Biology subjects

Lu, T.

Publications and source records attributed to Lu, T..

At least 19 recordsLinked to original sources

bioRxiv2026

A Cell-Autonomous Cold Sensor Promotes Lipolysis Through Phase Separation

Temperature is a fundamental determinant of mammalian metabolism. The canonical cold-adaptation pathway involves cold sensing by peripheral sensory neurons, which activate sympathetic outflow, {beta}-adrenergic signaling, and lipolysis in adipose tissue. In contrast, we report here a cell-intrinsic mechanism by which physiologically relevant cooling temperatures (27-31 oC) enhance lipolysis in subcutaneous adipose tissue. Mechanistically, cooling drives liquid-liquid phase separation (LLPS) of perilipin 1 (PLIN1), the dominant lipid droplet coat protein, promoting its condensation and the local recruitment of PKA to the droplet surface. This spatial reorganization selectively amplified PKA-mediated perilipin 1 phosphorylation, CGI-58 release, and ATGL-dependent lipolysis. The process depends on the intrinsically disordered region (IDR) of perilipin1, where four aromatic residues, conserved in endotherms but not in ectotherms, are required for cooling-induced LLPS and lipolysis in vivo. Notably, targeted delivery of an LLPS-competent perilipin 1 variant to the inguinal adipose tissue of mice enhanced local lipolysis and reduced depot mass. Together, these findings uncover a cell-intrinsic thermosensitive mechanism that can tune lipolytic responsiveness, offering a possible strategy for targeted depot-selective fat reduction.

cell biology↗

bioRxiv2026

Auto-sumoylation of UBC9 coordinates meiotic prophase and protects ovarian reserves

The small ubiquitin-like modifier SUMO regulates key events of meiosis, including pairing and crossing over between homologous chromosomes. Auto-sumoylation of the SUMO E2-conjugating enzyme UBC9 at lysine 14 alters its substrate selectivity in vitro, but the role of this modification in vivo is unknown. Here, we show that UBC9-K14 auto-sumoylation helps coordinate meiotic prophase and is important for maintenance of the ovarian reserve. Ubc9K14R/K14R knock-in mice show a variety of defects in meiotic prophase I, including altered assembly of DNA strand-exchange complexes, delayed and defective homolog synapsis, and unstable crossover recombination complexes. In spermatocytes, these defects are associated with reduced efficiency of crossing over between the X and Y chromosomes. Oocytes from Ubc9K14R/K14R females show related but distinct defects in recombination and synapsis. Moreover, maintenance of the primordial follicle reserve is defective in Ubc9K14R/K14R females, and their fecundity is reduced. Finally, we identify the meiosis-specific homolog-axis protein SYCP3 as a direct target of UBC9 in vitro and show that SYCP3 modification is strongly stimulated by K14 auto-sumoylation. We infer that auto-sumoylation enables UBC9 to modify a subset of targets in vivo, helping to coordinate key events of meiotic prophase and enhance the survival of primordial follicles to maximize fecundity.

genetics↗

bioRxiv2026

PCIF1 loss licenses antitumour immunity via cholesterol biosynthesis

Cholesterol biosynthesis is classically viewed as a tumour supportive metabolic programme, yet cholesterol-associated metabolic states can paradoxically coincide with improved responses to immune checkpoint blockade (ICB)1, highlighting an unresolved context-dependent relationship between cholesterol homeostasis and antitumour immunity. Here we identify PCIF1, a cap-specific RNA methyltransferase2, as a tumour-intrinsic brake on the inflammatory potential of cholesterol biosynthesis. PCIF1 depletion had limited effects on tumour cell proliferation in vitro, but suppressed tumour growth in immunocompetent, not immunodeficient, hosts and sensitized immune-refractory syngeneic tumours to ICB. Immune profiling by single-cell RNA sequencing, flow cytometry and multiplex immunofluorescence revealed increased CD8+ T cell infiltration in PCIF1-deficient tumours, and CD8+ T cell depletion restored tumour growth. Mechanistically, PCIF1 loss selectively enhanced translational engagement of SCAP, activated SREBP2-dependent cholesterol biosynthesis and promoted intracellular cholesterol accumulation. Unexpectedly, activation of this cholesterol programme induced a tumour cell inflammatory cytokine state enriched for T cell-recruiting chemokines, including CXCL10. Pharmacological perturbation of the SCAP-cholesterol axis attenuated this inflammatory programme, placing cholesterol sensing upstream of PCIF1 loss-induced tumour cell inflammatory activation. In ICB-treated patient cohorts, high PCIF1 expression in pre- and on-treatment samples was associated with poor clinical response, and across tumour types, PCIF1 expression inversely correlated with cytolytic immune signatures. These findings reveal PCIF1-mediated SCAP translational control as a tumour cell checkpoint that constrains the antitumour inflammatory activity of a canonical growth-associated cholesterol programme and shapes checkpoint therapy responsiveness.

