Biology subjects

Yu, H.

Publications and source records attributed to Yu, H..

At least 19 recordsLinked to original sources

bioRxiv2026

In situ loading of biologics on and tissue redirection of circulating extracellular vesicles with an anchoring nanobody

Extracellular vesicles (EVs) are promising nanocarriers of therapeutic macromolecules, but ex vivo production, cargo loading and quality control constrains clinical translation. Here we demonstrate that NEV3, an EV-anchoring nanobody identified by phage display, binds EVs from multiple sources through interactions involving fibronectin and loads diverse proteins onto EVs, resulting in enhanced intracellular delivery and biological activities in vitro and in vivo. Conjugating NEV3 to target proteins hijacks circulating EVs, prolonging circulation and altering tissue distribution in vivo. When fused with an GFP nanobody or protein A, NEV3 enabled simultaneous capture and loading of circulating GFP or immunoglobulin G (IgG) antibodies on endogenous EVs with enhanced intracellular delivery in vitro and redistribution to liver in vivo. Intravenous protein A-NEV3 significantly reduced circulating IgG and anti-dsDNA antibodies in systemic lupus erythematosus mice with improved survival and reduced pathologies. Intravenous anti-transferrin receptor-protein A-NEV3 redirected circulating EVs to muscle and alleviated muscle pathologies in dystrophin-deficient mdx mice. Our study demonstrates that NEV3 enables in situ loading of protein therapeutics and tissue-specific delivery of circulating EVs, providing a modular strategy for loading cargo onto and redirecting circulating EVs in vivo.

bioengineering↗

bioRxiv2026

Tumor SETDB1 expression predicts worse overall survival for bladder cancer patients

Aberrant activity of histone methyltransferases promotes tumorigenesis across multiple cancer types, making this class of enzymes attractive therapeutic targets. The histone methyltransferase SETDB1 is frequently amplified in bladder cancer and has been implicated in chemotherapy resistance. We examine whether SETDB1 protein expression is associated with overall survival and tumor characteristics in bladder cancer. To assess SETDB1 protein expression in patients, we performed immunohistochemistry in tissue microarrays (TMAs) containing 350 bladder cancer cases with well-annotated clinical information. Digital image analysis was used to quantify SETDB1 protein expression to produce a SETDB1 H-score, from which patients were categorized as SETDB1-high or SETDB1-low. Using these categories, we compared tumor characteristics, clinical outcomes, and gene expression profiles between SETDB1-high and SETDB1-low patients. Patients with high SETDB1 expression (n=143) had significantly shorter overall survival compared to SETDB1-low (n=125) patients (median survival months: SETDB1-high, 30.09; SETDB1-low, 87.85; p = 0.0006), and SETDB1 expression was independently associated with overall survival upon multivariate cox regression analysis (HR = 1.93, 95% CI = 1.08 - 3.45, p = 0.026). SETDB1-high patients were more likely to present with squamous (35% vs 20%, p = 0.026) and sarcomatoid (11% vs 3.6%, p=0.049) differentiation features, which are associated with poorer prognosis. Exploratory analyses identified transcriptional differences associated with SETDB1 expression, providing rationale for further exploration of the mechanisms by which SETDB1 may promote bladder cancer.

cancer biology↗

bioRxiv2026

A brain signature of vaping cue-elicited craving

E-cigarette use among young adults has grown rapidly, raising concerns about nicotine dependence and motivating a better understanding of the neurobiology of vaping. We used a cue-induced craving fMRI paradigm to develop and evaluate a brain signature of vaping cue-elicited craving, and to compare it with smoking- and food-craving signatures developed using the same analytic pipeline. Using LASSO-PCR with leave-one-subject-out cross-validation, we identified a distributed pattern of brain activity that reliably predicted normative vaping craving from an independent sample (hereafter, normative craving). The vaping-craving signature was also substantially distinct from smoking- and food-craving signatures, both spatially and functionally, and showed specificity relative to previously established craving signatures. Moreover, greater signature expression was associated with greater e-cigarette dependence severity and showed a positive, though modest, prospective association with daily puff counts over four weeks, demonstrating the signature's behavioral and clinical relevance. Together, these findings establish a cross-validated brain signature of vaping cue-elicited craving, demonstrate its differentiation from other craving-related neural patterns, and link its expression to e-cigarette dependence and real-world use. More broadly, they support the potential of this framework for developing reproducible neural markers of craving.

