Hepatology Communications

Background & Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a leading cause of chronic liver disease, and liver fibrosis is the principal determinant of disease progression and clinical outcome. We recently showed that non-apoptotic caspase-8 in hepatocytes is upregulated in MASH and leads to the induction and secretion of meteorin, which promotes hepatic stellate cell (HSC) activation and liver fibrosis. We address here the major mechanistic question arising from this study, namely how non-apoptotic caspase-8 induces pro-fibrotic meteorin in hepatocytes in MASH. Methods: Mechanistic studies were performed using AML12 hepatocytes; primary mouse and human hepatocytes; and diet-induced mouse models of MASH with hepatocyte-specific caspase-8 deletion. YY1 post-translational modifications were identified by LC-MS/MS and investigated using phosphorylation-defective and phosphomimetic YY1 mutants. Genetic and biochemical approaches were used to interrogate the caspase-8 DED-SRC-GSK3b; pathway, and its relevance was assessed in human MASH liver specimens. Results: In vitro mechanistic studies showed that the amino-terminal death effector domain (DED) of caspase-8 activated a SRC-GSK3b signaling cascade that promoted phosphorylation of YY1 at Ser247, resulting in YY1 nuclear localization and Metrn induction. Disruption of the conserved DED RXDLL motif impaired SRC activation, YY1 phosphorylation, and Metrn expression. Hepatocyte YY1 overexpression increased Metrn expression and fibrosis in wild-type MASH mice but not in hepatocyte-caspase-8-deficient mice. In contrast, phosphomimetic YY1-S247D restored these endpoints even when hepatocyte caspase-8 was lacking. Studies in primary mouse and human hepatocytes and human MASH liver specimens supported conservation of this signaling pathway. Conclusions: These findings identify YY1 Ser247 phosphorylation as a critical molecular event linking non-apoptotic hepatocyte caspase-8 signaling to METRN induction and MASH fibrosis and define a previously unrecognized DED-SRC-GSK3b-YY1 signaling axis with potential therapeutic relevance.

Background and aims: Cyclin E1 is a canonical regulator of cell cycle progression known to promote liver fibrosis and hepatocarcinogenesis. However, its precise contribution to hepatic immune responses remains poorly understood. We investigated whether Cyclin E1 regulates macrophage-associated inflammation and acute immune-mediated liver injury. Methods: Hepatic immune populations were analyzed in untreated and concanavalin A-treated wildtype and constitutive Ccne1-/- mice by flow cytometry. Liver injury was assessed by survival, histopathology, and caspase activation. Inflammatory mediators were quantified by qPCR and ELISA. Bone marrow-derived macrophages were examined during differentiation and pro-inflammatory polarization using flow cytometry, cytokine measurements, and RNA-sequencing. Lipid nanoparticle-mediated delivery of Ccne1-specific siRNA was evaluated in J774A.1 macrophage-like cells. Results: Under homeostatic conditions, Ccne1-/- mice exhibited reduced hepatic immune cell numbers, including Kupffer cells and CD4+ and CD8+ lymphoid cells. Following concanavalin A administration, Ccne1-/- mice showed improved survival, reduced hepatic necrosis, and diminished caspase-3 and caspase-8 activation. Protection was associated with reduced Kupffer and CD8+ lymphoid cell abundance and attenuated inflammatory mediator expression. Cyclin E1 deficiency did not substantially alter bone marrow-derived macrophage differentiation or cell cycle distribution. However, pro-inflammatory Ccne1-/- macrophages showed reduced interleukin-6 expression, attenuated inflammatory gene programs, and lower inferred IRF1 and NF-{kappa}B1 activity. Ccne1 knockdown similarly reduced interleukin-6 expression in pro-inflammatory J774A.1 cells. Conclusions: Cyclin E1 contributes to hepatic immune homeostasis and promotes macrophage-associated inflammatory signaling during immune-mediated hepatitis. Its inflammatory function is uncoupled from overt changes in macrophage cell cycle progression and may involve regulation of IRF1- and NF-{kappa}B-associated transcriptional programs. Targeting Cyclin E1 may therefore provide a strategy to attenuate selected inflammatory responses in liver disease.