cancer biology↗

bioRxiv2026

EDTA v2: enabling scalable TE annotation in animal genomes

The Extensive de-novo TE Annotator (EDTA) automates transposable element annotation in plant genomes but lacks direct LINE/SINE detection, limiting its applicability to animal genomes. We present EDTA v2, which integrates LINE and SINE detection, completely rewrites TIR-Learner for deployability and scalability, and accelerates structural detectors by up to two orders of magnitude. Tested in 30 animal genomes from the Vertebrate Genomes Project Phase I, EDTA v2 bridges the non-LTR detection gap that has prevented automated TE annotation in animals.

genomics↗

bioRxiv2026

A tunable receptor separates root barrier formation from nutrient signaling through ligand-perception states

In roots, the endodermis controls nutrient entry by forming a barrier known as the Casparian strip1-3. Yet how barrier-associated processes interface with systemic signaling remains unclear. Here, we show that the receptor kinase SCHENGEN3 links local barrier surveillance to systemic nutrient signaling, with outputs that depend on the effective state of ligand perception. Unlike in Arabidopsis thaliana, SCHENGEN3 activation in Lotus japonicus requires a distinct cellular competence state and cannot be triggered by exogenous ligands alone, revealing evolutionary divergence in pathway deployment. Cross-species complementation uncouples systemic nitrogen signaling from Casparian strip formation, while transcriptomic and phosphoproteomic analyses reveal largely non-overlapping signaling- and barrier-associated programs that differ between agar and agricultural soil conditions. Mechanistically, receptor-ligand comparisons, engineered receptor variants, and co-receptor mutant analyses show that systemic nitrogen signaling is retained in receptor-perception states that are insufficient to support full Casparian strip establishment. Together, these findings reveal how a shared receptor module can separate developmental and physiological outputs by linking receptor perception state to output specificity.

plant biology↗

bioRxiv2026

Selective knockout of PKA regulatory subunits reveal opposite catalytic and metabolic consequences with implications for Alzheimer's disease

cAMP-dependent Protein Kinase A (PKA) is a master regulator of cell signaling involved in energy metabolism, synaptic plasticity, and stress response. Dysregulated PKA signaling is implicated in diseases including neurodegeneration and cancer. PKA catalytic activity is regulated by two nonredundant regulatory subunits, Type I (RI/RI{beta}) and Type II (RII/RII{beta}), whose divergent functions are not fully understood. We generated double-knockout (KO) cell lines of RI/RI{beta} and RII/RII{beta} subunits and performed multiplexed MS-based proteomic and phosphoproteomic profiling under basal and glucose-perturbed conditions. We found that RI and RII loss drives distinct, and often opposite, remodeling of the cellular proteome and phosphoproteome. While both mutants blunted metabolic flexibility to glycolytic stressors and stimuli, RI and RII KO cells exhibited elevated and depressed glycolytic signaling, respectively. Interestingly, RI KO increased the abundance and kinase activity of the PKA catalytic subunit C isoform, leading to an increase in PKA substrate phosphorylation, whereas RII KO decreased the abundance, kinase activity, and substrate phosphorylation by the catalytic subunit C{beta} isoform. Notably, one of the most differentially affected PKA sites between RI and RII KOs maps to Tau, whose hyperphosphorylation is a hallmark of Alzheimers disease. Loss of RI increased Tau phosphorylation, which was not only caused by increased PKA catalytic activity, but also a higher binding affinity of Tau to RII subunits on the negatively-charged flexible linker region. Overall, the present study demonstrates that PKA RI and RII subunits play nonredundant roles in modulating PKA activity, metabolic flexibility, and phospho-regulation of key disease-associated substrates such as Tau.