neuroscience↗

bioRxiv2026

Cytoplasmic localization of 5hmC reveals RNA hydroxymethylation in breast cancer

DNA methylation (5-methylcytosine, 5mC) is a major repressive epigenetic mark that can be iteratively oxidized by ten-eleven-translocation (TET) enzymes to 5-hydroxymethylcytosine (5hmC); beyond DNA, modifications of RNA can also affect transcript stability and processing, yet RNA hydroxymethylation remains unexplored in breast cancer (BC). Here, we identify a quantifiable cytoplasmic 5hmC signal in BC cell lines and, more prominently, in human BC tissue, using immunoassays and digital pathology, and confirm with an adapted, RNase-sensitive, RNA-specific ELISA that this signal reflects hydroxymethylation of total RNA and mRNA. Cytoplasmic 5hmC was significantly enriched in triple-negative (ER-/PR-/HER2-) and grade 3 tumors, and correlated with increased cytoplasmic TET2/TET3 in tissue and TET3 upregulation in TCGA. As a leading candidate mechanism, TET3 expression was associated with RNA-processing/spliceosomal transcripts, and TET3 knockdown in three TNBC cell lines reduced a subset of these targets, independently supported across 70 DepMap breast cancer lines.

cancer biology↗

bioRxiv2026

A molecular clutch governs stator engagement and flagellar torque

Motile bacteria must continuously adjust their motility to navigate and colonize diverse environments, including mammalian hosts, yet the underlying mechanisms remain poorly understood. Here, using in situ single-particle cryo-electron microscopy, we determine near-atomic structures of the flagellar motor in the Lyme disease spirochete Borrelia burgdorferi and uncover a novel mechanism for controlling torque generation. We identify a distinct neutral state in which individual torque-generating MotA5B2 stator complexes remain incorporated in the motor but adopt an extended, tilted conformation disengaged from the rotor. Two conserved flagellar proteins, FliL and FlcA, form a transmembrane scaffold that retains 16 stator complexes, while FapA, a newly identified multidomain flagellar protein, binds the cytoplasmic base of the stator complex and stabilizes its rotor-disengaged conformation. Strikingly, rotor-engaged and rotor-disengaged stators coexist within individual motors, demonstrating that stator retention and torque engagement are separable states. Our findings establish a motor-retained neutral stator state and reveal a molecular-clutch mechanism that independently controls the engagement of individual stator units with the rotor, enabling rapid and precise regulation of flagellar torque and motility.

microbiology↗

bioRxiv2026

In situ cryo-EM reveals how a flagellar motor transmits high torque

Bacterial flagellar motors are diverse rotary machines that require robust mechanical coupling to transmit torque across components with distinct architectures, yet how such coupling is achieved remains poorly understood. Here, we determine near-atomic in situ structures of the periplasmic flagellar motor in the Lyme disease spirochete Borrelia burgdorferi, revealing how an expanded rotor is mechanically coupled to the conserved rod through symmetry breaking and local remodeling of the rotor rod interface. Surprisingly, pentameric FrbA forms a split washer-shaped brace around the rotor rod junction, providing a second layer of mechanical reinforcement. Deletion of frbA destabilizes distal flagellar structures and impairs motility, particularly under increased viscous load. FrbA homologues are broadly distributed across spirochetes, suggesting a conserved mechanism for reinforcing high-torque flagellar motors. Together, these findings reveal how symmetry breaking couples an expanded rotor to a conserved driveshaft, while a molecular split washer reinforces this interface to enable robust torque transmission and motility.

microbiology↗

bioRxiv2026

Towards Robust Automated Mouse Scratching Behavior Detection for Itch Research

Automated detection of mouse scratching behavior could reduce manual annotation and is essential for preclinical itch research. However, robust performance across laboratories remains a challenge for current methods. We present Scratch-AID+, a framework dedicated to automated mouse scratching detection, including a diverse and progressive set of approaches: classic ResNet-based detectors, task-adaptive fine-tuning of video foundation models, and in-context learning with advanced multimodal large language models. To simulate real-world use cases, we benchmark on a public dataset and utilize additional cross-laboratory video recordings for independent testing. We find that classic baselines suffered severe performance degradation on external data under the realistic setting with sparse scratching events. In contrast, fine-tuned video foundation models successfully bridged this generalizability gap, achieving significantly better detection performance. Furthermore, frozen multimodal large language models demonstrated promising out-of-the-box ability using only few-shot demonstrations. We also examined the effects of different video preprocessing strategies. Our findings highlight the improved performance and generalizability of the adapted foundation models for automated scratching behavior analysis and shed light on the real-world deployment of such detectors. Our code is publicly available at https://anonymous.4open.science/r/scratch aid-plus/.

