic liver disease (CLD), substantially impacting health-related quality of life (HRQoL). This study aimed to further understand CLD symptomatology, including fatigue, and its impact on HRQoL from a patient perspective. Methods: Abbott Global Assessment of Patients unmet needs (aGAP) was a multinational, cross-sectional survey in adults with compensated CLD in China, India and Mexico, conducted between July and November 2024. Adult participants who self-reported that they had physician-diagnosed CLD and were experiencing fatigue completed a quantitative survey to assess symptom burden and included three HRQoL patient-reported outcome (PRO) questionnaires (Patient-Reported Outcomes Measurement Information System [PROMIS]-29+2, Work Productivity and Activity Impairment - Specific Health Problem version 2.0 [WPAI: SHP], Multidimensional Fatigue Inventory [MFI]). Results: Overall, 505 participants (China: 200; Mexico: 105; India: 200) completed the study. Participants reported that their CLD-related fatigue sometimes, often or always affected their self-esteem/confidence (45.1%) and ability to maintain or acquire new employment (38.6%). Most participants reported moderate (51.3%) or serious (26.9%) fatigue, with 33.5% experiencing fatigue every day or almost every day. Many participants felt their social life was negatively impacted by their fatigue (47.3%) and that there were related financial difficulties (53.9%). Use of validated PRO tools demonstrated severe fatigue (MFI: overall mean [SD] 13.9 [3.4] general fatigue and 13.4 [3.6] physical fatigue) as well as substantial levels of work and activity impairment (WPAI: SHP overall mean [SD] 53.0 [26.4]) and high levels of anxiety, pain interference, depression and sleep interference (PROMIS T-scores [&ge;]54). Conclusions: Fatigue has a substantial impact on HRQoL among adults with CLD across several countries, highlighting a global unmet need for targeted interventions to effectively identify and manage the condition.

Alpha-1 antitrypsin deficiency (AATD) caused by the Z variant leads to hepatic accumulation of misfolded AAT polymers and liver disease. Although proteotoxic stress is well established, its impact on lipid metabolism, mitochondrial function, and organelle homeostasis remains incompletely understood. The effects of Z-AAT accumulation were investigated in Z-HepG2 cells and 3D patient-derived ZZ hepatic organoids through protein aggregation, lipid storage, mitochondrial structure and function, peroxisomal dynamics, and comprehensive transcriptomic and proteomic analyses. Z-AAT expression led to intracellular polymer accumulation and reduced secretion, together with lipid accumulation, mitochondrial structural abnormalities, increased mitochondrial number but impaired respiratory capacity. Metabolic profiling revealed reduced oxidative phosphorylation and partial reliance on glucose metabolism. Peroxisomes displayed increased mass, consistent with altered lipid handling. Multi-omics analysis demonstrated widespread transcriptional and proteomic reprogramming related to protein synthesis, lipid metabolism, and mitochondrial function. Proteomic analysis confirmed proteotoxic stress-induced mitochondrial dysfunction, impaired lipid handling, and activation of stress response, inflammatory and vesicular trafficking pathways. Importantly, lipid supplementation elicited adaptive mitochondrial transcriptional responses in control cells, whereas Z-HepG2 cells showed a blunted response to lipid challenge. In conclusion, Z-AAT accumulation disrupts hepatic lipid processing and impaired mitochondrial and peroxisomal homeostasis, producing diminished metabolic flexibility likely contributing to AATD-associated liver disease.