biochemistry↗

bioRxiv2026

Decoding Immune Reconstitution Failure in People Living with HIV through Single-cell Genomics

Immune non-responders (INRs), a subset of people living with HIV (PLWH), fail to achieve full immune reconstitution and remain at increased risk of morbidity, mortality, and non-AIDS-related illnesses. As the mechanisms underlying this impaired immune recovery remain poorly understood, we performed single-cell multi-omics profiling on peripheral blood mononuclear cells from 43 INRs, 47 immune responders and 53 healthy donors. Our dataset comprises 2,744,009 transcriptomes and 1,226,658 chromatin-accessibility profiles across 58 identified immune cell types. INRs exhibited markedly elevated inflammatory signaling, increased apoptotic activity, and dysregulation of immune-activating ligand-receptor interactions. Additionally, we identified 2,996 interaction cis-eGenes and 5,938 cis-caPeaks, and validated key cell type-specific cis-xQTL effects associated with immune reconstitution failure. Furthermore, we identified 3,551 sc-eQTLs and 872 cell-state interaction eQTLs (ieQTLs) in CD4+ T cells. A genotype-specific upregulation of OAS3 in INRs, likely regulated by STAT1, was identified based on these results. We developed scPRISM (single cell Predictive Reconstitution Immune Status Model), a multi-modal framework that integrates scRNA-seq and scATAC-seq data to perform the dual tasks of differentiating disease states and predicting gene expression from chromatin accessibility, thereby deciphering the cell type-specific cis-regulatory basis of the disease. Our study delineates the transcriptional and epigenomic landscape of immune dysregulation in INRs and provides new genetic insights into HIV-associated immune reconstitution failure. These findings offer a foundation for developing biomarkers and precision therapies to restore immune function in PLWH.

genetics↗

bioRxiv2026

A generalizable system for antigenic peptide targeting across HLA-I allotypes

T cell receptors (TCRs) and TCR-mimicking antibodies recognize peptide antigens in the context of specific Human Leucocyte Antigen (HLA-I) allotypes, and the extreme polymorphism of the HLA locus limits the breadth of immunotherapy development. Key barriers include divergent molecular surfaces on HLA proteins and differences in the peptide structure. As a result, existing modalities cannot confer therapeutic coverage across patients of divergent genetic backgrounds. Here, we develop an approach which combines a peptide conformational prediction tool, PepPred, with a cross-HLA binding protein engineering system, TRACeR-I1, to outline a generalized framework for developing binders (xTRACeRs) with compatibility across HLA allotypes while maintaining high levels of specificity towards the peptide antigen. We use our system to develop and validate xTRACeRs against clinically relevant, established peptide antigens presented across common alleles within five HLA-A/B/C supertypes2. Cryo-EM structures of xTRACeR-pHLA complexes for an oncofetal antigen from PRAME and a neuroblastoma-specific peptide from PHOX2B reveal effective mechanisms to navigate polymorphic HLA surface residues, and extensive interactions with the peptide. We implemented these two xTRACeRs as Chimeric Antigen Receptor (CAR) T cells and demonstrated their potent killing efficacy and specificity. Overcoming restriction across HLA supertypes lifts a key barrier in HLA-targeted immunotherapy by expanding patient coverage.

synthetic biology↗

bioRxiv2026

BAT protects against hepatic oxidative stress by remodeling the circulating metabolome

Brown adipose tissue (BAT) regulates systemic metabolism beyond thermogenesis, yet the circulating mediators through which BAT communicates with other organs remain less explored. Here, we performed comprehensive serum metabolomics and lipidomics in BAT-ablated mice and human cohorts with varying BAT activity to delineate how BAT activity shapes the circulating metabolome. By integrating datasets across serum, tissues, extracellular fluids, and conditioned media, we assembled BAT-linked circulating molecular signatures. The analyses support a critical role for BAT in the clearance of circulating branched-chain amino acids and triglycerides. We also identified a cold-inducible metabolite, 3-hydroxystearic acid (3-OHSA), produced primarily by BAT and released into circulation. 3-OHSA serves as a circulating readout of cold-activated BAT and acts on the liver to reduce mitochondrial membrane potential and reactive oxygen species production, thereby limiting oxidative stress. This work provides a framework for identifying BAT-derived mediators and uncovers a BAT-liver axis that coordinates adaptation to metabolic stress.

