Gasdermin D permeabilization of mitochondrial inner and outer membranes accelerates and enhances pyroptosis.

Gasdermin D (GSDMD)-activated inflammatory cell death (pyroptosis) causes mitochondrial damage, but its underlying mechanism and functional consequences are largely unknown. Here, we show that the N-terminal pore-forming GSDMD fragment (GSDMD-NT) rapidly damaged both inner and outer mitochondrial membranes (OMMs) leading to reduced mitochondrial numbers, mitophagy, ROS, loss of transmembrane potential, attenuated oxidative phosphorylation (OXPHOS), and release of mitochondrial proteins and DNA from the matrix and intermembrane space. Mitochondrial damage occurred as soon as GSDMD was cleaved prior to plasma membrane damage. Mitochondrial damage was independent of the B-cell lymphoma 2 family and depended on GSDMD-NT binding to cardiolipin. Canonical and noncanonical inflammasome activation of mitochondrial damage, pyroptosis, and inflammatory cytokine release were suppressed by genetic ablation of cardiolipin synthase (Crls1) or the scramblase (Plscr3) that transfers cardiolipin to the OMM. Phospholipid scramblase-3 (PLSCR3) deficiency in a tumor compromised pyroptosis-triggered anti-tumor immunity. Thus, mitochondrial damage plays a critical role in pyroptosis.

Operando studies reveal active Cu nanograins for CO2 electroreduction.

Carbon dioxide electroreduction facilitates the sustainable synthesis of fuels and chemicals1. Although Cu enables CO2-to-multicarbon product (C2+) conversion, the nature of the active sites under operating conditions remains elusive2. Importantly, identifying active sites of high-performance Cu nanocatalysts necessitates nanoscale, time-resolved operando techniques3-5. Here, we present a comprehensive investigation of the structural dynamics during the life cycle of Cu nanocatalysts. A 7 nm Cu nanoparticle ensemble evolves into metallic Cu nanograins during electrolysis before complete oxidation to single-crystal Cu2O nanocubes following post-electrolysis air exposure. Operando analytical and four-dimensional electrochemical liquid-cell scanning transmission electron microscopy shows the presence of metallic Cu nanograins under CO2 reduction conditions. Correlated high-energy-resolution time-resolved X-ray spectroscopy suggests that metallic Cu, rich in nanograin boundaries, supports undercoordinated active sites for C-C coupling. Quantitative structure-activity correlation shows that a higher fraction of metallic Cu nanograins leads to higher C2+ selectivity. A 7 nm Cu nanoparticle ensemble, with a unity fraction of active Cu nanograins, exhibits sixfold higher C2+ selectivity than the 18 nm counterpart with one-third of active Cu nanograins. The correlation of multimodal operando techniques serves as a powerful platform to advance our fundamental understanding of the complex structural evolution of nanocatalysts under electrochemical conditions.

Femtosecond laser writing of lithium niobate ferroelectric nanodomains.

Lithium niobate (LiNbO3) is viewed as a promising material for optical communications and quantum photonic chips1,2. Recent breakthroughs in LiNbO3 nanophotonics have considerably boosted the development of high-speed electro-optic modulators3-5, frequency combs6,7 and broadband spectrometers8. However, the traditional method of electrical poling for ferroelectric domain engineering in optic9-13, acoustic14-17 and electronic applications18,19 is limited to two-dimensional space and micrometre-scale resolution. Here we demonstrate a non-reciprocal near-infrared laser-writing technique for reconfigurable three-dimensional ferroelectric domain engineering in LiNbO3 with nanoscale resolution. The proposed method is based on a laser-induced electric field that can either write or erase domain structures in the crystal, depending on the laser-writing direction. This approach offers a pathway for controllable nanoscale domain engineering in LiNbO3 and other transparent ferroelectric crystals, which has potential applications in high-efficiency frequency mixing20,21, high-frequency acoustic resonators14-17 and high-capacity non-volatile ferroelectric memory19,22.

Disulfiram blocks inflammatory TLR4 signaling by targeting MD-2.