bioinformatics↗

bioRxiv2026

Decoupled recovery of soil physicochemistry, microbiome and methane sink function during secondary forest succession in the Eastern Himalayas

Secondary forests are increasingly important for biodiversity recovery and ecosystem functioning after disturbance. As vegetation regenerates, accompanying shifts in soil conditions and microbial communities regulate climate-relevant processes such as atmospheric methane (CH$_4$) uptake. Whether these belowground dimensions recover synchronously, however, remains unclear. Here we show that belowground recovery followed asynchronous trajectories across a forest chronosequence in Metok, Eastern Himalayas. Soil inorganic properties and microbial community composition converged progressively on the primary-forest state as soils acidified. By contrast, soil organic carbon, microbial diversity and abundance appeared to recover by 25 years but diverged again at 40 years. Co-occurrence network topology resembled the primary forest by 40 years, yet the keystone taxa underlying it were entirely stage specific. Atmospheric CH$_4$ uptake fluctuated: uptake recovered to primary-forest levels in 25-year stands but weakened in 40-year stands to rates comparable with the recently disturbed 5-year forest. Rather than canonical methane-cycling taxa, four amplicon sequence variants---three assigned to \textit{Mycobacterium}---tracked CH$_4$ flux closely while acting as hubs or connectors in the co-occurrence networks, warranting genomic and experimental work to resolve their function. Apparent recovery within the first 25 years therefore did not persist, cautioning against inferring restoration success from early succession alone and highlighting the need for longer chronosequences and repeated observation to resolve the dynamics of secondary-forest recovery.

microbiology↗

bioRxiv2026

Magel2 deficiency drives microglial synaptic engulfment and alters neuron-microglia interactions in Prader-Willi syndrome

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder characterized by hypothalamic dysfunction, developmental delay, hyperphagia, and cognitive impairment, yet the cellular mechanisms underlying circuit abnormalities remain unclear. Here, we show that loss of the imprinted gene Magel2 remodels microglial state and function in a mouse model of PWS. Three-dimensional morphometric analysis of hypothalamic microglia revealed reduced structural complexity and contracted territorial domains, emerging early in development and largely independent of obesity. Single-nucleus RNA sequencing of hypothalamic brain tissue identified extensive transcriptional reprogramming involving immune signaling, cellular communication, ribosome function, synaptic organization, and protein translation, with stronger effects in females. Magel2 deficiency also increased microglia-neuron contacts and synaptic engulfment, accompanied by reduced synaptic marker density, suggesting aberrant refinement of hypothalamic circuits. Analyses of human post-mortem hypothalamic tissue from individuals with PWS confirmed microglial structural alterations and increased complement engagement. Ligand-receptor inference further revealed reorganization of microglia-centered communication networks, including altered adhesion, trophic, and immune signaling consistent with enhanced phagocytic states. Together, these findings identify microglial remodeling and increased synaptic engagement as central features of Magel2 deficiency, implicate microglia in hypothalamic circuit dysfunction in PWS, and highlight excessive synaptic pruning as a novel dysfunctional mechanism of PWS.

neuroscience↗

bioRxiv2026

Accessing Enzyme Kinetic Data and Prediction Methods at Scale

Enzyme kinetic parameters inform metabolic models, yet experimental measurements are sparse. A growing body of work predicts them from protein and substrate features, but software fragmentation hinders adoption, so downstream tools lock into the most accessible method. We present OpenKinetics Predictor (at predictor.openkinetics.org), an open-source platform integrating thirteen methods in isolated environments behind one interface. The platform optionally reports similarity between query proteins and each method's training data to contextualise reliability. A common featurisation-prediction abstraction keeps it extensible, and independent parties, including original authors, contributed many methods. We pair it with a data portal (at data.openkinetics.org) that exposes CatLog, a curated kinetic dataset, with precomputed embeddings, predicted binding sites, and standardised splits. Both offer a web interface and an API, and the GECKO modelling toolbox already calls the predictor API. As a case study, we predict across an E. coli model and find inter-predictor agreement varies with metabolic context and data availability.

systems biology↗

bioRxiv2026

Lactate Receptor Activation Alleviates Senescence and Preserves Homeostasis of Aged Arteries