Background: To date, no precision oncology approach has been established for HCC. Despite the diverse underlying causes, HCC development exhibits a uniform pathophysiology characterised by chronic hyper-proliferation, resulting from hepatocyte apoptosis and compensatory liver regeneration. This chronic hyper-proliferative pressure, termed regeneration stress, drives genomic instability during HCC onset, yet its therapeutic potential remains poorly explored. This study aimed to identify targetable vulnerabilities tied to regeneration stress and establish clinically applicable markers for treatment stratification. Methods: Weighted gene co-expression network analysis (WGCNA) was applied on external bulk RNA-seq datasets to define a LIVer REgeneration Stress Signature (LIVRESS). The signature was functionally validated using HCC patient-derived organoids (HCC-Org), and vulnerabilities were mapped using mid-throughput drug screening, single-molecule and single-cell assays, and multi-omic integration. Results: High LIVRESS scores, characterised by enrichment in replication, mitotic and DNA damage repair pathways, identified a subset of HCC patients with aggressive disease and poorer survival across aetiologies. HCC-Org with high LIVRESS scores displayed exquisite sensitivity to multiple inhibitors of the checkpoint kinase ATR. Although HCC-Org models exhibited a baseline reduction in replication fork speed, sensitivity to ATR inhibitor (ATRi) was decoupled from replication fork dynamics and rather linked to intrinsic mitotic instability. ATR inhibition triggers mitotic failure and apoptosis in LIVRESSHigh HCC-Org. This killing effect was significantly potentiated by combining ATRi with PARPi or WEE1i. Multi-omic integration identified KPNA2 as a surrogate biomarker of ATRi sensitivity. Conclusion: Our findings demonstrate that a subset of HCC-Org, characterised by high liver regeneration-associated stress, is vulnerable to ATRi-based therapies. By focusing on a comprehensive regenerative stress model, we establish a framework to stratify HCC patients and implement biomarker-driven, ATR-based therapies for HCC patients with advanced disease. Impact and implications: Regeneration stress is a key factor that drives genomic instability in HCC, providing a basis for the LIVRESS to identify patients dependent on ATR-mediated checkpoints. These findings reveal a conceptual shift for researchers and trialists: ATRi efficacy is decoupled from replication fork dynamics and instead leverages mitotic fragility. Practically, the LIVRESS and its IHC surrogate marker (KPNA2) offer a scalable roadmap for physicians to improve patient stratification in ATRi-based precision oncology trials. While requiring prospective validation, these results pave the way toward biomarker-driven therapies for advanced HCC.

Alcohol exposure perturbs intracellular calcium (Ca2+) homeostasis in digestive organs, yet whether common or organ-specific mechanisms coordinate this response remains unclear. Using an acute ethanol paradigm in zebrafish, single-cell transcriptomics revealed broad up-regulation of Ca2+-signaling genes in hepatocytes and pancreatic acinar cells. In vivo Ca2+ buffering with SpiCee, a genetically encoded chelator, demonstrated a shared requirement for Ca2+ flux: in hepatocytes, lineage-restricted buffering was associated with pronounced cytoplasmic vacuolation composed of lipid-negative vesicles, consistent with stalled lysosomes or autophagosomes; in pancreatic acinar cells, it was associated with accumulation of aggregated/misfolded protein. Mechanistic experiments using pharmacological inhibitors implicated distinct molecular contributors in each tissue. In hepatocytes, inhibition of Pikfyve or its downstream effector, the lysosomal Ca2+ channel TRPML1, phenocopied Ca2+ buffering. While, in acinar cells, Pick1 inhibition produced analogous associations. These data position Pikfyve and Pick1 as organ-specific components linked to the Ca2+-coupled alcohol response. Notably, pharmacologic activation of TRPML1 in hepatocytes recapitulated alcohol-like Ca2+ dynamics but increased macrophage recruitment and cell death, indicating that Ca2+ signaling is required for the alcohol response yet can be detrimental when amplified. Together, our results support a model in which alcohol elicits a shared Ca2+ dynamics across liver and pancreas, modulated by tissue-specific molecular nodes.

More than two million deaths annually are attributed to liver-related conditions, making primary human hepatocytes (PHH) an invaluable in vitro model for studying liver pathophysiology and the molecular mechanisms underlying hepatic diseases. However, because PHH do not proliferate in culture, CRISPR gene editing has been highly inefficient. Here, we report lipofection- and lentivirus-mediated protocols for CRISPR-Cas9 delivery in mouse-passaged primary human hepatocytes (mpPHH), a system that enables PHH expansion in liver-humanized mice. We achieve robust gene editing efficiencies exceeding 90% in mpPHH while maintaining cell viability. We demonstrate the utility of these protocols by disrupting CYP3A4 to impair xenobiotic metabolism and by showing that edited mpPHH efficiently engraft and expand in mice, generating liver-humanized animals. We establish the feasibility of arrayed CRISPR screening in mpPHH using an 85-gene screen to identify host factors influencing hepatitis B virus (HBV) infection, and validate key findings in humanized mice by targeting the HBV entry receptor SLC10A1 (NTCP), which reduced viral infection in vivo. Our methodology enables scalable genetic manipulation of mpPHH, opening new avenues for HBV research and liver disease modeling.