Toll-like receptor 4 (TLR4) sensing of lipopolysaccharide (LPS), the most potent pathogen-associated molecular pattern of gram-negative bacteria, activates NF-κB and Irf3, which induces inflammatory cytokines and interferons that trigger an intense inflammatory response, which is critical for host defense but can also cause serious inflammatory pathology, including sepsis. Although TLR4 inhibition is an attractive therapeutic approach for suppressing overexuberant inflammatory signaling, previously identified TLR4 antagonists have not shown any clinical benefit. Here, we identify disulfiram (DSF), an FDA-approved drug for alcoholism, as a specific inhibitor of TLR4-mediated inflammatory signaling. TLR4 cell surface expression, LPS sensing, dimerization and signaling depend on TLR4 binding to MD-2. DSF and other cysteine-reactive drugs, previously shown to block LPS-triggered inflammatory cell death (pyroptosis), inhibit TLR4 signaling by covalently modifying Cys133 of MD-2, a key conserved residue that mediates TLR4 sensing and signaling. DSF blocks LPS-triggered inflammatory cytokine, chemokine, and interferon production by macrophages in vitro. In the aggressive N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease (PD) in which TLR4 plays an important role, DSF markedly suppresses neuroinflammation and dopaminergic neuron loss, and restores motor function. Our findings identify a role for DSF in curbing TLR4-mediated inflammation and suggest that DSF and other drugs that target MD-2 might be useful for treating PD and other diseases in which inflammation contributes importantly to pathogenesis.

Topological defect-mediated morphodynamics of active-active interfaces.

Physical interfaces widely exist in nature and engineering. Although the formation of passive interfaces is well elucidated, the physical principles governing active interfaces remain largely unknown. Here, we combine simulation, theory, and cell-based experiment to investigate the evolution of an active-active interface. We adopt a biphasic framework of active nematic liquid crystals. We find that long-lived topological defects mechanically energized by activity display unanticipated dynamics nearby the interface, where defects perform "U-turns" to keep away from the interface, push the interface to develop local fingers, or penetrate the interface to enter the opposite phase, driving interfacial morphogenesis and cross-interface defect transport. We identify that the emergent interfacial morphodynamics stems from the instability of the interface and is further driven by the activity-dependent defect-interface interactions. Experiments of interacting multicellular monolayers with extensile and contractile differences in cell activity have confirmed our predictions. These findings reveal a crucial role of topological defects in active-active interfaces during, for example, boundary formation and tissue competition that underlie organogenesis and clinically relevant disorders.

Large Field-of-View Nonlinear Holography in Lithium Niobate.

A nonlinear holographic technique is capable of processing optical information in the newly generated optical frequencies, enabling fascinating functions in laser display, security storage, and image recognition. One popular nonlinear hologram is based on a periodically poled lithium niobate (LN) crystal. However, due to the limitations of traditional fabrication techniques, the pixel size of the LN hologram is typically several micrometers, resulting in a limited field-of-voew (FOV) of several degrees. Here, we experimentally demonstrate an ultra-high-resolution LN hologram by using the laser poling technique. The minimal pixel size reaches 200 nm, and the FOV is extended above 120° in our experiments. The image distortions at large view angles are effectively suppressed through the Fourier transform. The FOV is further improved by combining multiple diffraction orders of SH fields. The ultimate FOV under our configuration is decided by a Fresnel transmission. Our results pave the way for expanding the applications of nonlinear holography to wide-view imaging and display.

Directional Cell Migration Guided by a Strain Gradient.

Strain gradients widely exist in development and physiological activities. The directional movement of cells is essential for proper cell localization, and directional cell migration in responses to gradients of chemicals, rigidity, density, and topography of extracellular matrices have been well-established. However; it is unclear whether strain gradients imposed on cells are sufficient to drive directional cell migration. In this work, a programmable uniaxial cell stretch device is developed that creates controllable strain gradients without changing substrate stiffness or ligand distributions. It is demonstrated that over 60% of the single rat embryonic fibroblasts migrate toward the lower strain side in static and the 0.1 Hz cyclic stretch conditions at ≈4% per mm strain gradients. It is confirmed that such responses are distinct from durotaxis or haptotaxis. Focal adhesion analysis confirms higher rates of contact area and protrusion formation on the lower strain side of the cell. A 2D extended motor-clutch model is developed to demonstrate that the strain-introduced traction force determines integrin fibronectin pairs' catch-release dynamics, which drives such directional migration. Together, these results establish strain gradient as a novel cue to regulate directional cell migration and may provide new insights in development and tissue repairs.

Characterizing the stabilization effects of stabilizers in protein-protein systems with end-point binding free energy calculations.

