Biology subjects

Yun, J.

Publications and source records attributed to Yun, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Quanta microscopy enables fluorescence imaging at the native bit-plane rate

Live-cell fluorescence microscopy remains constrained by limited photon budgets and exposure-integrated detection, imposing a fundamental trade-off between temporal sampling and image quality. Here we present quanta microscopy (QM), an optical-computational framework that integrates high-speed single-photon acquisition with self-supervised spatiotemporal reconstruction. QM transforms highly sparse binary photon-detection maps, or bit planes, into multibit grayscale fluorescence image sequences, generating one output frame per bit plane at the native 50 kHz sensor cadence without temporal binning. We demonstrate QM across biological specimens spanning diverse spatial and temporal characteristics, including subcellular organelles with distinct morphologies, rapid contractile dynamics in beating hiPSC-derived cardiomyocytes, and electrically stimulated calcium-response transients in primary cortical neurons. By uniting quanta-level acquisition with data-driven image formation, QM broadens the scope of live-cell fluorescence microscopy for quantitative analysis of rapid cellular physiology under photon-limited conditions.

bioengineering↗

bioRxiv2026

DTCAMD, a disease-associated truncated form of CRYAB in neovascular age-related macular degeneration: a potential driver of subretinal fibrosis

Abstract Purpose: Subretinal fibrosis causes irreversible vision loss in neovascular age-related macular degeneration (nAMD) and remains poorly controlled by anti-vascular endothelial growth factor (VEGF) therapy. We investigated the role of B-crystallin (CRYAB) in angiogenic and fibrotic pathology and evaluated disease-associated CRYAB truncation as a therapeutic target. Methods: Aqueous humor from patients with nAMD was analyzed by quantitative proteomics and receiver operating characteristic (ROC) analysis. CRYAB function was assessed using knockout and knockdown approaches in a choroidal neovascularization (CNV) mouse model. CRYAB truncation and associated proteins were examined by immunoblotting, co-immunoprecipitation, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). C-terminal truncation was further examined in mouse retinal tissue using antibodies generated against full-length CRYAB and its C-terminal region. Primary human retinal pigment epithelial (hRPE)-THP-1 co-cultures were used to examine CRYAB truncation under fibrosis-associated conditions. Pharmacological effects were tested using ({+/-})-threo-3-methylglutamic acid (T3MG) and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). Results: CRYAB was upregulated in the aqueous humor of patients with nAMD. Higher CRYAB levels were associated with persistent lesions after initial anti-VEGF treatment. CRYAB knockout or knockdown reduced CNV pathology and decreased VEGFA and -smooth muscle actin (SMA) expression, without a significant substrain effect between C57BL/6N and C57BL/6J mice. A Ser45-phosphorylated truncated CRYAB species was detected in mouse laser-induced CNV model. We termed this species disease-associated truncated CRYAB in AMD (DTCAMD). Peptide mapping showed reproducible loss of distal C-terminal peptide coverage in truncated CRYAB. Consistent with this finding, C-terminal antibody recognition was reduced in CNV lesions. In hRPE-THP-1 co-cultures, DTCAMD accumulated with increased SMA expression. Proteomic analysis identified Prss1 as a candidate CRYAB-associated serine protease linked to truncation under CNV conditions. T3MG and AEBSF reduced DTCAMD-associated signals and CNV pathology. Conclusions: CRYAB contributes to angiogenic and fibrotic pathology in CNV. Disease-associated CRYAB truncation, particularly Ser45-phosphorylated DTCAMD, represents a potential therapeutic target for fibrotic remodeling in nAMD.

pathology↗

bioRxiv2026

Endogenous SMN Heterogeneity Defines Dynamic States of Motor Neuron Vulnerability and Resilience