Background: The rising burden of metabolic dysfunction-associated steatotic liver disease (MASLD)-associated hepatocellular carcinoma (HCC) underscores the need for innovative therapeutic strategies. Methods: We integrated RNA-seq fusion detection, immunopeptidomics, and proteogenomics to systematically prioritize tumor-specific neoantigen candidates arising from gene fusions in MASLD-HCC. Results: We elucidated a landscape of private, clonally expressed fusions, and identified a previously unrecognized class of predicted phosphorylated fusion-neoepitopes. Cross-tumor proteomic analysis revealed that these phospho-motifs are present across malignancies, providing a broader context for their biological relevance. Importantly, fusion-positive tumors display immunosuppressive microenvironments, highlighting the need for future therapeutic strategies that combine fusion-targeted immunotherapy with approaches that overcome T-cell dysfunction. Conclusions: This study establishes a discovery pipeline and publicly available resource for fusion-derived phospho-neoepitopes in MASLD-HCC. The identified candidates provide a prioritized framework to guide and accelerate rigorous functional immunogenicity testing for future clinical validation.

Hepatocellular Carcinoma (HCC) arising from Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is an increasing public health burden with high mortality, highlighting the need for improved early detection strategies. Current surveillance tools, including Alpha-fetoprotein (AFP) and ultrasound, lack sufficient sensitivity for early-stage HCC detection. We analyzed serum samples from 131 patients, including 58 with cirrhosis and 73 with MASLD-related HCC (42 early-stage, 31 late-stage), using an nLC-stepped HCD-PRM-MS/MS workflow for targeted N-glycome profiling of glycopeptides derived from haptoglobin and vitronectin. Combining targeted glycopeptides with AFP significantly improved HCC detection compared with AFP alone. The optimal panel for all HCC versus cirrhosis (AFP + VTNC_169_A2G2F0S1 + VTNC_242_A3G3F2S2) achieved an AUC of 0.859 and 76.7% sensitivity at 90% specificity. For early-stage HCC, AFP + HP_184_A3G3F1S3 + VTNC_169_A2G2F0S1 yielded an AUC of 0.890 with 66.7% sensitivity at 1% specificity. A SHAP-selected Gaussian Naive Bayes model based on seven molecular/glycopeptide features, without demographic variables, further improved performance, achieving ROC-AUC values of 0.9985 in training and 1.0000 in independent testing cohorts, with accuracies of 98.1% and 100.0%, respectively.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent liver disorder worldwide, yet therapeutic options remain limited. TMPRSS6, a liver serine protease best known for its role in iron homeostasis, has recently emerged as a potential therapeutic target for MASLD. However, the molecular mechanisms linking TMPRSS6 to hepatic lipid metabolism remain incompletely understood. To identify novel TMPRSS6 substrates, we performed extracellular proteomic analyses of TMPRSS6-overexpressing cells. Among the proteins identified, {beta}-klotho (KLB), a co-receptor required for FGF19 and FGF21 signaling, emerged as a compelling candidate substrate. We demonstrate that TMPRSS6 interacts with KLB and promotes its proteolytic shedding in a catalytic activity-dependent manner. Functionally, TMPRSS6 reduced full-length KLB abundance at the cell surface and attenuated FGF19-dependent FGFR4 signaling in a heterologous expression system. Together, these findings identify KLB as a novel functional substrate of TMPRSS6, providing a mechanistic framework through which this protease may influence hepatic lipid metabolism. These results provide a rationale for investigating the regulation of KLB and other candidate substrates by TMPRSS6 in physiological models and further support its evaluation as a therapeutic target for MASLD.