Drug design targeting protein-protein interactions (PPIs) associated with the development of diseases has been one of the most important therapeutic strategies. Besides interrupting the PPIs with PPI inhibitors/blockers, increasing evidence shows that stabilizing the interaction between two interacting proteins may also benefit the therapy, such as the development of various types of molecular glues/stabilizers that mostly work by stabilizing the two interacting proteins to regulate the downstream biological effects. However, characterizing the stabilization effect of a stabilizer is usually hard or too complicated for traditional experiments since it involves ternary interactions [protein-protein-stabilizer (PPS) interaction]. Thus, developing reliable computational strategies will facilitate the discovery/design of molecular glues or PPI stabilizers. Here, by fully analyzing the energetic features of the binary interactions in the PPS ternary complex, we systematically investigated the performance of molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) and molecular mechanics generalized Born surface area (MM/GBSA) methods on characterizing the stabilization effects of stabilizers in 14-3-3 systems. The results show that both MM/PBSA and MM/GBSA are powerful tools in distinguishing the stabilizers from the decoys (with area under the curves of 0.90-0.93 for all tested cases) and are reasonable for ranking protein-peptide interactions in the presence or absence of stabilizers as well (with the average Pearson correlation coefficient of ~0.6 at a relatively high dielectric constant for both methods). Moreover, to give a detailed picture of the stabilization effects, the stabilization mechanism is also analyzed from the structural and energetic points of view for individual systems containing strong or weak stabilizers. This study demonstrates a potential strategy to accelerate the discovery of PPI stabilizers.

Tip-induced nanoscale domain engineering in x-cut lithium niobate on insulator.

Nanodomain engineering in lithium niobate on insulator (LNOI) is critical to realize advanced photonic circuits. Here, we investigate the tip-induced nanodomain formation in x-cut LNOI. The effective electric field exhibits a mirror symmetry, which can be divided into preceding and sequential halves according to the tip movement. Under our configuration, the preceding electric field plays a decisive role rather than the sequential one as in previous reports. The mechanism is attributed to the screening field formed by the preceding field counteracting the effect of the subsequent one. In experiment, we successfully fabricate nanodomain dots, lines, and periodic arrays. Our work offers a useful approach for nanoscale domain engineering in x-cut LNOI, which has potential applications in integrated optoelectronic devices.

Enhancing the Catalytic Efficiency of D-lactonohydrolase through the Synergy of Tunnel Engineering, Evolutionary Analysis, and Force-Field Calculations.

Computational design advances enzyme evolution and their use in biocatalysis in a faster and more efficient manner. In this study, a synergistic approach integrating tunnel engineering, evolutionary analysis, and force-field calculations has been employed to enhance the catalytic activity of D-lactonohydrolase (D-Lac), which is a pivotal enzyme involved in the resolution of racemic pantolactone during the production of vitamin B5. The best mutant, N96S/A271E/F274Y/F308G (M3), was obtained and its catalytic efficiency (kcat/KM) was nearly 23-fold higher than that of the wild-type. The M3 whole-cell converted 20 % of DL-pantolactone into D-pantoic acid (D-PA, >99 % e.e.) with a conversion rate of 47 % and space-time yield of 107.1 g L-1 h-1, demonstrating its great potential for industrial-scale D-pantothenic acid production. Molecular dynamics (MD) simulations revealed that the reduction in the steric hindrance within the substrate tunnel and conformational reconstruction of the distal loop resulted in a more favourable"catalytic" conformation, making it easier for the substrate and enzyme to enter their pre-reaction state. This study illustrates the potential of the distal residue on the pivotal loop at the entrance of the D-Lac substrate tunnel as a novel modification hotspot capable of reshaping energy patterns and consequently influencing the enzymatic activity.

Understanding the Structural Evolution of IrFeCoNiCu High-Entropy Alloy Nanoparticles under the Acidic Oxygen Evolution Reaction.