Arteries are among the first tissues to exhibit age-related dysfunction, yet the metabolic mechanisms driving vascular senescence remain poorly understood. Here, analysis of human aortic transcriptomic data identified HCAR1, encoding the lactate receptor GPR81, as one of the genes most significantly downregulated with age. We therefore investigated whether age-associated loss of GPR81 contributes to cellular senescence within the vessel wall. Senescent human endothelial cells and vascular smooth muscle cells accumulated neutral and oxidized lipids and exhibited increased labile iron and ferroptosis. Silencing GPR81 in early-passage cells recapitulated this metabolic phenotype together with multiple hallmarks of cellular senescence. Moreover, endothelial-specific deletion of GPR81 in young mice was sufficient to induce senescent cell accumulation, impaired lipid homeostasis, endothelial dysfunction, and elastin disorganization. Conversely, pharmacological activation of GPR81 with the agonist CHBA restored fatty acid metabolism, promoted glycolytic reprogramming, and attenuated ferroptotic stress and senescence-associated phenotypes. In lamin A knock-in (LAKI) progeroid mice, CHBA reduced arterial lipid accumulation and cellular senescence, shifted vascular cell composition toward a youthful state, improved endothelial integrity, and restored extracellular matrix homeostasis. Together, these findings identify age-associated loss of GPR81 as a driver of vascular metabolic dysfunction and cellular senescence and establish pharmacological GPR81 activation as a promising therapeutic strategy for preserving vascular homeostasis and mitigating age-associated cardiovascular disease.

bioengineering↗

bioRxiv2026

Preserved CETP Activity and Early Serum Amyloid A are Associated with Improved Outcomes in Sepsis-Associated ARDS

Background: Sepsis is a life-threatening condition characterized by organ dysfunction resulting from a dysregulated host response to infection. It is a leading cause of in-hospital mortality. The multicenter Acute Respiratory Distress Syndrome Network Statins for Acutely Injured Lungs from Sepsis (ARDSNet-SAILS) trial found that rosuvastatin (RS) did not improve clinical outcomes in patients with sepsis-associated ARDS and may increase hepatic and renal dysfunction. We evaluated associations of plasma cholesterol ester transfer protein (CETP) activity and Serum Amyloid A (SAA) levels with survival and clinical outcomes using plasma samples and clinical data from ARDSNet-SAILS trial. Methods: This secondary analysis included clinical data and plasma samples from 471 ARDSNet-SAILS participants, randomized to placebo (PL) or RS. Plasma CETP activities and SAA levels were measured at enrollment (Day 0, n=466) and Days 3 (n=425) and 6 (n=342). Treatment effects were assessed using linear mixed models, and associations with survival and outcomes using Cox, linear, beta-binomial, and zero-inflated negative binomial regression models. Results: CETP activities were significantly higher in the survivors compared to non-survivors on days 0, 3, and 6 of analysis, in both the treatment groups (p<0.01). Higher Day 0 and Day 3 CETP activities in PL patients independently predicted improved survival, with every 3-unit increase in activity associated with 18 to 19% lower mortality risk. In the RS treatment group, higher CETP at Day 0, Day 3, and Day 6 was significantly associated with reduced hazard of mortality (p=0.039, 0.005, and 0.022, and hazard ratios = 0.87, 0.82, and 0.80, respectively, for Days 0, 3, and 6). Higher CETP values on Days 3 and 6 were also significantly negatively associated with APACHE score and positively associated with increased renal dysfunction-free days (p=0.001). Plasma SAA levels decreased significantly over time in both treatment groups (p<0.0001). In the PL group, higher plasma SAA levels were associated with a lower hazard of mortality. Accordingly, survivors had significantly higher plasma SAA levels than non-survivors at Day 0 (p=0.005). Higher SAA levels at Days 0 and 3 were also inversely correlated with APACHE scores, while Day 0 SAA levels were positively associated with overall organ dysfunction-free days (p=0.002). In contrast, in the RS group, higher plasma SAA levels were associated with an increased hazard of mortality, reaching statistical significance at Day 6 (p<0.01). Despite this, higher SAA levels in the RS group were positively associated with renal dysfunction-free days (Day 3, p=0.004; Day 6, p=0.006), hepatic dysfunction-free days (Days 0, 3, and 6; all p[≤]0.001), and overall organ dysfunction-free days (Day 0, p=0.017; Days 3 and 6, both p<0.001). Analysis of SAA levels in FPLC fractions demonstrated higher SAA levels in the HDL fraction among survivors compared with non-survivors in the PL group on Days 0 and 3; however, this difference was not observed in the RS group. Conclusion: Taken together, these findings identify plasma CETP activities and early SAA levels as stable prognostic biomarkers associated with improved survival and reduced disease severity in sepsis. However, while both CETP activity and SAA levels were unaffected by RS treatment, it appeared to modify the association between SAA and mortality.