Background The detrimental impact of alcohol consumption on the gut microbiome is well-established. However, less is known about how alcohol exposure during pregnancy affects the maternal gut and vaginal microbiota, or how these microbial changes relate to subsequent infant diagnosis of fetal alcohol spectrum disorder (FASD). We therefore investigated associations between self-reported alcohol use during pregnancy, infant FASD diagnosis, and maternal gut and vaginal microbiota. Methods Fecal samples (n = 207) and vaginal swabs (n = 28) from pregnant participants recruited through antenatal clinics in the Western Cape Province of South Africa were profiled by 16S rRNA V1-V2 amplicon sequencing. Maternal alcohol use was assessed using Alcohol Use Disorder Identification Test (AUDIT) scores, and FASD was diagnosed in infants using revised Institute of Medicine criteria. Microbial diversity, taxonomic profiles and PICRUSt2-predicted functional pathways were analyzed using vegan, phyloseq and MaAsLin3. Results Maternal AUDIT scores were negatively associated with maternal gut microbiota richness, Shannon, and Inverse Simpson diversity (p < 0.05). Observed richness was also reduced in participants whose infants were diagnosed with FASD (p = 0.046). Gut microbiota community structure was not significantly associated with alcohol use or infant FASD diagnosis. However, taxonomic and functional analysis suggested gram-positive taxa depletion with alcohol use and FASD diagnosis, and impaired one-carbon metabolism among participants with infants diagnosed with FASD. Vaginal microbiota diversity and composition were not associated with alcohol use or infant diagnosis. Conclusions This is the first human study to investigate the maternal gut and vaginal microbiota in relation to alcohol use during pregnancy and infant FASD outcomes. Our findings suggest that alcohol use is associated with maternal gut microbiota disruption, with potential implications for FASD development in exposed infants. Further investigation of alcohol-associated maternal microbial disturbances may inform microbiota-targeted strategies to improve maternal and infant health linked to alcohol use.

Excessive alcohol intake is frequently associated with hypertriglyceridemia, a condition that increases the risk of severe complications including acute pancreatitis and cardiovascular disease. The very low-density lipoprotein (VLDL) receptor (VLDLR) promotes uptake of apoE-containing VLDL particles by peripheral tissues and plays an important role in maintaining plasma triglyceride (TG) homeostasis. Brown adipose tissue (BAT) is a major metabolic organ that contributes to circulating lipid clearance during thermogenic activation. It was reported that cold-induced thermogenesis upregulates VLDLR expression in BAT and reduces plasma TG via VLDL uptake. However, whether BAT VLDLR-mediated VLDL uptake regulates alcohol-induced hypertriglyceridemia remains unknown. Here, we generated BAT-specific fatty acid synthase (FASN) knockout mice (FASNBKO) and subjected them to binge and acute-on-chronic alcohol feeding paradigms. We found that BAT FASN deficiency enhanced thermogenic function and promoted VLDL uptake, resulting in attenuation of alcohol-induced elevations in plasma TG. Consistent with these findings, pharmacological inhibition of FASN by TVB3664 treatment in differentiated brown adipocytes (bADs) increased thermogenic gene expression and VLDL uptake under both control and alcohol-exposed conditions. In addition, FASNBKO mice were protected from alcohol-induced hepatic steatosis, which was accompanied by increased hepatic AMP-activated-protein kinase (AMPK) activation and enhanced {beta}-oxidation. Furthermore, FASNBKO mice exhibited upregulated FGF21 mRNA expression in the BAT and elevated circulating FGF21 levels. Similarly, TVB3664-treated differentiated bADs showed higher FGF21 expression and increased FGF21 content in culture medium. Taken together, these findings identify the important role of brown adipocyte FASN in regulating thermogenic function and TG homeostasis during alcohol exposure and suggest that enhancing thermogenic lipid utilization in BAT may represent a potential therapeutic strategy for mitigating alcohol-associated increases in plasma TG and hepatic fat accumulation.

The liver is organized into tightly regulated zones with distinct metabolic functions but zonation erodes with age. Cellular senescence contributes to aging and liver diseases, however, its impact on aging biology is ill-defined. As part of The Cellular Senescence Network Consortium, we used multiple spatial transcriptomics approaches (GeoMx, Visium, CosMx) with snRNA-seq to profile senescence signatures, zonation markers, and metabolic pathways in livers from wild-type (WT) mice of multiple ages. We observed a loss of canonical zone signatures in aged mouse livers characterized by "expansion" of midlobular (zone 2) marker gene expression, accompanied by diminished expression of zone 3 marker genes by middle-age (18 months), indicative of loss of cell identity. Multiple analytic approaches identified distinct age-, zone- and sex-specific senescence signatures, which were significantly associated with zonation markers changes. This was recapitulated in Ercc1 mutant models of accelerated senescence, supporting a causal role of senescent cells in liver aging. A "no-zone" hepatocyte-like cluster expanded with age and with the strongest Senescence-Associated Secretory Phenotype (SASP) profile. Gene expression profiles from senescent hepatocytes implicate decreased WNT signaling and increased BMP as contributing to age-related loss of zonation. Together, these data elucidate the role of senescent cells in driving aging biology in non-diseased liver through disruption of cell:cell signaling and the loss of metabolic and cell identity gene expression necessary for hepatocyte function.