High-entropy alloy (HEA) nanoparticles are promising catalyst candidates for the acidic oxygen evolution reaction (OER). Herein, we report the synthesis of IrFeCoNiCu-HEA nanoparticles on a carbon paper substrate via a microwave-assisted shock synthesis method. Under OER conditions in 0.1 M HClO4, the HEA nanoparticles exhibit excellent activity with an overpotential of ∼302 mV measured at 10 mA cm-2 and improved stability over 12 h of operation compared to the monometallic Ir counterpart. Importantly, an active Ir-rich shell layer with nanodomain features was observed to form on the surface of IrFeCoNiCu-HEA nanoparticles immediately after undergoing electrochemical activation, mainly due to the dissolution of the constituent 3d metals. The core of the particles was able to preserve the characteristic homogeneous single-phase HEA structure without significant phase separation or elemental segregation. This work illustrates that under acidic operating conditions, the near-surface structure of HEA nanoparticles is susceptible to a certain degree of structural dynamics.

4-Hydroxyphenylacetate 3-Hydroxylase (4HPA3H): A Vigorous Monooxygenase for Versatile O-Hydroxylation Applications in the Biosynthesis of Phenolic Derivatives.

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) is a long-known class of two-component flavin-dependent monooxygenases from bacteria, including an oxygenase component (EC 1.14.14.9) and a reductase component (EC 1.5.1.36), with the latter being accountable for delivering the cofactor (reduced flavin) essential for o-hydroxylation. 4HPA3H has a broad substrate spectrum involved in key biological processes, including cellular catabolism, detoxification, and the biosynthesis of bioactive molecules. Additionally, it specifically hydroxylates the o-position of the C4 position of the benzene ring in phenolic compounds, generating high-value polyhydroxyphenols. As a non-P450 o-hydroxylase, 4HPA3H offers a viable alternative for the de novo synthesis of valuable natural products. The enzyme holds the potential to replace plant-derived P450s in the o-hydroxylation of plant polyphenols, addressing the current significant challenge in engineering specific microbial strains with P450s. This review summarizes the source distribution, structural properties, and mechanism of 4HPA3Hs and their application in the biosynthesis of natural products in recent years. The potential industrial applications and prospects of 4HPA3H biocatalysts are also presented.

Chemical and Structural Evolution of AgCu Catalysts in Electrochemical CO2 Reduction.

Silver-copper (AgCu) bimetallic catalysts hold great potential for electrochemical carbon dioxide reduction reaction (CO2RR), which is a promising way to realize the goal of carbon neutrality. Although a wide variety of AgCu catalysts have been developed so far, it is relatively less explored how these AgCu catalysts evolve during CO2RR. The absence of insights into their stability makes the dynamic catalytic sites elusive and hampers the design of AgCu catalysts in a rational manner. Here, we synthesized intermixed and phase-separated AgCu nanoparticles on carbon paper electrodes and investigated their evolution behavior in CO2RR. Our time-sequential electron microscopy and elemental mapping studies show that Cu possesses high mobility in AgCu under CO2RR conditions, which can leach out from the catalysts by migrating to the bimetallic catalyst surface, detaching from the catalysts, and agglomerating as new particles. Besides, Ag and Cu manifest a trend to phase-separate into Cu-rich and Ag-rich grains, regardless of the starting catalyst structure. The composition of the Cu-rich and Ag-rich grains diverges during the reaction and eventually approaches thermodynamic values, i.e., Ag0.88Cu0.12 and Ag0.05Cu0.95. The separation between Ag and Cu has been observed in the bulk and on the surface of the catalysts, highlighting the importance of AgCu phase boundaries for CO2RR. In addition, an operando high-energy-resolution X-ray absorption spectroscopy study confirms the metallic state of Cu in AgCu as the catalytically active sites during CO2RR. Taken together, this work provides a comprehensive understanding of the chemical and structural evolution behavior of AgCu catalysts in CO2RR.

Direct Observation of Transient Structural Dynamics of Atomically Thin Halide Perovskite Nanowires.

Halide perovskite is a unique dynamical system, whose structural and chemical processes happening across different timescales have significant impact on its physical properties and device-level performance. However, due to its intrinsic instability, real-time investigation of the structure dynamics of halide perovskite is challenging, which hinders the systematic understanding of the chemical processes in the synthesis, phase transition, and degradation of halide perovskite. Here, we show that atomically thin carbon materials can stabilize ultrathin halide perovskite nanostructures against otherwise detrimental conditions. Moreover, the protective carbon shells enable atomic-level visualization of the vibrational, rotational, and translational movement of halide perovskite unit cells. Albeit atomically thin, protected halide perovskite nanostructures can maintain their structural integrity up to an electron dose rate of 10,000 e-/Å2·s while exhibiting unusual dynamical behaviors pertaining to the lattice anharmonicity and nanoscale confinement. Our work demonstrates an effective method to protect beam-sensitive materials during in situ observation, unlocking new solutions to study new modes of structure dynamics of nanomaterials.