Defining molecular patterns underlying pediatric end-stage liver disease and its treatment with liver transplantation requires rich datasets spanning a multitude of diagnoses and transplant outcomes. Here, we describe the spatial molecular landscape of approximately five million liver cell profiles from 98 liver tissue samples. The samples span donor liver, used here as a reference tissue, and end-stage diseases including biliary atresia, progressive familial intrahepatic cholestasis, hepatoblastoma, acute liver failure, and gestational alloimmune liver disease. Post-transplant liver allograft biopsies include those without and with T-cell-mediated and antibody-mediated rejection, mixed pathology, and outcome groups from immunosuppression withdrawal studies. Samples were profiled using the CosMx Human Universal Cell Characterization RNA Panel (1K) and the CosMx Human 6K Discovery Panel (6K). Preliminary exploratory analyses include cellular composition and gene abundance and variability across clinical cohorts. A twelve-gene signature comprising the genes with the highest variance implicates acute-phase response genes (e.g., SAA1) and profibrotic genes (e.g., COL1A1) as a shared link across these otherwise distinct conditions. This injury-repair-remodeling gene network warrants further investigation through higher-resolution, zone-specific spatial mapping to help clarify disease pathogenesis.

Alcohol-associated liver disease (ALD) is a leading cause of liver-related morbidity and mortality, yet its pathogenesis remains incompletely understood. While neural regulation is critical for hepatic homeostasis, how autonomic and neurotrophic signaling is regulated in the liver during ALD progression remains largely unexplored. Here, we performed an integrative analysis of bulk and single-cell transcriptomic datasets of human livers across the spectrum of ALD. We identified stage-dependent remodeling and coordinated expression patterns of autonomic and neurotrophic genes. Autonomic signaling exhibited a consistent decline, with reduced expression of sympathetic (DBH, TH) and the parasympathetic (ChAT) markers. In contrast, pan-neuronal and neuroplasticity-associated genes (UCHL1, TUBB3, STMN2) were upregulated, indicating active neural remodeling. In parallel, factors such as MANF, GDNF, and NRG2, with the receptor NTRK1, were upregulated and positively correlated with clinical indicators of disease severity, inflammation and fibrosis. In contrast, trophic factors including NTF3 and CDNF, and receptors such as GFRA1 and GFRA3, were downregulated and inversely associated with these parameters. Single-cell analyses revealed that neurotrophic factors are predominantly expressed by hepatic stellate cells, supporting their role as resident neuroglia-like cells in the liver. In another independent cohort, we observed a coordinated downregulation of NTF3 and sympathetic nerve markers in alcohol-associated hepatitis (AH), suggesting impaired NTF3-mediated neurotrophic support may contribute to sympathetic dysfunction in AH. Collectively, these findings uncover previously unrecognized neuro-regulatory dimensions in ALD and suggest that dysregulated autonomic and neurotrophic signaling may contribute to disease progression. Conflict of interestThe authors have declared that no conflict of interest exists.

Hospital readmission is used in cirrhosis research as if a return to hospital meant the disease had worsened: it is a trial outcome, a quality indicator and a prediction target. We asked what those readmissions were. Sixty-one adults with alcohol-related cirrhosis, selected because they had been hospitalized more than once, contributed all 326 later admissions to one department. A clinician read each full record and classified it for acute decompensation and for admission mode, blinded to diagnostic codes and structured flags. Fewer than two in five admissions (125, 38.3%) were decompensations. One in three (114) had been booked in advance; none of these was a decompensation, yet they used 29.1% of all bed-days. Among 124 returns within 30 days, planned admissions outnumbered decompensations, 48 to 43. Counting all admissions tracked a patients decompensations only moderately (correlation 0.50) whereas counting urgent admissions tracked them closely (0.93). In crude analysis the sicker patients had fewer admissions, not more, because planned care goes to those well enough to receive it. A liver severity score, computable for 165 admissions, did not separate planned from urgent admissions or decompensations from other stays, even within one patient. Readmission was not the same thing as decompensation.