Background & AimsCurrent nucleos(t)ide analogs efficiently suppress hepatitis B virus (HBV) replication but have limited effects on viral transcription from covalently closed circular DNA (cccDNA) and integrated HBV DNA. We aimed to identify clinically applicable compounds that directly inhibit HBV transcription by screening FDA-approved drugs. Approach & ResultsScreening of 1,470 FDA-approved compounds using an HBV enhancer I/X promoter reporter system identified vorapaxar and aripiprazole as potent inhibitors of viral promoter activity. Both compounds suppressed HBV replication in HBV-producing cells, HBV-infected HepG2-hNTCP cells, and primary human hepatocytes. Aripiprazole reduced hepatocyte nuclear factor 4 (HNF4) protein levels through an ERK/JNK-dependent pathway and inhibited HBV core promoter activity, whereas vorapaxar acted independently of HNF4. Both compounds suppressed enhancer I/X promoter activity through inhibition of STAT3 signaling. Vorapaxar inhibited PAR-1-mediated SRC, EGFR, and STAT3 activation, while aripiprazole suppressed SRC-STAT3 signaling independently of EGFR. PAR-1 activation enhanced HBV transcription, whereas PAR-1 knockdown reduced promoter activity and viral RNA expression. Both compounds also reduced HBV replication in human liver chimeric mice at clinically relevant exposure levels without apparent severe toxicity. ConclusionsVorapaxar and aripiprazole suppress HBV transcription and replication through distinct host signaling pathways. These findings identify PAR-1-STAT3 signaling as a previously unrecognized regulator of HBV transcription and suggest that host-targeting approaches may complement current therapies by suppressing viral gene expression from both cccDNA and integrated HBV DNA. Impact and implicationsCurrent nucleos(t)ide analogues effectively suppress HBV reverse transcription but have limited effects on viral transcription from cccDNA and integrated HBV DNA, highlighting the need for therapies targeting viral gene expression. We identify PAR-1- STAT3 signaling as a previously unrecognized regulator of HBV transcription and demonstrate that two clinically approved drugs, vorapaxar and aripiprazole, suppress HBV replication through distinct host signaling pathways. These findings are relevant to researchers developing host-targeting antivirals and to clinicians seeking complementary therapeutic strategies beyond current nucleos(t)ide analogue therapy. Although further clinical validation and combination studies are required, our results provide a rationale for repurposing approved drugs and for developing transcription-targeting therapies that may complement existing treatments for chronic hepatitis B. HighlightsO_LIVorapaxar and aripiprazole suppress HBV through distinct host pathways. C_LIO_LIBoth drugs inhibit HBV replication in vitro and in humanized liver mice. C_LIO_LIPAR-1 inhibition reduces HBV transcription by blocking SRC/EGFR/STAT3 signaling. C_LIO_LIPAR-1-STAT3 signaling is a novel regulator of HBV transcription. C_LIO_LIHost-targeting antiviral therapy complements current HBV treatment. C_LI