Fabrication of 100-nm-period domain structure in lithium niobate on insulator.

Lithium niobate on insulator (LNOI) is a powerful platform for integrated photonic circuits. Recently, advanced applications in nonlinear and quantum optics require to controllably fabricate nano-resolution domain structures in LNOI. Here, we report on the fabrication of stable domain structures with sub-100 nm feature size through piezoelectric force microscopy (PFM) tip poling in a z-cut LNOI. In experiment, the domain dot with an initial diameter of 80 nm and the domain line with an initial width of 50 nm can survive after a storage of more than 3 months. Particularly, we demonstrate the successful fabrication of 1D stable domain array with a period down to 100 nm and a duty cycle of ∼50%. Our method paves the way to precisely manipulate frequency conversion and quantum entanglement on an LNOI chip.

Purification and characterization of aspartic protease from Aspergillus niger and its efficient hydrolysis applications in soy protein degradation.

BACKGROUND: Adding acid protease to feed can enhance protein digestibility, boost feed utilization, and stimulate the growth of animals in breading industry. In order to obtain an acid protease with high hydrolysis efficiency to plant protein, in this study, an aspartic protease from Aspergillus niger was heterologous expressed in Pichia pastoris (P. pastoris). The enzymatic properties and application in soybean protein degradation were also studied. RESULTS: In our investigation, the high aspartic protease (Apa1) activity level of 1500 U/mL was achieved in 3 L bioreactor. After dialysis and anion exchange chromatography, the total enzyme activity and specific enzyme activity were 9412 U and 4852 U/mg, respectively. The molecular weight of the purified protease was 50 kDa, while the optimal pH and temperature were 3.0 and 50 °C, respectively. It was stable at pH 2.0-5.0 and 30-60 °C. Apa1 was used to hydrolyze soybean isolate protein (SPI) at 40 °C and pH 3.0, and a high hydrolysis degree (DH) of 61.65% was achieved. In addition, the molecular weight distribution of SPI hydrolysis products was studied, the result showed that the hydrolysis products were primarily oligopeptides with molecular weights of 189 Da or below. CONCLUSIONS: In this study, Apa1 was successfully expressed in P. pastoris and high expression level was obtained. In addition, the highest protein hydrolysis rate to SPI degradation so far was achieved. The acid protease in this study provides a new protease that is suitable for the feed industry, which will be very helpful to improve the feed utilization and promote the development of the breeding industry.

Fe(II) Oxidation Shaped Functional Genes and Bacteria Involved in Denitrification and Dissimilatory Nitrate Reduction to Ammonium from Different Paddy Soils.

Microbial nitrate reduction can drive Fe(II) oxidation in anoxic environments, affecting the nitrous oxide emission and ammonium availability. The nitrate-reducing Fe(II) oxidation usually causes severe cell encrustation via chemodenitrification and potentially inhibits bacterial activity due to the blocking effect of secondary minerals. However, it remains unclear how Fe(II) oxidation and subsequent cell encrustation affect the functional genes and bacteria for denitrification and dissimilatory nitrate reduction to ammonium (DNRA). Here, bacteria were enriched from different paddy soils with and without Fe(II) under nitrate-reducing conditions. Fe(II) addition decelerated nitrate reduction and increased NO2- accumulation, due to the rapid Fe(II) oxidation and cell encrustation in the periplasm and on the cell surface. The N2O accumulation was lower in the treatment with Fe(II) and nitrate than that in the treatment with nitrate only, although the proportions of N2O and NH4+ to the reduced NO3- were low (3.25% ∼ 6.51%) at the end of incubation regardless of Fe(II) addition. The dominant bacteria varied from soils under nitrate-reducing conditions, while Fe(II) addition shaped a similar microbial community, including Dechloromonas, Azospira, and Pseudomonas. Fe(II) addition increased the relative abundance of napAB, nirS, norBC, nosZ, and nirBD genes but decreased that of narG and nrfA, suggesting that Fe(II) oxidation favored denitrification in the periplasm and NO2--to-NH4+ reduction in the cytoplasm. Dechloromonas dominated the NO2--to-N2O reduction, while Thauera mediated the periplasmic nitrate reduction and cytoplasmic NO2--to-NH4+ during Fe(II) oxidation. However, Thauera showed much lower abundance than the dominant genera, resulting in slow nitrate reduction and limited NH4+ production. These findings provide new insights into the response of denitrification and DNRA bacteria to Fe(II) oxidation and cell encrustation in anoxic environments.