physiology↗

bioRxiv2026

Colocalization and discordance between plasma and brain protein quantitative trait loci

Studies of protein quantitative trait loci (pQTLs) provide opportunities to interpret complex trait genetics and identify potential biomarkers and therapeutic targets. Circulating proteins are commonly used in pQTL studies due to the accessibility of blood-based measurements, but their levels may not always reflect regulation in disease-relevant tissues. We assessed colocalization and discordance between plasma and dorsal prefrontal cortex cis-pQTLs using data from four large-scale studies and investigated their implications for downstream analyses. Across the proteins examined, at most 80% of the cis-pQTLs showed evidence of colocalization. Among the colocalized loci, approximately 20% exhibited opposite directions of genetic effects. We characterized tissue-specific gene expression profiles based on data from the Genotype-Tissue Expression project. Proteins with colocalized cis-pQTLs were more likely to have high gene expression levels in systemic tissues and immune cells, whereas the remaining proteins were more likely to have high expression in brain tissues. We conducted Mendelian randomization (MR) analyses using neuroticism as an illustrative outcome to compare effect estimates derived using instruments from different pQTL studies. MR analyses identified 13 proteins significantly associated with neuroticism, including six with opposite effect directions between plasma and dorsal prefrontal cortex, highlighting the importance of tissue context. Overall, circulating pQTLs remain informative for proteins from systemic and immune pathways, while incorporating tissue-specific data may provide additional insight for proteins with more localized expression. Considering multiple tissue contexts may refine the interpretation of protein-trait associations and may improve the prioritization of candidate targets.

genetics↗

bioRxiv2026

Physics-Guided Deep Neural Networks: Correcting Physical Distortions in Protein Phase Separation Prediction

BackgroundIn computational biology, embedding known physical laws into deep learning models to construct "Physics-Informed Neural Networks" (PINNs) is a mainstream paradigm for enhancing model interpretability and extrapolation capability. However, in complex multi-physics coupling problems, there is a risk of competitive imbalance between the physical term and the flexible artificial intelligence (AI) residual term, causing the model to degenerate into a "black-box" fit and lose the original purpose of being physics-driven. MethodsIn this study, targeting the problem of predicting protein liquid-liquid phase separation (LLPS) behavior in response to environmental factors (temperature, salt concentration), we identified physical distortions, gradient vanishing, and numerical instability in the initial physics-AI hybrid model. Three core correction strategies were proposed: (1) Weight Allocation Logic Reconstruction: Force the physical trunk weight to 1.0 at the output layer, suppressing the AI residual term to the perturbation level of 0.05~0.1, ensuring physics dominance; (2) Robust Physics Formula Construction: Abandon the unstable power function and introduce a combination of Softplus and logarithmic functions to stably simulate the nonlinear effects of charge shielding; (3) Gain Compensation Alignment: Apply gain compensation to the weak signal branch (temperature) to ensure its effective participation in optimization. ResultsThe optimized model maintained a fitting accuracy of R2{approx}0.62 on the test set, while physical consistency was significantly enhanced. The model successfully restored the monotonic increase in solubility with temperature characteristic of UCST-type phase diagrams and correctly captured the nonlinear charge shielding features in the salt concentration response. The weights of key physical parameters (e.g., hydrophobic contribution w_h, net charge contribution w_ncpr) increased from <10-3 to the 10-2 magnitude, demonstrating the reactivation of the physical branch. ConclusionsThe weight control, formula stabilization, and signal gain alignment strategies proposed in this study effectively address the classic problem of "AI hijacking" physics in physics-AI hybrid models. This work provides a universal solution for constructing biophysical predictive models that combine high fitting accuracy with strong physical interpretability.

cell biology↗

bioRxiv2026

Temporal AI model predicts drivers of cell state trajectories across human aging

Foundational AI models have recently shown promise for predicting the impact of perturbations on cell states. However, current models typically consider only one cell state at a time, limiting their ability to learn how cellular responses unfold over time, particularly across long trajectories such as diseases of aging. Here, we develop a temporal AI model, MaxToki, trained on nearly 1 trillion gene tokens including cell state trajectories across the human lifespan to generate cell states across long timelapses of human aging. MaxToki generalized to unseen trajectories through in-context learning and predicted novel age-modulating targets that were experimentally verified to influence age-related gene programs and functional decline in vivo. MaxToki represents a promising strategy for temporal modeling to accelerate the discovery of interventions for programming therapeutic cellular trajectories.

