Biology subjects

Cao, Y.

Publications and source records attributed to Cao, Y..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Hepatobiliary Progenitor-like Reprogramming in Liver Metastases

Metastatic progression requires cancer cells to adapt to the unique constraints of distant organ microenvironments, yet the mechanisms that drive organ-specific adaptations remain poorly understood. Here, we show that the liver actively rewrites metastatic cancer cell identity, driving tumor cells toward a hepatobiliary progenitor-like state. Through integrated transcriptomic, proteomic, metabolomic, and epigenomic analyses of patient-derived rapid-autopsy samples and experimental models, we identify this state as selectively enriched in liver metastases. It is characterized by co-activation of hepatic and biliary/progenitor regulators HNF4A and SOX9 and is observed across multiple epithelial cancers, indicating a conserved response to the hepatic niche. Mechanistically, hepatocyte-derived TGF-{beta} and hypoxia converge to activate a HIF-1-ACLY axis, increasing nuclear acetyl-coenzyme A availability and histone acetylation at hepatic lineage regulatory elements to drive hepatobiliary reprogramming. This coordinated niche-response program can be captured transcriptionally and is associated with inferior overall survival. Disruption of this pathway suppresses hepatic reprogramming and impairs liver metastatic fitness. These findings identify the liver as an active determinant of metastatic cell fate, linking microenvironmental signaling to metabolic and chromatin remodeling programs that enable lineage plasticity. More broadly, they reveal organ-specific reprogramming as a fundamental principle of metastasis and a therapeutic vulnerability in liver metastases.

cancer biology↗

bioRxiv2026

Tirzepatide attenuates atherosclerosis through weight loss-independent anti-inflammatory mechanisms

BackgroundAtherosclerosis is a chronic inflammatory vascular disorder with persistent residual inflammation even after standard lipid-lowering therapy. Mounting evidence from bench to bedside suggests that diabetes and obesity accelerate atherosclerosis development. Tirzepatide (TZP), a dual Glucagon-Like Peptide-1 Receptor/Glucose-Dependent Insulinotropic Polypeptide Receptor (GLP-1R/GIPR) agonist approved for treating diabetes and obesity, has demonstrated proven cardiometabolic efficacy in large cardiovascular outcome trials. However, it remains largely uncertain whether TZP attenuates atherosclerosis independent of its anti-diabetic and anti-obese effects through direct actions on the vasculature. MethodsWe established atherosclerotic mouse models under diabetic, obese, and non-diabetic/non-obese conditions. Analysis of covariance (ANCOVA) and pair-feeding experiments were applied to experimentally decouple weight-dependent metabolic improvement from intrinsic vasculoprotection. Molecular and cell biological assays in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were performed to dissect the underlying signaling mechanisms. ResultsTZP markedly reduced aortic plaque burden and inflammation, restrained necrotic core enlargement, and improved plaque stability across all experimental mouse models. Both ANCOVA and pair-feeding experiments confirmed that these atheroprotective effects were independent of food intake and body weight loss. Furthermore, TZP attenuated systemic and vascular inflammation in Tumor Necrosis Factor- (TNF)-treated C57BL/6J mice, and this protection occurred without changes in body weight or blood glucose levels. Mechanistically, TZP directly targeted endothelial cells and activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/endothelial nitric oxide synthase (eNOS) pathway, increased eNOS phosphorylation and nitric oxide bioavailability, consequently downregulating the expression of the pro-inflammatory adhesion molecules Vascular Cell Adhesion Molecule-1 (VCAM-1) and Intercellular Adhesion Molecule-1 (ICAM-1). ConclusionsTZP arrests atherosclerosis progression through weight loss-independent anti-inflammatory mechanisms. These findings implicate TZP as a promising therapeutic drug for mitigating residual vascular inflammation in patients with atherosclerotic cardiovascular disease (ASCVD), irrespective of glycemic status or obesity. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LITirzepatide exerts direct anti-atherosclerotic effects in preclinical mouse models of atherosclerosis under diabetic, obese, and non-obese conditions. C_LIO_LITirzepatide directly targets endothelial GLP-1R/GIPR and downstream cAMP/PKA/eNOS signaling pathway to suppress NF-{kappa}B-driven vascular inflammation, thereby uncovering a previously unrecognized vasculoprotective mechanism underlying its cardiovascular benefits C_LI What Are the Clinical Implications?O_LITirzepatide exerts direct vascular protective effects independent of body weight reduction, suggesting that its cardiovascular benefits may extend beyond glycemic control and obesity management. C_LIO_LITirzepatide may represent a promising therapeutic drug for addressing residual vascular inflammation in ASCVD patients, including those without overt diabetes or obesity C_LI