Alcohol use disorder (AUD) disrupts the gut-liver-brain axis, yet mechanistically grounded and therapeutically actionable targets within this network remain poorly defined. To identify microbial modulators of alcohol-induced tissue pathology, longitudinal advanced diffusion MRI and fecal 16S rRNA profiling were integrated across Marchigian Sardinian alcohol-preferring rats evaluated at baseline, after four weeks of voluntary alcohol intake, and following six weeks of abstinence. Machine learning, specifically random forest models combining neuroimaging and microbiota data, improved phase classification and identified Akkermansia as the microbial feature most strongly associated with alcohol-related white matter microstructural abnormalities. Alcohol exposure induced widespread white matter alterations alongside gut dysbiosis characterized by reduced microbial diversity. To evaluate functional relevance, Akkermansia muciniphila was administered during the abstinence phase. Supplementation with A. muciniphila restored intestinal mucus, reduced liver injury markers, and elevated myelin basic protein levels within affected white matter regions. Collectively, these findings highlight Akkermansia as a critical modulator of alcohol-induced gut-liver-brain pathology and provide experimental support for a causal contribution of specific gut bacteria to persistent white matter damage in AUD. More broadly, this work establishes a robust multimodal framework for microbiome-based target discovery with clear translational relevance for disorders characterized by dysfunction along the gut-liver-brain axis. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSAlcohol use disorder (AUD) is associated with gut dysbiosis, impaired intestinal barrier function, liver injury, and persistent white matter abnormalities. Previous studies in patients and animal models have linked alcohol exposure to reduced microbial diversity, altered gut permeability, and white matter microstructural damage, particularly during abstinence. Other work has shown that microbiota-derived interventions can ameliorate peripheral consequences of alcohol exposure, especially in the gut and liver. However, the specific microbial features linked to alcohol-induced brain pathology remain poorly defined, and no prior study has integrated longitudinal microbiota and neuroimaging data to identify candidate microbial modulators of alcohol-related white matter damage and then functionally test them in vivo across the gut-liver-brain axis. Added value of this studyWe developed a multimodal framework that integrates longitudinal advanced diffusion MRI with fecal microbiota profiling and machine learning in alcohol-preferring rats. This approach identified Akkermansia as the microbial feature most strongly associated with alcohol-induced white matter abnormalities. Guided by this result, we administered Akkermansia muciniphila during abstinence and observed coordinated beneficial effects across multiple organs, including restoration of intestinal mucus, reduction of liver injury markers, and recovery of myelin basic protein in affected white matter regions. To our knowledge, this is the first study to combine longitudinal microbiota-MRI integration with experimental validation of a microbiota-based intervention that mitigates alcohol-induced pathology across the gut-liver-brain axis while restoring central white matter integrity. Implications of all the available evidenceOur findings support a mechanistic contribution of specific gut bacteria to persistent alcohol-induced tissue damage and identify Akkermansia as a candidate modulator of gut-liver-brain axis dysfunction in AUD. More broadly, this study establishes a generalizable strategy for integrating microbiota and neuroimaging data to discover biologically meaningful and therapeutically actionable targets in complex disorders involving coordinated peripheral and central pathology.

Background and aims Model systems for bile duct disorders are needed for testing therapeutic interventions. Current models have poor human relevance or limited potential for recreating the complex bile duct microenvironment at scale. We aimed to generate a humanized microphysiological system to model and treat cholangiopathies. Methods An in vitro bile duct was created using 3D printed microfluidic chips containing a collagen-embedded canal seeded with patient-derived primary human cholangiocytes. Barrier permeability and compound transport across the epithelium was measured, and disruption of the barrier was performed with lipopolysaccharide treatment. The duct was challenged with the known hepatotoxicant Chlorpromazine. Biliatresone was used to model biliary-atresia and treated using N-acetyl-L-cysteine. Results Cholangiocytes in the bile duct chip established a tight, polarized epithelial barrier. Verapamil and Linerixibat inhibited transport of rhodamine 123 and cholyl-lys-fluorescein respectively with 66 % (p = 0.0004) and 57 % (p = 0.03) reduction. 10 g/mL lipopolysaccharide led to a loss of epithelial barrier integrity, measured by an increase of over 1000 % in leakage of both 3 kDa (p = 0.0002) and 10 kDa dextran (p = 0.0001) along with upregulation of cytokines. Chlorpromazine displayed dose-dependent toxicity with EC50 values of 84, 140 and 96 M for three patient lines. Biliatresone induced a dose-dependent abnormal phenotype with loss of viability. The induced phenotype could be treated with N-acetyl-L-cysteine, improving viability from 23 % to 59 % (p < 0.0001) with treatment of 2 g/mL Biliatresone. Conclusions Our novel platform allows complex studies of bile duct biology, testing of off-target effects from drugs and treatment of a disease phenotype.