biochemistry↗

bioRxiv2026

Sequential assembly in vivo of the bacteriophage P22 genome translocation channel

Bacteriophage P22 ejects four internal proteins into infected cells to assemble a channel for genome translocation across the cell envelope. However, how these proteins cooperate to build the trans-envelope channel remains enigmatic. Using in situ single-particle cryogenic electron microscopy, we determined near-atomic in vivo structures of P22 infection intermediates, revealing how the tail hub protein gp10 templates the sequential assembly of three internal proteins gp7*, gp20 and gp16 into a ~50-nm trans-envelope channel that extends from the virion into the host cytoplasm. Functional analyses explain why phage DNA can be detected in supernatant fluids during infection by channel-defective mutants. Our structures also provide a mechanistic basis for the long-standing observation that a P22 virion lacking gp16 can be complemented, extra-cytoplasmically, by a co-infecting virion lacking gp20, thereby allowing the genome in the gp16-deficient particle to enter the host cytoplasm.

microbiology↗

bioRxiv2026

Large parallel deletions are associated with massive gene losses and virulence acquisition in Meloidogyne incognita

The root-knot nematode Meloidogyne incognita is a major constraint to crop production worldwide. The resistance gene Mi-1.2 has been widely deployed in tomato varieties to control this parasite. However, virulent nematode isolates, overcoming the Mi-1.2-mediated resistance, have increasingly emerged in fields. Understanding the genetic mechanisms underlying resistance circumvention is therefore essential towards sustainable crop protection. From two geographically distinct M. incognita avirulent isolates, previous experimental evolution assays have allowed producing two virulent M. incognita isolates that escaped Mi-1.2 resistance in tomato. Here, we investigated the transcriptomic and genomic changes in the two virulent M. incognita isolates, as compared to their avirulent progenitor isolates controlled by the resistance. First, our transcriptomic analyses revealed hundreds of strongly downregulated genes, in common, in both virulent isolates. Most of these downregulated genes are clustered in two main genome regions. Long-read whole-genome sequencing of the four isolates revealed parallel structural variations in both virulent isolates. This included two large deletions spanning ca. 1.3 Mb and 0.6 Mb. The two big deletions encompass clusters of downregulated genes, suggesting they have in fact been lost in both virulent isolates. Short-read genome sequencing and PCR validation confirmed the two deletions in the two virulent isolates. The largest deletion includes predicted effector and orphan genes. The parallel large-scale structural variations identified here are likely to underlie the rapid adaptation of M. incognita to Mi-1.2-mediated resistance. These findings highlight genome structural plasticity as one of the potential key drivers of virulence acquisition in these asexually reproducing parasites.

evolutionary biology↗

bioRxiv2026

Paternal regulation of H3K4 methylation supports tumor suppressor networks in mammals intergenerationally

Paternally-inherited epigenetic information can influence phenotype in offspring (1). Here, we identify a critical mechanistic contribution of KDM6A (UTX), an X-linked histone modifier and tumor suppressor, in regulating transmissible epigenetic information in mammalian sperm. Paternal loss of KDM6A increases cancer risk in genetically wild type offspring, but how Kdm6a knockout sperm transmit this effect at the molecular level is unknown (2). We find that KDM6A functions in spermatogenesis to promote methylation of histone H3 lysine 4 (H3K4) via selective interaction with the COMPASS complex methyltransferase KMT2C (MLL3). KMT2C and KDM6A are coordinately recruited to promoters of active genes in spermatogenic cells, contrasting with recruitment to intergenic enhancers in other cell types (3, 4). Loss of KDM6A disrupts H3K4 methylation at promoters of tumor suppressor genes in spermatogonia, and some of these defects persist in epididymal sperm and correspond to impaired expression in preimplantation embryos. These genes are also misregulated in normal and malignant hematopoietic tissue of genetically wild type offspring, indicating that impaired H3K4 methylation in KDM6A-deficient male germ cells may preferentially alter regulation of tumor suppressor gene networks in development across generations.