Selective neuronal vulnerability, whereby some neurons degenerate while others remain resilient despite a shared genetic and disease context, is a defining feature of neurodegenerative diseases, yet the intrinsic mechanisms underlying these divergent fates remain poorly understood. Here, we identify naturally occurring heterogeneity in endogenous Survival Motor Neuron (SMN) protein abundance as a determinant of dynamic motor neuron (MN) vulnerability states that predict neuronal fate. Longitudinal live-cell imaging of human SMN-Clover reporter MNs shows that neurons with high endogenous SMN survive significantly longer than neighboring low-SMN neurons across healthy, spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) genetic backgrounds. These vulnerability states are not fixed: a substantial proportion of MNs progressively increase endogenous SMN abundance, transitioning toward a more resilient state associated with enhanced survival. Low-SMN neurons also exhibit increased spontaneous activity, linking endogenous SMN abundance to neuronal physiology and survival. In vivo, selectively vulnerable MN populations display corresponding shifts in endogenous SMN abundance during disease progression, supporting the relevance of SMN-defined vulnerability states beyond in vitro models. Integrated transcriptomic and proteomic analyses reveal distinct molecular programs associated with resilient and vulnerable states, including RNA metabolism, proteostasis, cytoskeletal organization, neuronal activity and stress responses. Among conserved molecular differences, the anti-apoptotic regulator LMO3 emerged as a candidate mediator of resilience. LMO3 overexpression reduced expression of p53-responsive genes, increased MN survival and shifted neurons toward higher endogenous SMN states. Together, these findings establish endogenous protein heterogeneity as a determinant of dynamic neuronal vulnerability and reveal neuronal resilience as a plastic molecular state that can be identified and therapeutically reinforced.

neuroscience↗

bioRxiv2026

Aquaporin-3 amplifies arsenite genotoxicity as a dose-dependent gene-by-environment interaction in human cells

Chronic exposure to inorganic arsenic affects more than 200 million people, and disease outcome varies widely among individuals with comparable intake. Explanations have centered on arsenic metabolism, which acts on arsenite that is already inside the cell. Uptake sits upstream of metabolism and sets how much arsenite reaches the genome, yet whether it acts as a gene-by-environment modifier of genotoxicity has been asserted far more often than it has been measured. We expressed human aquaporin-3 (AQP3), its point mutants and the paralogues AQP7, AQP9 and AQP10 in three human cell lines, and measured DNA damage as {gamma}H2AX by flow cytometry against a damage threshold re-derived from mock-transfected cells in every experiment. In lung fibroblasts, AQP3 raised the {gamma}H2AX-high fraction 2.36-fold at 5 M arsenite. Because a gene-by-environment interaction widens the genotype gap with dose while an independent genotoxin would not, the combination exceeds what the two factors would produce if they simply added by 6.36 percentage points, which is an interaction. The response required the AQP3 sequence, was suppressed by N-acetylcysteine in proportion to arsenite dose, was absent in RKO cells, and was shared with AQP9 and AQP10, which created an arsenite dose-response in a cell line that had none of its own. Low-input RNA sequencing of 135 ten-cell pools located the effect inside the cell: at an identical 1 M applied dose and within a single batch, AQP3 raised HMOX1 (induced when trivalent arsenic modifies KEAP1 thiols, and so a report of intracellular rather than applied exposure) 3.3-fold, a shift equivalent to 40% of the 1-to-10 M interval on the measured dose-response, while AQP3 without arsenite was indistinguishable from untreated. Duplex sequencing detected no consistent change in somatic mutation frequency from arsenite alone at any dose to 10 M, in an assay that resolved a 26-fold response to N-ethyl-N-nitrosourea; the arsenite-induced mutation frequency at the EGFR locus nonetheless differed 1.61-fold by AQP3 genotype. The interaction is therefore not unique to AQP3, and aquaglyceroporin complement belongs alongside metabolism among the host factors that set arsenic susceptibility.

cell biology↗

bioRxiv2026

Acute head shaking precedes chronic corpus callosum deficits during repeated cocaine exposure in common marmosets