A missense variant in TM6SF2 (transmembrane 6 superfamily member 2, TM6SF2E167K) is a major risk factor for steatotic liver disease1, while protecting against coronary artery disease2. TM6SF2 is a polytopic resident protein of the smooth endoplasmic reticulum (ER) and ER-Golgi intermediate compartment that promotes lipidation of hepatic ApoB-containing lipoproteins before secretion into the circulation. Here, we used cryo-electron microscopy (cryo-EM) to determine the structures of TM6SF2 and TM6SF2E167K at 3.64 [A] and 3.58 [A] resolution, respectively. TM6SF2 comprises 10 transmembrane helices that bind a single cholesterol molecule within a transmembrane cavity. The protein assembles into homodimers and homotetramers that interact with ApoB. Structural and biochemical analyses show that the E167K substitution reduces cholesterol binding and ApoB interaction without disrupting overall protein structure. Expression of wild-type, but not mutant, TM6SF2 restores hepatic triglyceride secretion in TM6SF2-deficient hepatocytes. Together, these findings establish the first structural framework for the bulk lipidation step in hepatic lipoprotein biogenesis, the principal pathway for hepatic triglyceride and cholesterol export into the circulation.

Background Alcohol-associated liver disease (ALD) has emerged as a major cause of chronic liver disease and liver-related mortality in China. This study aimed to project the future burden of ALD in Chinese adults from 2020 to 2050, including prevalence of ALD, number of alcoholic steatohepatitis (ASH) cases, incident hepatocellular carcinoma (HCC) cases, liver transplantation (LT) demand, liver-related deaths, and disability-adjusted life years (DALYs). Methods We developed an agent-based state-transition microsimulation model with yearly cycles and a lifetime horizon. The model simulated 5,678,912 representative Chinese adults (mean age 36.2 years, 51.2% male). Health states included no steatosis, alcohol-associated steatotic liver, ASH, fibrosis stages F0-F4, decompensated cirrhosis, HCC, LT, and liver-related death. Model inputs were derived from the China Kadoorie Biobank, Global Burden of Disease Study 2021, China's national surveys, published meta-analyses, and transplant registry data. Projections incorporated demographic shifts, alcohol consumption trends, and calibrated transition probabilities. Uncertainty was assessed via 1,000 Monte Carlo simulations generating 95% uncertainty intervals. Results ALD prevalence was projected to increase from 4.8% (55 million individuals) in 2020 to 8.5% (94 million individuals) by 2050. ASH cases rose from approximately 18 million to 20 million. Annual incident HCC cases nearly doubled from 20,500 in 2020-2025 to 45,200 by 2046-2050. LT demand quadrupled from 2,300 to 9,800 cases. Liver-related deaths increased from 50,000 in 2020 to 85,000 in 2050, while DALYs rose from 1.5 million to 2.6 million. Conclusions In the absence of strengthened alcohol control policies, ALD will impose a substantial and growing burden on China's health system by 2050, with marked increases in HCC incidence, LT demand, and liver-related mortality.

Environmental enteropathy (EE) is a chronic, subclinical disorder of the small intestine common in low- and middle-income countries (LMICs), where access to sanitation and exposure to enteric pathogens vary greatly by socioeconomic status (SES). Systemic immune cell activation by enteric microbial exposure is a suspected but poorly characterized driver of EE severity. We hypothesised that adults from Low-SES communities would have more severe EE than adults from High-SES communities and that this would be associated with distinct circulating immune cell phenotypes. We enrolled clinically healthy adults from High- (n=26) and Low-SES (n=76) communities in Lusaka, Zambia. Duodenal biopsies from these adults were used for microscopic morphometry assessments, while plasma and stool biomarkers of epithelial damage, intestinal inflammation, microbial translocation, and systemic inflammation were measured by ELISA. Circulating monocyte, neutrophil and T cell phenotypes were characterised in buffy coat cells by flow cytometry. Compared with the High-SES group, adults from Low-SES communities had higher duodenal villus width and crypt depth and lower epithelial surface area, indicative of more severe EE pathology, and higher levels of plasma biomarkers associated with microbial translocation and systemic inflammation. The Low-SES group also had higher expression of activation markers (CD86 and TLR4) and lower expression of HLA-DR on circulating classical monocytes and neutrophils, higher percentages of gut-homing (4{beta}7+) and activated/exhausted (PD-1+) T cells, including gut-homing (4{beta}7+) regulatory T cells. Principal Component Analysis identified key patterns of immune cell phenotypes across SES groups. Confounder-adjusted linear regression models showed that Principal Component 1 (monocyte/neutrophil activation) was inversely associated with duodenal villus height and epithelial surface area across SES groups. These findings indicate that EE severity varies by SES within LMIC and suggest that monocyte and neutrophil activation is linked to greater duodenal remodelling in adults with EE.