genetics↗

bioRxiv2026

TRAPPC8 Is an Endogenous Brake on UFMylation That Suppresses Tauopathy

UFMylation, a ubiquitin-like protein modification, drives tau spread through the brain, but what keeps this process in check has remained unclear. We show that TRAPPC8 acts as a natural brake on UFMylation, binding directly to the E1 enzyme UBA5 to dampen pathway activity. In Alzheimer's disease brain tissue, TRAPPC8 is reduced while UFMylation is elevated, suggesting this brake fails as disease progresses. Sustaining UFMylation in human iPSC-derived neurons increased tau aggregation and spread while disrupting lysosomal function and lipid balance. Restoring TRAPPC8-UBA5 binding reversed these defects through the lysosomal protein CLN8, which restored lysosomal function and reduced tau pathology. Notably, expressing just the UBA5-binding region of TRAPPC8 was enough to suppress tau pathology in vivo, marking it as a promising therapeutic target.

neuroscience↗

bioRxiv2026

The human papillomavirus L2 protein in the capsid is an ensemble of related structures poised to exit the virus particle

The human papillomavirus (HPV) capsid contains 360 molecules of the L1 protein, which assembles into pentamers that form the capsid shell, and up to 72 molecules of the L2 protein. The C-terminus of the L2 protein must emerge from the capsid, insert into the endosome membrane, and protrude into the cytoplasm to bind to cellular proteins that mediate trafficking of the virus to the nucleus during virus entry. Computational analysis predicts that much of L2 is disordered, but the stoichiometry, structure and location of L2 in the capsid remain elusive. Here, we use cryo-EM single particle analysis of HPV16 pseudovirus capsids with and without the L2 protein combined with AlphaFold3 predictions and molecular dynamics simulations to develop a structural model of L2 within the capsid. Our results revealed that a single molecule of L2 associates with the inner surface of most L1 pentamers. The relatively rigid structure of the pentamer forces the long intrinsically disordered segments of L2 to adopt numerous related, compact, stable conformations in the capsid, whereas the structured elements of L2 remain folded but adopt varied positions in space largely beneath the pentamer. A finger-like projection of L2 extends into the central pore of the pentamer, but many different segments of the disordered domains of L2 can form fingers. In some models the C-terminus of L2 extends through the pore to the exterior surface of the capsid positioned to insert into the endosomal membrane. Consistent with these models, analysis of purified pseudoviruses showed that the C-terminus of multiple L2 molecules is exposed on the surface of each capsid. These findings provide a structural basis for understanding how L2 is organized in the capsid poised to initiate its action during virus entry.

microbiology↗

bioRxiv2026

Chemically programmed multistage morphogenesis in coacervate microdroplets

Chemical reaction-driven morphogenesis enables adaptive membraneless organelles to translate biochemical inputs into coordinated structural and functional responses autonomously, but achieving non-equilibrium morphogenesis in synthetic coacervates remains challenging. Here we report an oxidation programmed multistage morphogenesis in coacervate microdroplets composed of polyethyleneimine (PEI) and sodium thioctate (ST). Coacervate microdroplets form through electrostatic complexation between PEI and ST, together with hydrophobic association among the dithiolane motifs of ST. Hydrogen peroxide converts these dithiolane motifs into more polar species, progressively weakening hydrophobic clustering, increasing hydration within the coacervate phase, and driving the microdroplets away from their initial equilibrium state. This reaction-induced compositional imbalance drives initially homogeneous microdroplets to evolve into multivacuolated intermediates, hollow structures, and finally contracted microdroplets. Experimental and simulation results support a reaction-induced secondary phase separation in which ST oxidation promotes secondary phase separation, osmotic water uptake, vacuole growth, coalescence, and shell remodeling. By recruiting glucose oxidase (GOx) into the coacervate phase to generate H2O2 in situ, we further establish an enzyme-driven route in which glucose autonomously actuates a similar sequence of multistage morphogenesis. Coupling the GOx/glucose pathway with the horseradish peroxidase/Amplex Red cascade reaction further linked glucose-triggered morphogenesis to fluorescent signal generation, enabling coacervate microdroplets to integrate biochemical sensing, structural remodeling, and optical readout. Overall, this work establishes a reaction-induced secondary phase separation for programming life-like multistage morphogenesis in membraneless microcompartments.

biochemistry↗