BACKGROUND:Hepatic ischemia/reperfusion injury (IRI) is a major complication occurring in hemorrhagic shock, liver resection, and transplantation. Big mitogen-activated kinase-1 (BMK1), a member of the mitogen-activated protein kinases family, is essential for regulating endothelial cell integrity and organ development. METHODS:We explored the putative role of BMK1 in human liver transplant recipients and in a murine model of partial warm liver IRI. RESULTS:Liver biopsies from adult transplant patients (n = 60) revealed that increased intrahepatic BMK1 expression was associated with well-preserved histology and improved graft function. In mice, the ischemia insult (90 min) induced hepatocellular BMK1 expression, peaking at 6 h of reperfusion. Inhibiting intrahepatic BMK1 (siRNA/chemically) made livers more vulnerable to IR stress, as evidenced by elevated serum alanine aminotransferase levels, damaged hepatic architecture, increased necrosis, and inflammation. Conversely, BMK1-overexpression protected livers from IR stress in a Krüppel-like factor 4 (KLF4)-dependent manner. Further in vivo and in vitro experiments confirmed that BMK1 overexpression upregulated hepatic yes-associated protein (YAP) mRNA/protein levels and promoted YAP dephosphorylation, whereas YAP silencing abrogated the protective effects of BMK1. The hypoxia-reoxygenation in vitro assay indicated that hepatocellular BMK1 regulated YAP function via itchy E3 ubiquitin protein ligase-related ubiquitination to large tumor suppressor homolog 1. Hypoxia-reoxygenation stress in BMK1-overexpressed hepatocytes resulted in increased intracellular itchy E3 ubiquitin protein ligase and decreased phosphorylated large tumor suppressor homolog 1, whereas no change in the phosphorylation level of mammalian sterile 20-like protein kinase-1. Furthermore, dual hepatocyte immunofluorescence staining revealed that BMK1 relied on the nuclear YAP-KLF4 cooperation, as hepatic deficiency of either BMK1, YAP, or KLF4 enhanced the severity of hepatic IRI. CONCLUSIONS:This study documents the crucial role/underlying mechanism of BMK1 signaling and points to a novel target to minimize hepatocellular dysfunction in IR-stressed liver transplantation.

Translating in situ dynamic changes of key signaling molecules into actionable clinical readouts remains a formidable challenge for noninvasive diagnostics. Here, focusing on reactive oxygen species (ROS) as pivotal signaling mediators, we developed defect-programmed DNA origami ROS sensors (DOSs) for portable urinalysis of localized oxidative stress. Using triangular DNA origami (DO) nanostructures as two-dimensional synthetic soft crystals, we programmed the number of discontinuity defects between adjacent staple strands and established a positive correlation between defect number and ROS-triggered degradation kinetics. To transform this programmable degradation into a diagnostic function, we then engineered DOSs via orthogonal assembly of targeting and signaling modules onto DO. In a murine model of acute liver injury (ALI), DOSs selectively accumulated in the liver and underwent ROS-triggered fragmentation into renal-clearable debris, converting hepatic ROS levels into quantifiable urinary signals. Notably, this transformation efficiency depended positively on defect number in DOSs, enabling portable urinalysis that detected ALI onset at least 4 h earlier than conventional alanine aminotransferase (ALT) testing, with a maximum area under the curve of 0.94. This defect-engineering strategy establishes a generalizable platform for early, noninvasive diagnosis of ROS-related diseases.

Mammalian organic solute transporter α/β (OSTα/β) is crucial for the enterohepatic circulation of bile acids and the homeostasis of steroid conjugates, mediating their movement across membranes as an obligate heterodimer. Here we present high-resolution cryo-EM structures of human OSTα/β in apo, substrate-bound and inhibitor-bound states, revealing a tetrameric organization as a homodimer of heterodimers that is required for membrane activity. Substrates bind within a surface-exposed tunnel formed by transmembrane helices 5 and 6, which is unexpectedly sealed by multiple palmitoyl chains covalently attached to a conserved intracellular loop IL2. Two chemically distinct inhibitors, fidaxomicin and ethinylestradiol, disrupt transport by both competing for the substrate-binding pocket and sterically occluding the tunnel. Together with biochemical and evolutionary analyses, our work defines a distinctive class of solute carriers that uses palmitoylation to facilitate substrate transport, a mechanism conserved across eukaryotes.

BackgroundHuman liver tissue-derived organoids recapitulate key hepatic phenotypes but are commonly maintained under static conditions, whereas microfluidic organ-on-chip systems provide controllable perfusion and mass transport. Scalable integration of human liver tissue-derived organoids into a perfused, human-relevant Liver-on-Chip remains limited.ResultsWe combined healthy human liver tissue-derived organoids with a high-throughput three-lane OrganoPlate microfluidic format to establish a perfused organoid Liver-on-Chip (HepLoC) featuring 3D luminal tubules under continuous flow. After hepatocyte-directed differentiation under perfusion, bioengineered HepLoC formed mature hepatocyte-like architectures with increased mature hepatocyte marker proteins, enrichment of hepatic transcriptomic signatures, and functional bile canaliculi. As a proof-of-concept for drug-induced liver injury, troglitazone induced dose-dependent hepatocyte injury accompanied by tight-junction disruption, MRP2 mislocalization, and impaired bile acid export, recapitulating key features of cholestatic liver injury. To model metabolic liver disease, free fatty acids triggered lipid droplet accumulation, increased triglycerides and reactive oxygen species, and upregulated lipogenic and inflammatory genes while largely preserving viability, consistent with early-stage metabolic dysfunction-associated fatty liver disease. The high-throughput HepLoC format further enabled parallel testing of reference hepatotoxic drugs and curcumin liposomes by reduced lipid accumulation in fatty-acid-treated HepLoC with minimal hepatotoxicity.ConclusionsOur perfused, organoid-based microfluidic Liver-on-Chip recapitulates essential human liver structure and function and enables integrated, parallel evaluation of hepatotoxicity and optimization of nanotherapeutic strategies, which deciphers the mechanisms of liver diseases, bridging the gap between preclinical research and clinical translation.

Lipid nanoparticles (LNPs) are the most advanced non-viral platform for hepatic mRNA delivery, yet the functional role of helper phospholipids, particularly under repeated dosing regimens required for chronic protein replacement therapies, remains poorly understood. Here, we systematically investigated six helper phospholipids varying in alkyl chain length, headgroup, and unsaturation, evaluating LNP properties and in vivo efficacy after single and repeated intravenous administration (weekly, biweekly, or triweekly). DSPE-based LNPs consistently achieved the highest mRNA expression in vitro and in vivo. Mechanistically, DSPE’s superior performance involves a two-step cooperative process: enhanced ApoE adsorption promoting cellular uptake and optimal pKa (6.6) for pH-responsive endosomal protonation. Notably, DSPC induced the highest IL-6 levels under the weekly dosing regimen, suggesting that vaccine-approved lipids may be suboptimal for low-inflammation therapies requiring frequent administration. Importantly, substituting DSPC with DSPE consistently enhanced expression across multiple ionizable lipid platforms (ALC-0315, MC3, and Lipid 5), and significantly improved PCSK9 gene editing efficiency as well as exogenous OTC protein expression. These findings redefine helper phospholipids as active regulators of repeated-dose mRNA delivery and identify DSPE as a promising candidate for chronic hepatic protein replacement.