bioinformatics↗

bioRxiv2026

An interferon independent innate immune response to double stranded RNA in embryonic stem cells

In vertebrate early embryos and embryonic stem cells, the interferon (IFN)-centered double-stranded RNA (dsRNA) sensing and signaling pathway is markedly suppressed, implying the existence of an alternative repertoire of dsRNA sensors in these stages. We recently reported that dsRNA treatment triggers translation inhibition using Prkra as a sensor in zebrafish and mouse early embryos. Independently, here we show that dsRNA stimulates the expression of a subset of interferon-stimulated genes (ISGs) in the absence of IFN production, establishing a defensive state in mouse embryonic stem cells (mESCs). Upon dsRNA stimulation, the multifunctional DExD/H-box RNA helicase Dhx9 is recruited into dsRNA-induced condensates, where it promotes the recruitment and functional suppression of the Mdm2/Cul4A ubiquitin ligase machinery by excluding their substrate adaptor Ddb1, thereby stabilizing p53 and Stat1. Dhx9, p53 and Stat1 then cooperate to stimulate an ISG response. This signaling is important for the defense against ZIKV infection in mESCs as well as other dsRNA stresses. Such a cascade is conserved in human ESCs, while in zebrafish embryos, p53 but not Stat1 is required for the transcriptional response. Our findings define an immediate, cell-autonomous innate immune pathway operating in ESCs and vertebrate embryos.

immunology↗

bioRxiv2026

Eco-tank Housing Maintains Wild-Type Microbiota and Rewilds the Laboratory Mouse Gut Microbiome to Restore Natural Immune Tone

Laboratory mice housed under individually ventilated cage (IVC) conditions harbor simplified gut microbiota and immune phenotypes that diverge substantially from those shaped by environmental exposure, limiting translational relevance. To reintroduce controlled ecological complexity while maintaining biosafety and reproducibility, we developed the Eco-tank, a pathogen-monitored semi-natural housing system incorporating environmental substrates and dietary diversity. Longitudinal 16S rRNA sequencing revealed that even wild-caught Mus musculus rapidly lose microbial richness and predicted metabolic breadth under IVC housing. Eco-tank conditions stabilized diversity and preserved elements of wild-associated community structure during extended captivity. In parallel, standardized C57BL/6 mice housed in Eco-tanks underwent rewilding-like restructuring, with increased richness and community shifts toward a wild-associated configuration. Functional inference analyses indicated expansion of predicted pathways linked to short-chain fatty acid production, amino acid metabolism, and environmental substrate utilization. Eco-tank housing enhanced baseline resistance to pulmonary Pseudomonas aeruginosa (Pa) infection without compromising vaccine-induced protection, indicating that restoration of environmental microbial signals does not impair adaptive immunity. Together, these findings identify housing ecology as a dominant determinant of microbiome structure and functional potential. The Eco-tank provides a scalable and tractable framework for integrating environmental microbial complexity into laboratory models to better align preclinical immunology with ecologically conditioned immune systems. ImportanceLaboratory mice are foundational models for immunology, yet their specific pathogen-free rearing and housing environments impose ecological constraints that reshape the gut microbiome and immune tone. This study introduces a scalable, pathogen-monitored Eco-tank system that restores environmental microbial complexity while preserving experimental control. By demonstrating that housing ecology reshapes microbiome functional potential and modulates baseline immune resistance without compromising vaccine responsiveness, this work highlights environmental context as a critical experimental variable in preclinical immunology and offers a tractable framework for improving translational relevance.