The aim of this study was to determine whether the frequency of metabolic dysfunction-associated steatotic liver disease (MASLD) and the severity profile of inflammatory bowel disease (IBD) differ across body mass index (BMI) categories. A retrospective analysis was performed using 400 inpatient medical records from 2023-2024 with verified ulcerative colitis or Crohn's disease and sufficient information on anthropometry, hepatic status, and intestinal disease activity. The cohort comprised 118 patients with normal BMI, 176 with overweight, and 106 with obesity. MASLD status was reconstructed from routine clinical documentation and required hepatic steatosis in the appropriate cardiometabolic context. Ultrasonography was the principal source of evidence for steatosis; laboratory findings, non-invasive indices, and elastography were considered supportive when available. Ulcerative colitis activity was assessed with the Mayo Score and Crohn's disease activity with the Crohn's Disease Activity Index; disease-specific results were harmonized into mild, moderate, and severe categories for pooled comparisons. MASLD was identified in 181 of 400 patients (45.3%). Its frequency increased from 20.3% in the normal-BMI group to 49.4% in the overweight group and 66.0% in the obesity group (chi2=49.29; p<0.001). The largest absolute increase, 29.1 percentage points, occurred between normal BMI and overweight. The proportion of severe IBD rose in the same order, from 6.8% to 14.8% and 32.1% across the three BMI strata (chi2=39.45; p<0.001). In unadjusted pairwise comparisons, the odds of MASLD were higher with overweight than with normal BMI (OR=3.83; 95% CI 2.24-6.55) and higher with obesity than with normal BMI (OR=7.62; 95% CI 4.17-13.91); obesity also differed from overweight (OR=1.99; 95% CI 1.21-3.28). These findings demonstrate an ordered BMI-related pattern in which MASLD becomes more frequent and severe IBD represents a larger share of cases. However, MASLD was still documented in approximately one fifth of patients with normal BMI, indicating that BMI is useful for clinical prioritization but not for excluding steatotic liver disease. Because the study was retrospective, liver assessment reflected routine care, and the effect estimates were not adjusted for treatment exposure, disease duration, or other metabolic covariates, the observed relationships should be interpreted as associations rather than causal effects.

Reliance on invasive liver biopsy endpoints is a major limiting factor in new therapeutic development for metabolic dysfunction-associated steatohepatitis (MASH). While liver fat accumulation is considered a driver of early disease, liver fat reduction is not recognized as a surrogate endpoint by health authorities. Here, we conducted the largest-to-date genome-wide association study (GWAS) of liver fat in 79,642 people with magnetic resonance imaging and developed a polygenic score with liver fat-lowering alleles across 55 genomic loci. In a separate set of 714,886 individuals, a 30% relative liver fat reduction due to the polygenic score was associated with 39% lower odds of liver cirrhosis and 41% lower odds of hepatocellular carcinoma, consistent with a strong causal relationship between lifelong liver fat differences and advanced liver clinical outcomes. A systematic review and meta-analysis of 20 randomized controlled trials including a total of 3,502 MASH patients showed that treatment-induced liver fat reductions were associated with MASH resolution (meta-regression slope in % units of the outcome for each 1% relative reduction in liver fat from baseline, {beta} = -0.70%, p<0.001) and fibrosis improvement ({beta} = -0.24%, p<0.001). These findings demonstrate the profound etiologic impact of liver fat in MASH and provide compelling evidence for liver fat reductions as a reasonably likely surrogate endpoint for future clinical development.

Background: The gut-liver axis is involved in Metabolic dysfunction-Associated Steatotic Liver Disease (MASLD), but the host molecular actors involved in such dialogue have not yet been fully characterized. Objective: We investigated the sex-dependent role of the Constitutive Androstane Receptor (CAR) as a hepatic sensor of microbial signals in MASLD pathophysiology. Design: Male and female Car+/+ and Car-/- mice were fed a western diet (WD) for 4, 14 or 35 weeks. We performed plasma biochemistry, liver histology, bile acid and cholesterol quantification, untargeted multi-omics approach combining liver RNA-sequencing and 16S rRNA sequencing, together with co-housing, fecal microbiota transplantation and analysis of publicly available transcriptomics data in human cohorts. Results: Short-term WD revealed CAR-dependent protection against steatosis, together with perturbations in the transcriptional signatures of multiple transcription factors involved in hepatocyte identity in males. After 14 weeks, CAR deletion increased WD-induced liver damage, inflammation and fibrosis, and disrupted bile acid, cholesterol and gut microbiota homeostasis, in males only. Co-housing and fecal microbiota transplantations revealed that a Car+/+ gut microbiota protected against WD-induced liver damage, while a Car-/- microbiota was reshaped by a Car+/+ host. No CAR-dependent phenotype was observed in females. Finally, in human cohorts, hepatic CAR expression decreased in advanced clinical stages of MASLD. Conclusion: CAR is a sex-dependent protective nuclear receptor against MASLD progression, acting both as a transcriptional regulator of hepatocyte identity and as a mediator of the host-microbiota crosstalk.