physiology↗

bioRxiv2026

Rett syndrome lifespan extension in mice via AI-guided ADAR editing

Rett syndrome is a severe neurodevelopmental disorder primarily caused by mutations in the MECP2 gene. A significant subset of severe cases are driven by nonsense mutations that generate premature stop codons, leading to loss of functional MeCP2 protein. Here, we describe a novel therapeutic strategy that uses endogenous adenosine deaminase acting on RNA (ADAR) enzymes to correct the R168X mutation at the RNA level. Using generative artificial intelligence trained on large empirical datasets, we engineered guide RNAs (gRNAs) that recruit endogenous ADAR to convert the mutant stop codon (UGA) into a tryptophan (UGG) to restore full-length MeCP2. Once incorporated into an optimized expression system based on endogenous small nuclear RNA regulatory elements and packaged into adeno-associated virus, these gRNAs enabled precise RNA editing at the target site with minimal off-target activity across the transcriptome while restoring full-length MeCP2 protein expression in patient-derived induced pluripotent stem cell neurons. Delivered intravenously to an R168X mouse model, the gRNAs achieved ~70% targeted RNA editing and substantially restored MeCP2 throughout the brain resulting in markedly improved Rett-like phenotypes and significantly extended lifespan. These findings demonstrate that AI-guided ADAR-mediated RNA editing is a precise and efficient technology for correcting nonsense mutations, with therapeutic potential for Rett syndrome and other genetic diseases.

neuroscience↗

bioRxiv2026

Dual HER2/HER3 Targeting with Antibody-Drug Conjugates Reveals a Novel Therapeutic Strategy for HER2-Mutant Lobular Breast Cancer

Activating HER2 mutations are significantly enriched in both primary and metastatic invasive lobular breast cancer (ILC), with large public datasets of primary breast tumors linking them to a worse prognosis in ILC. Despite their oncogenic role, no FDA-approved therapies currently target HER2-mutant breast cancers. While the HER2-directed antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd) has shown efficacy in HER2-mutant non-small cell lung cancer, its activity in HER2-mutant ILC remains unknown. Using the Caris real-world database, one of the largest cohorts with survival data in advanced breast cancers, we confirmed that HER2 mutations are more prevalent in advanced ILC than in invasive breast cancer of no special type (NST) tumors, are associated with worse survival in both histologies, yet predict improved response to T-DXd across subtypes, highlighting the need for mutation-directed, histology-informed therapies. Using endogenous HER2-mutant ILC cell lines (UACC3133-S310F, BCK4-L755S) and CRISPR-engineered isogenic ILC models with clinically relevant HER2 mutations (S310F, V777L), we found these mutations drive HER2/HER3 hyperactivation and downstream signaling, conferring increased sensitivity to HER2 tyrosine kinase inhibitors (TKIs) and T-DXd. Mechanistically, HER2 mutants showed enhanced receptor ubiquitination, internalization, and lysosomal degradation upon T-DXd treatment, explaining the observed drug sensitivity. While combining T-DXd with neratinib or the HSP90 inhibitor ganetespib yielded synergistic effects in long-term growth assays, accompanied by increased HER2 ubiquitination, the concurrent hyperactivation of HER3 in HER2-mutant cells suggested that co-targeting HER3 could provide an effective alternative strategy. Accordingly, HER2-mutant ILC exhibited enhanced sensitivity to the HER3-directed ADC patritumab deruxtecan (P-DXd) or LJM716, a HER3-targeting antibody. We further uncovered a previously unrecognized mechanism of P-DXd beyond HER3 ligand blockade and payload delivery: P-DXd promotes HER2/HER3 association, increases HER2 ubiquitination, and enhances T-DXd internalization, resulting in potent synergy with T-DXd. Mechanistically, we identified HER3 extracellular domains I and II as essential for P-DXd binding and for mediating P-DXd-induced HER2/HER3 association, establishing a structural basis for this activity. In vivo, both T-DXd and P-DXd suppressed UACC3133 and BCK4 xenograft growth, with combination therapy trending toward greater efficacy and prevented regrowth of tumors. Extending these findings beyond HER2-mutant ILC, combination treatment with T-DXd and P-DXd demonstrated synergistic activity across multiple breast cancer models, including (i) HER2-amplified NST patient-derived organoids (PDOs) harboring hotspot HER2 mutations, (ii) HER2-wild-type NST PDOs with clinically intrinsic or acquired T-DXd resistance, and (iii) isogenic HER2-mutant ILC PDOs with experimentally induced resistance after prolonged T-DXd exposure. Collectively, these findings support HER2 as an actionable target in HER2-mutant ILC and position T-DXd-based regimens, particularly in combination with HER3 inhibition, as a promising therapeutic strategy for this underserved patient population.