microbiology↗

bioRxiv2026

No evidence for sleep-dependent memory generalization in a large online sample

Memory generalization allows individuals to extract and apply information from prior experiences to novel situations, supporting flexible learning and efficient decision-making. Theoretical models suggest that sleep should facilitate generalization, yet the literature examining its role in promoting generalization is mixed. We recruited 137 participants via Prolific to complete an image-location memory task over two sessions spaced 12 hours apart. Participants were randomly assigned to the Wake group (learning in the morning) or the Sleep group (learning in the evening). In Session 1, participants learned the location of stimuli on the screen and were tested on their memory five minutes later. Twelve hours later, in Session 2, they were tested on their memory again. Stimuli consisted of 160 images from eight semantic categories and were strategically positioned on-screen to test the effects of generalization on retrieval (i.e., category-based memory distortions and biases). After the delay, retrieval was less accurate and demonstrated more generalization. However, these effects were mostly independent of Group, with some evidence for enhanced generalization following a period of wakefulness over sleep. Generalization was also driven by time of day, with more generalization in the evening relative to the morning. Taken together, our results, based on a large online sample, do not support a role for sleep in promoting memory generalization. Significance StatementBehavior is often guided by memories of previous experiences. However, for behavior to be adaptive and flexible (e.g., when encountering never-before-seen stimuli), regularities about the world must be extracted from these memories. This process, termed memory generalization, has been hypothesized to rely on sleep. We used a large online sample to test sleeps role in generalization and found no support for this hypothesis. Our results suggest that sleep and wakefulness contribute to generalization equally, with the latter potentially having a larger contribution.

neuroscience↗

bioRxiv2026

Lmna deficiency promotes EPHX2 nuclear translocation to ameliorate cardiac dysfunction in mice

BackgroundCardiovascular diseases are often associated with altered protein subcellular localization. As a major cause of inherited cardiomyopathy, LMNA deficiency could trigger nuclear envelope rupture and broadly impair the localization of nuclear and cytoplasmic proteins. Systemic approaches to identify, dissect and manipulate the localization of endogenous proteins are important for mechanistic and therapeutic investigation. MethodProximity proteomics of the nuclear lamina was performed specifically in cardiomyocytes in Lmna-deficient murine models. AAV-mediated Cas9-based gene silencing and subcellular gene upregulation were conducted via the nuclear localization signal (NLS) and nuclear export signal (NES). Cas9-based somatic mutagenesis was supplemented with the single-strand DNA templates of AAV to achieve robust homology-directed repair (HDR) and targeted NLS knock-in, which translocated cytoplasmic proteins into nuclei. ResultIn vivo proximity proteomics detected increased epoxide hydrolase 2 (EPHX2) in cardiomyocyte nuclei in mice carrying germline or cardiac-specific Lmna truncating variants. This phenotype was associated with ruptured nuclear envelope. Cas9-mediated Ephx2 knockout in cardiomyocytes ameliorated cardiac dysfunction in Lmna-deficient mice. Strikingly, overexpression of NLS-EPHX2, but not NES-EPHX2, also mitigated cardiac dysfunction. The cardiac protective EPHX2 substrates, epoxyeicosatrienoic acids (EETs), did not alter upon NLS-EPHX2 overexpression. By contrast, the Lmna-related DNA damage marker {gamma}-H2AX was reduced. The EPHX-D333A mutant lacking hydrolase activity recapitulated the effects of wildtype EPHX2 in nuclei. AAV-Cas9-based HDR achieved efficient NLS knock-in and EPHX2 nuclear translocation in more than 60% cardiomyocytes, which improved cardiac function. ConclusionLmna deficiency leads to the nuclear translocation of EPHX2, which ameliorated cardiac dysfunction in a hydrolase-independent manner. AAV-HDR-mediated somatic gene editing provides an efficient approach to manipulate the subcellular localization of endogenous proteins in cardiomyocytes in vivo. What is Known?O_LICardiovascular diseases are regulated by the changes in protein subcellular localization. In particular, LMNA-related cardiomyopathy is associated with nuclear rupture and impaired separation between nuclear and cytoplasmic proteins. C_LIO_LIEpoxide hydrolase 2 (EPHX2) is a cytoplasmic hydrolase that catalyzes the hydrolysis of cardioprotective epoxyeicosatrienoic acids (EETs) and aggravates an array of heart diseases including myocardial infarction and heart failure. C_LIO_LICRISPR/Cas9-mediated homology-directed repair (HDR) exhibits uniquely high gene editing efficiency in cardiomyocytes with an AAV-based DNA donor, which is suitable for somatic genetic knock-in of small DNA fragments in vivo. C_LI What New Information Does This Article Contribute?O_LILamin-targeted proximity proteomics specifically in cardiomyocytes uncovers novel proteins undergoing subcellular localization changes upon Lmna deficiency. C_LIO_LILmna deficiency leads to EPHX2 nuclear translocation that ameliorates cardiac dysfunction by both mechanisms of cytoplasmic reduction and nuclear induction. C_LIO_LIAAV-HDR-mediated knock-in provides a robust platform to manipulate subcellular localization of endogenous proteins specifically in cardiomyocytes in vivo. C_LI Cardiovascular diseases are often associated with altered protein subcellular localization, but systemic approaches to identify, study and manipulate subcellular localization remain incomplete. This study established an in vivo proximity proteomics approach to identify novel proteins undergoing localization changes relative to the nuclear lamina. In murine models of LMNA-related cardiomyopathy, this approach uncovered EPHX2, a classic cytoplasmic hydrolase that aggravates cardiovascular diseases, as a new protein that translocated into cell nucleus and exerted a cardiac protective effect. CRISPR/Cas9-based cardiomyocyte gene editing with an AAV-based DNA donor efficiently achieved NLS knock-in into the Ephx2 gene, promoted EPHX2 protein nuclear translocation and mitigated cardiac dysfunction with Lmna deficiency. These findings indicated a novel avenue to identify and manipulate the subcellular localization changes of endogenous proteins for basic and translational cardiology.