BACKGROUND Primary sclerosing cholangitis (PSC) is characterized by multifocal biliary strictures on MRI/MRCP. However,we have observed that young PSC patients often displayed a marked intrahepatic bile duct visibility,sometimes with gallbladder enlargement, before developing biliary strictures. This study aimed to evaluatewhether this imaging pattern was associated with subsequent diagnosis of PSC. PATIENTS AND METHODS This retrospective two-center study included patients younger than 21 years who had a baseline liverMRI/MRCP, without biliary strictures. The endpoint was a definite diagnosis of large duct PSC during follow-up. The association of baseline imaging findings with the endpoint was evaluated by multivariable logisticregression, Cox models, and Kaplan-Meier analyses. RESULTS Ninety-four patients were included: 54% were women, the mean age was 16.7 years, and 23% had IBD. Overa mean follow-up of 7.75 years, 21 patients were subsequently diagnosed with large duct PSC after a mediantime of 5.60 years. Marked intrahepatic bile duct visibility and gallbladder enlargement displayed goodperformance to predict the subsequent diagnosis of PSC (AUC, 0.77 and 0.69, respectively). The presence ofboth signs displayed a specificity of 1.00. In multivariable analyses, marked intrahepatic bile duct visibility andgallbladder enlargement were independently associated with subsequent PSC diagnosis (OR 17.00 (95% CI,3.10-93.24; p = 0.001) and 16.08 (95% CI, 2.29-113.02; p = 0.005) respectively) and shorter time to diagnosis(HR 9.42 (95% CI, 2.50-35.59; p < 0.001) and 7.59 (95% CI, 2.03-28.41; p = 0.003) respectively). Survivalanalyses confirmed the association between these two imaging signs and the subsequent PSC diagnosis (HR7.78 (95% CI, 2.62-23.16; p < 0.001) and 3.85 (95% CI, 1.64-9.08; p < 0.001) respectively). CONCLUSIONS In young patients without biliary strictures, marked intrahepatic bile duct visibility and gallbladder enlargementon MRI/MRCP were strongly associated with subsequent diagnosis of large duct PSC.

Introduction: Adipose tissue fibrosis has been associated with metabolic dysfunction-associated steatotic liver disease (MASLD) and suboptimal weight loss after bariatric surgery. The objective was to characterize changes in adipose tissue fibrosis and extracellular matrix (ECM) components in adipose tissues from two abdominal fat compartments, subcutaneous and omental, in response to bariatric surgery; and determine the effect of weight loss on the association between adipose tissue fibrosis and MASLD feature changes with weight loss. Methods: Liver and adipose tissue biopsy samples were obtained before and after weight loss from patients who underwent two-stage bariatric surgery. Adipose tissue pericellular fibrosis was evaluated histologically with picrosirius red staining for collagen quantification and normalized to adipocyte count per microscope field. Transcript levels of ECM remodeling and inflammatory genes were measured by RNA sequencing in subcutaneous adipose tissue pre- and post-surgery. Results: At baseline, adipose tissue pericellular fibrosis did not associate with MASLD features, metabolic dysfunction-associated steatohepatitis or hepatic fibrosis in patients with severe obesity. Changes in liver histology after surgery were unrelated to changes in adipose tissue fibrosis. Transcripts for most genes involved in ECM remodeling and inflammation were downregulated in subcutaneous fat one year after bariatric surgery. Omental, but not subcutaneous, adipose tissue fibrosis decreased after surgery when adjusted for cell number (mean {+/-} SD: 0.41 {+/-} 0.12 vs 0.32 {+/-} 0.08 {micro}m2/cell, p<0.005). Higher subcutaneous adipose tissue fibrosis correlated with reduced weight loss (estimate {+/-} SE: -2.04 {+/-} 0.99, p<0.05) and BMI loss (-0.73 {+/-} 0.35, p<0.05), while increased omental adipose tissue fibrosis after weight loss was associated with worsening hepatic fibrosis (0.31 {+/-} 0.14, p<0.05). Conclusion: In individuals with severe obesity undergoing bariatric surgery, adipose tissue fibrosis is poorly correlated with MASLD features. Notably, pericellular fibrosis in subcutaneous abdominal, but not omental, adipose tissue was associated with a reduced weight-loss response to surgery.