Background: Stimulant exposure can induce acute stereotyped behaviors and chronic alterations in brain connectivity and white matter integrity. However, the temporal sequence and molecular changes linking these cocaine-induced phenotypes remain unclear. Methods: We combined LabGym-based behavioral analysis, longitudinal brain imaging, and cross-species molecular analysis in 11 common marmosets (Callithrix jacchus; seven male, four female): four underwent behavioral and imaging studies, six provided molecular data, and one provided immunohistochemical data. Male Cryab knockout and wild-type mice underwent functional studies. Results: Acute cocaine (5 mg/kg, intraperitoneally) induced rapid, repetitive lateral head movements, defined as head shaking. After 1 and 10 months of repeated exposure, resting-state functional connectivity was altered in sensory, parietal, prefrontal, motor, and hippocampal regions. Changes were detected at 1 month, whereas diffusion tensor imaging showed reduced fractional anisotropy and axial diffusivity in the corpus callosum splenium at 10 months, with no significant genu changes. Cross-species transcriptomic and proteomic comparison identified DPYSL2 and DNM3 as shared axon-associated molecules. Western blotting showed reduced DPYSL2 in brain tissue from cocaine-treated marmosets. CRYAB localized to O4-positive callosal oligodendrocytes and increased following chronic cocaine exposure. Cryab knockout mice had reduced corpus callosum thickness, increased forced-swim immobility, and reduced open-field distance traveled, supporting a role for CRYAB in corpus callosum integrity and depression-related behavior. Conclusions: These findings show that acute cocaine-induced stereotyped head shaking precedes functional connectivity changes and later corpus callosum deficits. Reduced DPYSL2 may be associated with axonal dysfunction, whereas increased CRYAB may represent a response to cocaine-induced white matter stress.

pharmacology and toxicology↗

bioRxiv2026

Integrated pharmacological and structural profiling reveals CB1 residue interaction patterns associated with synthetic cannabinoid receptor agonist efficacy

Background and Purpose: Among new psychoactive substances, synthetic cannabinoid receptor agonists (SCRAs) encompass considerable structural diversity and show wide variation in cannabinoid receptor 1 (CB1) potency and efficacy, but the molecular and receptor-interaction characteristics underlying these differences remain unclear. We assessed the pharmacological profiles of 16 SCRAs and examined structural features associated with differences in CB1 potency and efficacy. Experimental Approach: CB1 agonist activity was measured in a CB1-G15-based Ca2+ assay, and cataleptic effects were assessed in mice. Ligand-receptor interactions were characterized by molecular docking to an active-state CB1 structure. Principal component analysis (PCA) was applied to residue-level interaction profiles, and the resulting scores were tested for associations with CB1 potency and efficacy. Key Results: Potency and efficacy varied widely across cellular and animal assays, with several SCRAs exhibiting different pharmacological responses across the two experimental systems. PCA identified an interaction pattern whose PC1 scores were significantly associated with Emax but not EC50, with contributions from activation-related CB1 residues including the PHE200-TRP356 toggle switch. Molecular descriptor analysis likewise identified structural features significantly associated with in vitro and in vivo Emax. Conclusion and Implications: Our findings suggest that CB1 efficacy is associated with distinct receptor-interaction patterns among structurally diverse SCRAs. Combining functional pharmacology with receptor-level interaction analysis provides structural insight into SCRA efficacy and may help prioritize emerging compounds for further pharmacological and behavioral evaluation.

pharmacology and toxicology↗

bioRxiv2026

Restoration of circulating 2-arachidonoylglycerol levels attenuates cocaine self-administration through endocannabinoid-dopamine interactions in common marmosets

To date, no pharmacotherapy has been approved for cocaine use disorder. Endocannabinoid signaling is closely related to dopamine-dependent reinforcement and may regulate cocaine-related behaviors. In this study, we used common marmosets (Callithrix jacchus) to investigate whether 2-arachidonoylglycerol (2-AG), an endogenous cannabinoid lipid, reduces cocaine reinforcement in a non-restraint oral self-administration model. Marmosets performed oral cocaine self-administration under a fixed-ratio 1 schedule, and cocaine intake was confirmed using plasma benzoylecgonine detection. Dopamine transporter (DAT)-related positron emission tomography (PET) signal, DAT and G protein-coupled receptor 55 (GPR55)-associated fluorescent signals, GPR55/DAT immunofluorescence, and synaptosomal dopamine responses were assessed using 18F-N-(3-fluoropropyl)-2{beta}-carboxymethoxy-3{beta}-(4-iodophenyl) nortropane (18F-FP-CIT) PET imaging, ex vivo fluorescent ligand imaging, confocal microscopy, and dopamine quantification. Marmosets acquired oral cocaine self-administration with preferential active lever responding. Repeated cocaine self-administration reduced striatal DAT-related 18F-FP-CIT PET signal in vivo. In striatal slices, cocaine decreased fluorescent false neurotransmitter (FFN102)-associated DAT signal and increased T1117-associated GPR55 signal. Systemic 2-AG pretreatment reduced cocaine self-administration while restoring diminished circulating 2-AG levels. GPR55 and DAT immunofluorescent signals were colocalized in marmoset brain sections, and 2-AG enhanced cocaine-induced dopamine elevation in synaptosomal preparations. Taken together, these findings suggest that 2-AG attenuates cocaine-taking behavior and may be associated with GPR55/DAT-related dopaminergic responses in common marmosets.