pathology↗

bioRxiv2026

Synergistic integration of inducible RNA switches enhances the manipulation of vector expression

Precise manipulation of gene expression is pivotal for gene function studies and the optimization of gene therapy. RNA-based gene switches are attractive tools due to their robust tunability by FDA-approved small molecules, the absence of exogenous immunogenic proteins, and the small size for gene delivery vectors such as adeno-associated virus (AAV). However, existing RNA switches only target a single step of gene expression such as transcription or RNA splicing, exhibiting intrinsic limitations in gene regulation. To overcome this issue, this study integrated the aptamer-based polyA regulator (pA), the drug-elicitable alternative splicing module (DreAM) and an engineered translation modulator with conditional upstream open reading frames (uORFs) to construct the DreAM-plus RNA switch. The pA-DreAM concatenation led to 1.5[~]5.0-fold and 1.2[~]4.4-fold increase of inducible fold changes than pA and DreAM, respectively. The uORF module further enhanced the switching performance by 1.4[~]6.3-fold. DreAM-plus-mediated transient transgene expression demonstrated a temporal resolution of about 24 hours and high tissue specificity to liver or heart. Critically, DreAM-plus achieved transient expression of an array of gene editors (SpCas9, SaCas9, Un1Cas12f1, OsCas12f1, AcCas12n, IsDra2 TnpB etc.) that significantly mitigated off-target effects by 1.4[~]2.8 folds in plasmids, lentivirus and AAV. In a new mouse model with lipid-nanoparticle-delivered pre-existing immunity, DreAM-plus attenuated AAV-delivered Cas-specific CD8 T cell immune toxicity in the liver and the heart. Therefore, multiple RNA switches could be synergistically integrated to build more sophisticated genetic cassettes for enhanced manipulation of gene expression.

bioengineering↗

bioRxiv2026

Converting Lysosomes into Photothermal Organelles Enables Nanoparticle-Free Tumor Ablation via Intracellular Vapor Bubbles

Photothermal nanomaterials enable precise tumor ablation but face limitations in biodistribution, tissue penetration, toxicity, and biodegradability. Here, we present a unique concept for nanoparticle-free photothermal therapy based on the lysosomal entrapment of cationic amphiphilic small molecular dyes for spatially controlled vapor bubble (VB)-mediated tumor cell ablation. This strategy, which exploits a universal biological and physical effect, employs intracellular pH gradients for extensive local dye enrichment in acidified organelles, transforming them into transient endogenous nanosized photothermal reactors for subsequent light activation. Using sunitinib, a clinically approved lysosomotropic anticancer drug, and the commercially available dye LysoTracker Deep Red, lacking intrinsic anticancer activity, we demonstrate pulsed laser-induced VB formation specifically from dye-loaded lysosomes, leading to selective photomechanical disruption of various cancer cell models across 2D cultures, 3D spheroids, patient-derived neuroblastoma tumoroids and tumor fragments from an ovarian carcinoma patient. This approach allows precise, low-fluence and wavelength-tunable cancer tissue ablation without the need for synthetic photoresponsive nanoparticles.

bioengineering↗