Background. Activation of de novo lipogenesis (DNL) is a major hallmark of steatotic liver disease (SLD), a highly prevalent group of diseases ranging from a harmless steatosis to more severe steatohepatitis, cirrhosis, and hepatocellular carcinoma. We interrogated herein the early alterations in liver proteome, transcriptome, lipidome, inflammation, fibrosis, and oxidative status in a mouse model of progressive SLD induced by constitutive activation of PI3K-AKT signaling, the major inducer of DNL. Methods. Male, 8 weeks-old mice bearing phosphatase and tensin homolog (Pten) deletion and therefore constitutive activation of PI3K-AKT in hepatocytes (albumin-Cre) and littermate controls were evaluated for liver steatosis, injury, lipid peroxidation, metabolism, proteome, lipidome and transcriptome either upon feeding with a chow or high-fat diets supplemented or not with linoleic acid or after treatment with vehicle or acetyl-CoA carboxylase (ACC) inhibitor (ND-630, 20 mg/kg/day, i.p.) Results. Mice bearing Pten deletion and therefore constitutive activation of PI3K-AKT in hepatocytes display steatohepatitis characterized by enhanced DNL, severe steatosis, hepatocyte ballooning, inflammation, fibrosis and hepatomegaly, which, unexpectedly, was associated with reduced lipid peroxidation, increased GSH content and enhanced rates of triacylglycerol turnover, fatty acid {beta}-oxidation, tricarboxylic acid cycle flux, and mitochondrial respiration, altogether promoting a robust lipidome remodeling mainly defined by enrichment of DNL-derived fatty acids in detriment of a broad depletion of essential fatty acids (linoleic and -linolenic acids) and cardiolipins. Dietary restoration of liver linoleic acid content mildly attenuated liver inflammation, without however affecting lipid peroxidation and improving steatosis and fibrosis, whereas pharmacological acetyl-CoA carboxylase (ACC) and DNL inhibition markedly reduced steatosis, inflammation and fibrosis, despite further lowering hepatic linoleic acid content. Conclusions. Collectively, these findings support a harmful role of essential fatty acids depletion fomenting liver inflammation and identify DNL as a major culprit of steatohepatitis induced by constitutive PI3K-AKT activation.

Biliary Atresia (BA) is the leading cause of pediatric liver transplantation. Given the heterogeneity of the disease, outcomes after diagnosis remain unpredictable. A multiscale atlas of BA hepatic tissue established early lymphatic dysplasia in infants that later proceed toward liver failure and transplantation (severe BA). An architecturally distorted, immature and CCL21-high lymphatic vasculature marked severe BA, along with an expanding cholangiocyte compartment pivoting toward immune-reactive MMP7/SOX9-high states. Hepatocyte-to-biliary transdifferentiation emerged broadly across BA- and non-BA-cholestatic injuries, while dedifferentiation and acute-phase-reactions were specific to severe BA. Functionally, lymphatic endothelial tissue, isolated from severe BA livers, induced an analogous LGR5- and SOX9-positive epithelial progenitor response when assembled with cholangiocytes as organoids. Further experiments revealed that lymphatic endothelium signals ectopically on liver epithelium via CCL21-CCR7-axis after BA-specific inflammatory priming. CCL21-depletion strategies mitigated the BA-specific MMP7-program. In sum, a maladaptive lymphatic endothelial dysplasia perturbs intrahepatic epithelium in severe BA and offers CCL21 as a possible target for epithelial stability.

Hepatic stellate cells (HSCs) are the key cellular drivers of liver fibrosis. During liver injury and chronic inflammation HSCs adopt an activated phenotype and secrete fibrotic extracellular matrix (ECM) components such as collagen 1. Mechanical cues derived from the fibrotic ECM drive and support the activation of HSCs, via mechanisms that involve integrins and the mechano-sensitive transcriptional regulator YAP1. It is not yet well understood how external mechanical cues are translated into a molecular response that alters YAP1 nuclear shuttling. There is evidence that suggests the PDZ and LIM domain protein (PDLIM) 5 can regulate YAP1 shuttling in human epithelial cells. We therefore investigated whether PDLIM5 is expressed in HSCs and contributes to YAP1 associated HSC mechano-activation. PDLIM5 protein was detected in HSCs in fibrotic human and mouse liver. PDLIM5 transcript and protein were expressed by primary human and mouse HSCs and by the immortalised HSC LX-2 cell line. PDLIM5 localised with actin stress fibres suggesting a role in HSC adhesion. Co-immunoprecipitation and proximity ligation in LX-2 cells support an association between PDLIM5 and YAP1. We used pharmacological (paclitaxel) and genetic (siRNA and CRISPRi) approaches to inhibit PDLIM5 in HSCs. Inhibiting PDLIM5 reduced YAP1 nuclear localisation and fibrotic gene (COL1A1, ACTA2) expression in LX-2 cells. Overall, these data support a role for PDLIM5 in regulating YAP1 localisation and fibrogenic gene expression in HSCs.