pharmacology and toxicology↗

bioRxiv2026

A mutation-agnostic and allele-specific ASO strategy demonstrates potent functional rescue and retinal preservation in RHO-linked retinitis pigmentosa

Autosomal dominant retinitis pigmentosa (adRP) caused by RHO mutations is a leading form of inherited retinal degeneration. Extensive allelic heterogeneity of RHO pathogenic variants limits the translational applicability of mutation-specific gene therapies. To address this, we developed SNARE (SNP-guided Silencing of Aberrant RHO Expression), a mutation-independent, allele-specific antisense oligonucleotide (ASO) strategy. SNARE selectively suppresses mutant RHO transcripts by targeting the common, benign c.-26A/G single-nucleotide polymorphism (SNP) as an allelic discriminator. Candidate gapmer ASOs were screened in engineered reporter lines and validated in patient-derived retinal organoids, identifying RHOligo-A as the lead c.-26A-targeting candidate. In vitro, RHOligo-A achieved robust, preferential knockdown of the target allele, improving RHO localization in retinal organoids, and demonstrated a favorable safety profile with minimal transcriptomic off-target effects and no detectable immunostimulatory activity. Subsequent validation in a novel, humanized RHOP347L/WT mouse model, achieved sustained c.-26A-linked allele-selective suppression, retinal structure preservation, and significantly restored visual function, upon a single intravitreal administration. These findings establish RHOligo-A and SNARE as a scalable, mutation-independent therapeutic platform with strong translational potential and substantial clinical reach for RHO-associated adRP.

genetics↗

bioRxiv2026

Spatial second-order features predict glioma malignant transformation

Isocitrate dehydrogenase (IDH) mutant gliomas often transform from low to high grade aggressive tumors. The genetic and molecular drivers of this Malignant Transformation (MT) are poorly understood, and predicting whether a patient will undergo MT is an unmet challenge of high clinical relevance. To stratify patients by MT risk, we applied integrated spatial DNA and RNA profiling to biopsies from 18 retrospective glioma patients which will either remain stable, undergo MT or have already transformed. The resulting dataset consisted of >600,000 single cells, measuring 962 DNA loci, 1150 RNAs, and their spatial locations. Using this dataset, we found that genetic copy number alterations (CNAs), cell types compositions, and cellular neighborhoods did not predict MT. Instead, second order effects i.e. pairwise interactions, are highly predictive of future transformation. First, we identified abnormal chromosomal contact patterns that clearly separate future stable versus future MT samples. Second, we identified 24 ligand-receptors (LR) pairs cross-expressed in neighboring cells as the main molecular factors predictive of transformation and recurrences. We then validated a cross-expressing pair of ENPP2-LPAR1 interactions with a separate cohort of patient samples. In addition, using the LR+ cell pairs as an anchor, we identified cell signaling-specific gene expression programs that can predict from bulk or single cell RNAseq data the time to recurrence. We used a cell-interaction-based foundation model (CIFM) optimized on the spatial RNA data in forward simulations and identified potential myeloid signaling factors involved in MT. Lastly, we analyzed the effect of detection sensitivity on the ability to capture pertinent LR+ neighboring cells by down-sampling transcript and showed that the ability to detect cross-expressing signaling LR transcripts (typically <10 copies per cell) decays rapidly with lower sensitivity, but is more robust to down-sampling of the areas of the tissue imaged. The importance of second-order features suggests that increased depth and dimensionality of data on a smaller quantity of samples can provide valuable insight, and that high-sensitivity and multiple-modalities spatial approaches can help identify markers to risk-stratify patients, aid in therapeutic decision making, and uncover potential therapeutic targets.

cancer biology↗

bioRxiv2026

Complementary remodeling strategies distinguish human subcutaneous and omental adipose tissue

BackgroundSubcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) differ in their metabolic risk, but whether they retain distinct transcriptional identities and remodeling programs in adult humans remains unclear. MethodsBulk RNA sequencing was performed on 29 adipose specimens from 19 patients, including 10 paired SAT-VAT samples, along with baseline CT-derived depot area and attenuation measurements. The findings were compared with Masson trichrome staining and CD68 histology in an independent cohort of 30 patients and validated using GTEx adipose tissue data and an external human single-nucleus atlas. ResultsSAT and VAT showed distinct transcriptomic identities. SAT was enriched for a mesenchymal patterning program characterized by TBX15 and SHOX2, whereas omental VAT exhibited a mesothelial-stromal signature marked by UPK3B. These depot-specific identity signals remained significant after adjusting for BMI, age, and measured cellular signatures. SAT area correlated with extracellular matrix remodeling, whereas VAT area correlated with vascular-hypoxia signaling. These associations were attenuated after BMI adjustment, indicating that remodeling was linked to overall adiposity. In an independent histological cohort, SAT exhibited substantially greater fractional fibrosis than VAT, whereas VAT demonstrated a markedly higher storage-to-scaffold index. Depot-associated transcriptional effects were independently reproduced in the external datasets. ConclusionsHuman SAT and omental VAT retain distinct tissue identities and exhibit complementary remodeling strategies. SAT preferentially adopts a mesenchymal-ECM scaffold program, whereas VAT favors mesothelial-stromal and vascular remodeling programs. These findings support a storage-versus-scaffold framework for adaptation of human adipose tissue to chronic excess energy.

developmental biology↗

bioRxiv2026

Image-Guided Injectable Niche for Hepatocyte Transplantation

Liver transplantation remains the standard of care for end-stage liver failure, yet it is constrained by donor scarcity, surgical complexity, and restricted access for many patients. Cell-based therapies offer a potential alternative, yet their translation has been hindered by low engraftment, poor localization, and a lack of delivery strategies that are both effective and minimally invasive. To address these challenges, we developed a new approach termed INSITE (Injected Self-assembled Image-guided Tissue Ensembles), an injectable platform composed of primary human hepatocytes and hydrogel microspheres that can be delivered by image-guided injection and assembled in situ into supportive, vascularizable scaffolds. In vivo, ultrasound-guided delivery into an ectopic site enabled precise graft localization, persistent visibility under noninvasive imaging, and vascular integration. Hepatocytes within these niches remained confined to the scaffold and maintained long-term functional activity. Furthermore, tuning material properties allowed control over scaffold remodeling and vascular recruitment, providing a means to enhance graft function. By integrating image-guided delivery with a modular and supportive scaffold, INSITE establishes a clinically compatible strategy for advancing minimally invasive cell therapies.

bioengineering↗

bioRxiv2026

Classical enhancers couple cis-regulatory logic with transcriptional condensates and 3D genome architecture

Deciphering the regulatory logic of enhancers remains a central question in understanding cell- and tissue-specific gene expression in multicellular organisms. This is particularly pertinent at multipartite enhancer clusters such as super-enhancers, where multiple enhancers contribute to the expression of a single gene. Gene expression has been studied largely through sequence-dependent recruitment of transcription factors (TF) and co-activators, whereas 3D chromatin structure has been attributed to architectural proteins such as cohesion and CTCF1-3. However, the contribution of DNA sequence encoded in enhancers to shaping higher-order genome organization remains poorly understood. Here we show that classical enhancers, embedded within multipartite super-enhancer structures, act as determinant regulatory elements that initiate the gene regulatory cascade by linking DNA sequence recognition to 3D chromatin architecture. Classical enhancers are more evolutionarily conserved and display stronger regulatory activity than facilitator elements, which lack intrinsic enhancer activity but potentiate classical enhancer function. We show that classical enhancers are selectively bound by specific TFs with strong intrinsically disordered regions, such as NFE2L2 in liver cancer cells, capable of driving transcriptional condensate formation through phase separation. NFE2L2 depletion reduced enhancer activity and induced widespread chromatin reorganization, characterized by increased cohesin and CTCF occupancy at super-enhancer boundaries and beyond. This "cohesin clogging" impaired DNA loop extrusion, led to formation of smaller topologically associated domains, and weakened enhancer-promoter contacts. These findings highlight that sequence-specific TFs have multifaceted roles beyond transcriptional control, establishing a direct mechanistic link between enhancer sequence, TF binding, condensate formation, and 3D genome organization, with the regulatory logic being encoded in the DNA sequence itself.