Cancer Biology↗

bioRxiv2026

A Stress-Adaptive Lipid Kinase Axis Defines Metabolic Vulnerabilities in Neuroendocrine Prostate Cancer

Neuroendocrine prostate cancer (NEPC) persists in a profoundly hypoxic microenvironment, yet the mechanisms enabling tumor adaptation to this metabolically challenging niche remain undefined. Here, we identify the lipid kinase PIKfyve as overexpressed in NEPC, functioning as a central node in a stress-lipid kinase axis that drives adaptation to persistent endoplasmic reticulum (ER) stress. Mechanistically, NEPC requires PIKfyve-mediated lysosomal degradation and lipid recycling to maintain metabolic homeostasis under hypoxia. PIKfyve inhibition disrupts lysosomal function, leading to ER stress accumulation and activation of a compensatory, sterol regulatory element-binding protein (SREBP)-dependent de novo lipogenesis program essential for NEPC survival. This stress-lipid axis creates a synthetic vulnerability between PIKfyve and fatty acid synthase (FASN), where dual inhibition synergistically amplifies ER stress, triggers the terminal unfolded protein response, and induces tumor cell death. These findings reveal a metabolic adaptation in NEPC and provide preclinical evidence that co-targeting PIKfyve and FASN can overcome hypoxia-associated stress adaptation.

cancer biology↗

bioRxiv2026

Generalisable tissue-wide molecular reconstruction from histology

Spatial transcriptomics technologies measure gene expression within intact tissues but remain difficult to scale across large tissue sections and patient cohorts. Consequently, many studies rely on tissue microarrays (TMAs) or sparse spatial profiling designs, where molecular measurements are available for only limited tissue regions and are often generated using heterogeneous gene panels. Existing H&E to spatial gene expression prediction methods remain challenged by sparse molecular measurements, partially overlapping gene panels and tissue-wide reconstruction across heterogeneous spatial datasets. Here, we present GHIST+, a framework for tissue-wide reconstruction of single-cell molecular states from H&E histology. GHIST+ integrates cellular morphology, local tissue context and shared tissue representations to extend sparse molecular measurements into tissue-wide molecular maps across heterogeneous spatial datasets. Across multiple cancer types and GTEx breast tissues, GHIST+ reconstructs biologically meaningful tissue-wide molecular organisation from sparse TMA-derived measurements while preserving spatial tissue structure, cell-type organisation and age-associated tissue states across cancer and non-cancer settings. GHIST+ establishes a scalable framework for transforming sparse spatial profiling experiments into tissue-wide molecular maps, enabling cohort-scale molecular reconstruction from routine histology under heterogeneous spatial transcriptomic settings.

bioinformatics↗

bioRxiv2026

Lineage tracing and live-cell imaging reveal that NeuroD1 does not reprogram microglia into neurons