Objective: Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) frequently present as overlap syndromes, characterized by shared sexual dimorphism and elevated intestinal Veillonella dispar (V. dispar) abundance, yet exhibiting distinct hallmark pathological manifestations. This study aimed to elucidate the shared pathogenesis, phenotypic differentiation mechanisms and sex-specific predisposition of AIH and PBC induced by V. dispar. Methods: Clean-grade and germ-free (GF) mice were employed to establish AIH and PBC models. Model induction involved oral gavage of V. dispar and intraperitoneal injection of either liver-specific antigen S100 (for AIH) or a pyruvate dehydrogenase complex E2 subunit (PDC-E2) mimetic peptide (for PBC). Single-cell RNA sequencing, metabolomics, transmission electron microscopy, western blotting, and flow cytometry were utilized to elucidate the underlying mechanisms. Results: V. dispar successfully induced autoimmune liver disease-like features in mice, characterized by significant intrahepatic B cell infiltration. Mechanistically, V. dispar caused systemic arginine reduction, leading to intrinsic apoptosis of gonadal stromal cells and a subsequent reduction in testosterone. This decreased testosterone weakened its inhibitory effect on B cell activating factor (BAFF) secretion, thereby amplifying B cell-mediated immune responses. Furthermore, in the presence of S100 or PDC-E2 mimetic peptide, V. dispar also facilitated Th17 cell activation, which guided the phenotypic differentiation into AIH or PBC, respectively. Consistent with these findings, our clinical observations revealed that patients with AIH and PBC tended to harbor enriched V. dispar, along with lower serum arginine and testosterone. In addition, elevated serum levels of BAFF and IL-17 were also observed in these patients. Conclusion: V. dispar induces reduced testosterone levels and upregulation of BAFF, leading to intrahepatic B cell expansion, which serves as the common pathogenic basis for AIH and PBC. The presence of S100 and PDC-E2 subsequently drives further maturation of these expanded B cells and promotes Th17 cell activation, thereby determining the phenotypic divergence between AIH and PBC.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a common, clinically heterogeneous condition associated with liver-related and cardiometabolic morbidity. Sex differences in MASLD prevalence and progression are well recognized, but how they extend across the liver and visceral adipose tissue (VAT), a major source of lipid and endocrine signals to the liver, remains unclear. Here, we profiled paired liver and VAT biopsies from women and men with and without MASLD, generating single-nucleus datasets of 34 liver samples and 40 VAT samples, including 32 patient-matched pairs. In liver, women with MASLD showed a higher Kupffer-cell triglyceride-catabolism score (1.30-SD female-male difference; FDR=0.0022), accompanied by directionally consistent hepatocyte and endothelial programs. In VAT, women with MASLD showed higher adipocyte lipid and oxidative metabolism (0.87-1.15 SD; FDR<0.05 across seven pathways) together with macrophage immune activation, whereas men with MASLD showed stronger stromal communication routes. Across matched patients, gene modules linked VAT adipocyte and macrophage states to liver hepatocyte stress and lipid-receipt states. Bulk RNA-seq module scoring and paired liver-VAT analyses in 293 patients supported hepatic stress in both sexes, lower adipocyte signaling in women with increasing steatosis, and cross-tissue adipocyte-hepatocyte coupling (r=0.30; FDR=2.1e-6). Together, these findings identify distinct sex-associated transcriptional states in liver and VAT in MASLD and show that these states are coordinated across both tissues within the same individuals.