genomics↗

bioRxiv2026

Spatiotemporal Mapping of Tertiary Lymphoid Structure Heterogeneity Shapes Immune Niches and Clinical Outcomes in Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal malignancy with limited therapeutic options. The spatial architecture and functional diversity of tertiary lymphoid structures (TLSs) in iCCA remain unclear. Here, we present a multimodal spatial atlas of TLSs and identified Intra-tumoral TLSs (iTLSs) as independent prognostic markers. Bulk proteomic profiling of 214 discovery and 155 validation cases identified a four-tier TLS-based TME classification system and supported development of a TLS-predictive random forest classifier. Imaging mass cytometry revealed that iTLS tumors harbor structured immune architectures, where M1-like tissue-resident macrophages (RTMs), dendritic cells, and CXCL13 CD4 T cells co-localize to form antigen-presenting neighborhoods (apc-CNs) spatially coupled to TLS core regions (TLScore-CNs). Single-cell spatial transcriptomics further resolved 61 TLSs into 14 spatial niches and defined a pseudo-temporal maturation continuum: aggregated, activated, and post-activated. Intra-niche communication, primarily mediated by ifnCAFs, iCAFs, and CXCL12 macrophages, evolved dynamically with maturation. Single-nucleus RNA-sequencing combined with Tangram-based spatial mapping revealed CXCL12 macrophages and iCAFs forming a peripheral band in aggregated TLSs, while ifnCAFs infiltrated TLS interiors during activation. These findings define TLS heterogeneity and provide insights for stroma-directed immunotherapy. TeaserAn atlas of tertiary lymphoid structures reveals immune-stroma interactions in Intrahepatic cholangiocarcinoma.

immunology↗

bioRxiv2025

A unified derivative-like dopaminergic computation across valences

Dopamine activity in the brain affects decision-making and adaptive behaviors. A wealth of studies indicate that dopamine activity encodes discrepancy between actual and predicted reward, leading to the reward prediction error (RPE) hypothesis. Specifically, it has been claimed that mesolimbic dopamine activity conforms to temporal-difference reward prediction error (TD RPE), a teaching signal in machine learning algorithms. Recently, there is growing evidence suggesting that dopamine is also involved in learning during aversive situations. However, the fundamental computation of dopamine activity in aversive situations is still unknown. A plausible but untested hypothesis is that dopamine activity in aversive situations also encodes TD RPE. Here, we tested this hypothesis by using mice in virtual reality. Mice were trained to avoid electrical tail shocks by running out of a virtual shock zone. Using probe conditions with speed manipulation or teleportation, we revealed that the dopamine signal in the ventral striatum follows the temporal derivative form of a value function. Delivering a reward at the end of the track enabled us to observe the integration of aversion and reward in a derivative form. Moreover, the value functions unbiasedly estimated from the recorded signal is consistent with the initial hypothetical form, with a realistic reflection of a received shock distribution. Taken together, our results show that mesolimbic dopamine activity can operate as a unified teaching signal in natural situations with positive and negative valences.

neuroscience↗

bioRxiv2025

Maladaptive inflammatory signaling in old mice impairs colonic regeneration by promoting a sustained fetal-like epithelial state

Aging is associated with a decline in the regenerative capacity of many tissues. Central to this decline is a complex interplay between inflammation and stem cell function. How these two processes are linked and influence regenerative capacity remains unclear. Here, we undertake a comprehensive assessment of age-related changes in the mouse colon at single-cell resolution. A survey of immune and epithelial compartments revealed a hyperactivated inflammatory state in the colon of old mice characterized by the induction of an interferon {gamma} (IFN{gamma}) response signature in immune cells. This does not result in increased inflammation under homeostasis, but triggers a disproportionate inflammatory response, disrupting regeneration after challenge with the enteropathogen Citrobacter rodentium. Colons of old mice exhibit higher production of IFN{gamma} by T and innate lymphoid cells (ILCs) that are associated with reduced Lgr5+ stem cells and decreased epithelial proliferation. Interestingly, we find aged intestinal epithelial cells to be hypersensitive to IFN{gamma} signaling, inducing a regeneration-associated fetal-like gene expression signature that, in turn, renders these cells more sensitive to IFN{gamma}-induced apoptosis. Our findings reveal an age-related imbalance in the interaction between the immune and epithelial compartments in the colon, priming the system for excessive inflammatory responses and the emergence of a hypersensitive epithelial cell state thus derailing proper repair of the intestinal epithelium after injury.