Immobilization of Actinobacillus succinogenes on nano- and micro-fiber membranes for efficient and robust production of succinic acid.

This work aimed to study the efficiency of nano- and micro- fiber membranes in immobilizing Actinobacillus succinogenes CCTCC M2012036 for succinic acid production. Among the four kinds of electrospun nanofiber membranes of cellulose acetate, chitosan, poly(vinyl alcohol) (PVA) and chitosan-PVA, the cellulose acetate nanofiber membrane-immobilized cells performed the best with a succinic acid concentration and yield to be 27.3 ± 3.5 g/L and 70.9 ± 5.8%. The cell-immobilized viscose microfiber membrane presented good reuse stability, and 17 batches of fermentation without activity loss were realized with the highest succinic acid yield of 83.20%. A microfiber membrane bioreactor was further constructed with the cell-immobilized viscose microfiber membrane to perform fermentation on a larger scale, and the concentration, yield and productivity of succinic acid were 73.20 g/L, 86.50% and 1.49 g/(L⋅h) using a fed-batch strategy, which were 124.30%, 127.60% and 124.2% of those obtained in the traditional fermenter. This study provided an approach for improving the practicality of biological succinic acid production.

Microwave-Frequency Scanning Gate Microscopy of a Si/SiGe Double Quantum Dot.

Conventional transport methods provide quantitative information on spin, orbital, and valley states in quantum dots but lack spatial resolution. Scanning tunneling microscopy, on the other hand, provides exquisite spatial resolution at the expense of speed. Working to combine the spatial resolution and energy sensitivity of scanning probe microscopy with the speed of microwave measurements, we couple a metallic tip to a Si/SiGe double quantum dot (DQD) that is integrated with a charge detector. We first demonstrate that the dc-biased tip can be used to change the occupancy of the DQD. We then apply microwaves through the tip to drive photon-assisted tunneling (PAT). We infer the DQD level diagram from the frequency and detuning dependence of the tunneling resonances. These measurements allow the resolution of ∼65 µeV excited states, an energy consistent with valley splittings in Si/SiGe. This work demonstrates the feasibility of scanning gate experiments with Si/SiGe devices.

PBMC transcriptomics identifies immune-metabolism disorder during the development of HBV-ACLF.

OBJECTIVE: Hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) pathophysiology remains unclear. This study aims to characterise the molecular basis of HBV-ACLF using transcriptomics. METHODS: Four hundred subjects with HBV-ACLF, acute-on-chronic hepatic dysfunction (ACHD), liver cirrhosis (LC) or chronic hepatitis B (CHB) and normal controls (NC) from a prospective multicentre cohort were studied, and 65 subjects (ACLF, 20; ACHD, 10; LC, 10; CHB, 10; NC, 15) among them underwent mRNA sequencing using peripheral blood mononuclear cells (PBMCs). RESULTS: The functional synergy analysis focusing on seven bioprocesses related to the PBMC response and the top 500 differentially expressed genes (DEGs) showed that viral processes were associated with all disease stages. Immune dysregulation, as the most prominent change and disorder triggered by HBV exacerbation, drove CHB or LC to ACHD and ACLF. Metabolic disruption was significant in ACHD and severe in ACLF. The analysis of 62 overlapping DEGs further linked the HBV-based immune-metabolism disorder to ACLF progression. The signatures of interferon-related, neutrophil-related and monocyte-related pathways related to the innate immune response were significantly upregulated. Signatures linked to the adaptive immune response were downregulated. Disruptions of lipid and fatty acid metabolism were observed during ACLF development. External validation of four DEGs underlying the aforementioned molecular mechanism in patients and experimental rats confirmed their specificity and potential as biomarkers for HBV-ACLF pathogenesis. CONCLUSIONS: This study highlights immune-metabolism disorder triggered by HBV exacerbation as a potential mechanism of HBV-ACLF and may indicate a novel diagnostic and treatment target to reduce HBV-ACLF-related mortality.