Background: Hepatitis B virus (HBV) basal core promoter (BCP) and precore (PC) mutations occur during chronic HBV infection with the BCP-mutant being associated with increased hepatocellular carcinoma (HCC) risk. Objectives: The effects of these mutants on viral replication, hepatocyte biology, and carcinogenesis remain poorly defined. To address this, we characterized BCP- and PC-mutants using human hepatocyte chimeric mice and asked whether the resulting infection-induced transcriptomes correspond to subsets of human HBV-related hepatocellular carcinomas (HBV-HCCs). Design: Isogenic wild-type (WT), BCP- and PC-mutants of HBV genotypes A, C, and D (HBV-A, -C, -D) were generated from recombinant covalently-closed circular DNA to infect chimeric mice. HBV-HCC transcriptomic datasets from The Cancer Genome Atlas (TCGA) were used to stratify association of HBV variants with HCC subtypes. Results: WT, BCP-mutant, and PC-mutant HBV sequences remained genetically stable. The BCP mutation, but not the PC mutation, accelerated the rise in serum viremia in HBV-D- and HBV-A-infected chimeras; in HBV-C, acceleration required both (PC+BCP) mutations. Further comparisons of HBV-D variants revealed that the BCP-mutant increased intrahepatic viral DNA, viral protein expression, and upregulated cancer-related pathways, including transcripts associated with a subset of HBV-HCCs. Analysis of HBV-HCC samples from the TCGA revealed that tumors often harbor a mixture of WT and mutant transcripts, and that WT- and BCP-mutant-associated HCCs exhibit distinct transcriptional profiles. Conclusions: The HBV BCP-mutant directly perturbs hepatocyte homeostasis via virus-intrinsic mechanisms, selectively activating cancer-related pathways and defining a molecularly distinct subset of HBV-HCC. These findings suggest that HBV variants form distinct subcategories of HBV-HCCs.

Pancreatic ductal adenocarcinoma (PDAC) arises within an extensively remodeled microenvironment, yet tissue markers that distinguish invasive carcinoma from precursor lesions remain limited. Integrating four single cell RNA sequencing atlases comprising 324,662 cells from 26 patients, we identified cancer associated fibroblast expansion, accumulation of C1QA+ immunoregulatory macrophages, and depletion of a dendritic cell compartment expressing plasmacytoid markers. Fibroblasts were enriched for extracellular matrix and PI3K AKT programs and macrophages for an antigen-presentation program. We then examined the protein level correlates of these populations in a tissue proteomic reference framework of 356 samples spanning non diseased pancreas, normal-adjacent tissue, low and high grade intraductal papillary mucinous neoplasms (IPMNs), IPMN associated invasive carcinoma, and PDAC, prioritizing proteins that changed monotonically across the six histopathologic reference groups or marked tumor enriched cell types. Despite depletion of plasmacytoid dendritic cells, the principal source of type I interferon, the interferon stimulated protein ISG15, expressed by both fibroblasts and macrophages, increased monotonically toward PDAC. In exploratory cross-validated benchmarking of PDAC versus low and high grade IPMN, excluding IPMN-associated invasive carcinoma (145 versus 56 samples), CATB+EPIPL achieved an AUC of 0.98, with a sensitivity of 92% at an achieved specificity of 92%, exceeding protein abundance models of diagnostic IHC-associated markers (S100P+IMP3+maspin+MSLN, 0.79) and refitted published IPMN progression combinations (<0.91). Against non diseased pancreas, candidate and comparator panels performed similarly (AUC 0.99 1.00). These biologically informed tissue protein panels warrant independent validation, including immunohistochemical evaluation of EUS guided fine needle biopsy specimens and measurement in blood.

Epidemiological studies associate replacing animal-sourced with plant-sourced protein with lower colorectal cancer (CRC) risk, but controlled feeding studies have rarely tested how this substitution alters the gut microbiota and CRC-related biomarkers. In a 12-week randomized controlled feeding study, 136 healthy adults consumed diets providing 30 energy percent (E%), 50 E%, or 70 E% of protein from plant sources. Change in gut microbiota composition was the primary outcome; stool characteristics, fecal N-nitroso compounds (NOCs), and fecal and plasma bile acids (BAs) were secondary outcomes. The 70% plant-protein diet significantly restructured the gut microbiota, increasing fiber-associated Lachnospiraceae and Roseburia while reducing Methanobrevibacter and several asaccharolytic taxa. Microbial functional potential shifted toward plant-carbohydrate degradation, with lower amino acid degradation and methanogenesis. Fecal pH decreased, stool characteristics suggested faster colonic transit, and potentially carcinogenic total and haem-derived NOCs declined progressively with plant-protein replacement. The 70% plant-protein diet also reduced fecal secondary and taurine-conjugated BAs, including the CRC-associated BAs DCA and LCA. Plasma glucuronidated BAs increased, expanding the circulating pool of readily excreted BA conjugates. Higher insoluble fiber intake was associated with greater microbiota restructuring and lower fecal NOCs. Methanogen carriers had higher baseline fecal NOCs and features of slower colonic transit, and showed weaker microbiota and less favorable BA responses to the 70% plant-protein diet. Baseline methanogen carriage thus could be exploited for participant stratification in future dietary interventions.