immunology↗

bioRxiv2025

A 3D in vitro model of the human hepatobiliary junction

Cholestasis, or disruption in bile flow, is a common yet poorly understood feature of many liver diseases and injuries. Despite this, many engineered human tissue models of liver disease fail to recapitulate physiological bile flow. Here, we present a 3D multicellular spheroid-based model of the human hepatobiliary junction, the interface between hepatocytes and cholangiocytes often disrupted in liver disease that is required for directing bile excreted by hepatocytes into the biliary ductal system. Building on advances in organoid and spheroid engineering, we co-aggregate human hepatocytes and intrahepatic cholangiocytes into adult hepatobiliary organoids (aHBOs) that structurally connect and functionally transport bile. aHBOs directionally transport bile from hepatocyte bile canaliculi to cholangiocyte-lined ductules, which we visualize through a high-throughput imaging assay. Hepatobiliary junction formation and bile flow dynamics are quantified over time using fluorescent bile acid analogs and AI-assisted image analysis. When subjected to hypoxia-reoxygenation, aHBOs recapitulate features of biliary dysfunction that mimics the cholestasis and ischemia-reperfusion injury that complicates liver transplant. Our findings suggest that 1) a reversible reduction in hepatocyte canalicular function under hypoxia, followed by 2) selective cholangiocyte death upon reoxygenation, are processes that potentially contribute to biliary dysfunction upon ischemic injury. This human-derived, scalable platform provides a phenotypically-relevant in vitro model for dissecting biliary pathophysiology and lays the groundwork for a therapeutic discovery platform for post-transplant ischemic cholangiopathy and other cholestatic liver diseases.

bioengineering↗

bioRxiv2025

Enhancing the protein fitness of interferon-lambda through computational design and glyco-engineering for prophylactic nasal drugs against respiratory viruses

Interferon-{lambda} (IFN-{lambda}), a type III interferon that selectively targets epithelial cells, holds strong potential as an intranasal antiviral due to its ability to suppress respiratory virus replication without inducing systemic inflammation. However, clinical translation of human IFN-{lambda}3 (hIFN-{lambda}3) is hindered by limited thermostability, protease susceptibility, and rapid mucosal clearance. In this study, instability-prone elements in hIFN-{lambda}3 are eliminated through artificial intelligence (AI)-based backbone remodeling and targeted surface hydrophobic patch engineering. A protease-sensitive loop is replaced with a de novo -helix, which shields neighboring hydrophobic patches and forms a new hydrophobic core, yielding an engineered variant (hIFN-{lambda}3-DE1) with enhanced thermostability (Tm >90{degrees}C), protease resistance, and preserved antiviral activity and structural integrity even after extended heat stress (two weeks at 50{degrees}C). Further glyco-engineering introduces an N-linked glycan at a site distant from receptor-binding interfaces, improving solubility, production yield, and diffusion through synthetic nasal mucus. Intranasal administration of the resulting variant (G-hIFN-{lambda}3-DE1) enables effective mucosal penetration and provides robust prophylactic protection against influenza A virus infection in an in vivo mouse model. These findings highlight a robust and versatile strategy that combines AI-driven structural design with glyco-engineering to develop scalable, bioavailable, and functionally enhanced nasal biologics for respiratory virus prophylaxis.

biochemistry↗

bioRxiv2025

Gene syntax defines supercoiling-mediated transcriptional feedback

Gene syntax--the order and arrangement of genes and their regulatory elements--shapes the dynamic coordination of both natural and synthetic gene circuits. Transcription at one locus profoundly impacts the transcription of nearby adjacent genes, but the molecular basis of this effect remains poorly understood. Here, using integrated reporter circuits in human cells, we show that the reciprocal effects of transcription and DNA supercoiling, which we term supercoiling-mediated feedback, regulates expression of adjacent genes in a syntax-specific manner. Using a suite of chromatin state assays, we measure syntax-and induction-dependent formation of chromatin structures in human induced pluripotent stem cells. Applying syntax as a design parameter and without altering sequence or copy number, we built compact gene circuits, tuning the expression mean, noise, and stoichiometry across diverse delivery methods and cell types. Integrating supercoiling-mediated feedback into models of gene regulation will expand our understanding of native systems and enhance the design of synthetic gene circuits.

synthetic biology↗