The role of aberrant glycosylation in pancreatitis remains an under-investigated area of research. Here, we determined that the ST6GAL1 sialyltransferase, which adds 2-6-linked sialic acids to N-glycoproteins, was upregulated in pancreatic tissues from patients with acute (AP) and chronic (CP) pancreatitis, and in mice with experimental AP. Within these tissues, ST6GAL1 was selectively expressed in acinar cells undergoing acinar to ductal metaplasia (ADM), a process whereby injured acinar cells de-differentiate and re-enter the cell cycle to enable tissue repair. To study the functional role of ST6GAL1 in pancreatitis, we used HPNE pancreatic epithelial cells with modulated ST6GAL1 expression, along with pancreatic organoids from mice with transgenic expression of ST6GAL1 (SC mice). In these models, ST6GAL1 activity promoted the activation of EGFR (a well-known ADM-driver), ERK, and AKT. ST6GAL1-expressing cells also displayed an ERK-dependent upregulation of the anti-apoptotic proteins, Mcl-1 and Bcl-xL, and impaired apoptotic signaling by the TNFR1 and Fas death receptors. Unbiased kinomics profiling revealed that ST6GAL1 induced the activation of many receptor tyrosine kinases associated with cell survival and proliferation (e.g., EGFR, PDGFR, MET). Furthermore, ST6GAL1 enhanced the survival and proliferation of cells exposed to stress-inducing conditions such as serum withdrawal. Based on these findings, we hypothesized that the pro-survival phenotype imparted by ST6GAL1 would facilitate tissue healing following a bout of pancreatitis. Accordingly, we induced AP in wild type and SC mice via L-arginine injection and found that SC mice had more rapid and efficient tissue repair, evidenced by accelerated restoration of the acini, diminished acinar apoptosis, and reduced immune cell infiltration. Together, these findings highlight a novel glycosylation-dependent mechanism that drives cell survival, positioning ST6GAL1 as a key adaptive mediator of pancreatic regeneration.

PURPOSEPancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with poor prognosis. A transcriptomic-based algorithm called Purity Independent Subtyping of Tumors (PurIST) relies on expression profiling of a defined set of genes to stratify PDAC into classical and basal-like molecular subtypes and assigns a confidence level to the subtypes as either "strong," "likely," or "lean." In this study, we apply PurIST to Appalachian and non-Appalachian patient cohorts and assess demographic variables between groups that may influence treatment outcomes. PATIENTS AND METHODSThis 13-site study includes 780 PDAC patients. Patients were divided into Appalachian and non-Appalachian cohorts according to Appalachian Regional Commission guidelines, and differences in PurIST classification, tobacco use, and alcohol use were analyzed. RESULTSIndividuals from Appalachian regions were more likely to receive a subtype classification from PurIST with "strong" confidence as opposed to "likely" or "lean" (P=0.0175; odds ratio=1.66). We did not observe any significant difference between basal and classical subtype classification in Appalachia (P=0.1457), yet an exploratory analysis showed that individuals aged [&ge;]85 were significantly more likely to have basal-like tumor classifications (P=0.005817; odds ratio=3.79). Appalachian patients were more likely to have a history of smoking (59.6% vs 49.9%; P=0.01756) and less likely to report alcohol consumption (50.5% vs 65.6%; P=0.000229). CONCLUSIONWe conducted the largest comparison of the PurIST algorithm in Appalachia to date and found PurIST is more likely to have higher confidence in molecular subtype classifications for Appalachian PDAC tumors compared to non-Appalachian tumors. If PurIST is to be considered as a prognostic or decision-making tool for PDAC patients, we must account for variability in different populations and model the likelihood that tumors will have a strong molecular signature.