In situ glia-to-neuron conversion induced by a single transcription factor is a promising regenerative strategy for treating central nervous system injuries and neurodegenerative diseases. Previous studies reported that NeuroD1 can induce microglia-to-neuron cross-lineage conversion and improve recovery after brain injury. However, these findings remain controversial. To examine the ability of NeuroD1 in inducing microglia-to- neurons conversion, we employed virus-free genetic lineage tracing systems to specifically induce NeuroD1 expression in microglia, and track the cell fate of NeuroD1-expressing cells at multiple timepoints. Meanwhile, we longitudinally monitored NeuroD1-expressing microglia via two-photon imaging, and characterized their transcriptional profile by single-cell RNA sequencing. All observations across these methods revealed that NeuroD1-expressing cells retained their microglia identity, and cannot convert into neurons, no matter under physical and injury conditions. Instead, sustained expression of NeuroD1 facilitated microglia apoptosis in vivo. Collectively, our findings provide strong evidence that NeuroD1 alone is sufficient to induce microglia-to neuron conversion. This study further highlighted the importance of rigorous lineage-tracing and cell fate mapping strategies for validating in situ glia-to-neuron conversion.

neuroscience↗

bioRxiv2026

Lack of ancestral SARS-CoV-2 imprinting promotes BA.3.2.2 infection in children

The ongoing evolution of SARS-CoV-2 is heavily constrained by population-level immune imprinting. Recent genomic surveillance reveals an unexpected demographic shift: the highly mutated BA.3.2.2 sublineage is significantly enriched in paediatric populations globally, unlike concurrent variants XFG and NB.1.8.1. Here, the evidence demonstrates that this paediatric susceptibility arises from the absence of ancestral Wuhan-strain immune imprinting. Serological analyses reveal a profound failure to neutralise BA.3.2.2 in young children with no ancestral strain exposure. Concurrently, single-B-cell sequencing analyses demonstrate that BA.3.2.2 completely evades Omicron-specific Class 1/4 neutralising antibodies, encoded by IGHV2-5 and IGHV5-51, which dominate the repertoires of weakly imprinted individuals such as young children. Conversely, BA.3.2.2 remains uniquely susceptible to broadly cross-reactive Class 1 antibodies, encoded by IGHV3-53/66, typically enriched in adults with strong ancestral imprinting, such as mRNA vaccine recipients. Critically, sustained transmission of BA.3.2.2 in paediatric populations may catalyse the emergence of secondary variants that combine the paediatric-evading features of BA.3.2.2 with adult-evading Class 1 mutations. This could allow the lineage to breach the imprinted immunity of the adult population, potentially driving widespread global transmission. Together, these findings show that legacy immune imprinting paradoxically provides superior protection against a highly divergent saltation variant, directly explaining the age-biased transmission of BA.3.2.2, and informing future paediatric vaccination strategies.

immunology↗

bioRxiv2026

Tracing the Evolutionary Origin of Interferon to Basal Chordates and Unveiling Its Antiviral Functionality in Jawless Vertebrates

Interferons (IFNs) are essential mediators of antiviral defense in vertebrates, having gradually replaced the RNA interference (RNAi) antiviral mechanism that predominates in invertebrates and plants. To date, IFNs have been identified exclusively in jawed vertebrates, leaving the origin of IFN-based antiviral mechanisms largely mysterious. In this study, by conducting a genome-wide screening of IFN homologs accross various species, we successfully identified seveal IFN homologs from agnatha and lancelet, but not other invertebrates. Notably, both agnatha and lancelet IFN homologs have ability to induce a set of interferon-stimulated genes (ISGs)-like genes, thus tracing the origin of IFN to basal chordate. Using VSV infected peripheral blood mononuclear cells (PBMCs) of Japanese lampreys, we found that lamprey IFNs have antiviral functionality by inducing the expression of hundreds of ISGs through interacting with a heterodimeric complex composed of CRFB7 and CRFB14. In addition to robustly mediating antiviral responses in monocytes, lamprey IFNs exert their effects on variable lymphocyte receptor B (VLRB)+ cells by remodeling the cytokine/chemokine networks to orchestrate antiviral innate and adaptive immunity. Furthermore, cross-species functional comparison of Dicer revealed that changes in residues essential for dsRNA processing occurred concurrently with the evolution of the IFN system. Collectively, these findings uncover the evolutionary origin of IFN and underscore its ancient roles in antiviral response and immune regulation, especially in the takeover of the RNAi antiviral mechanism during the early evolution of vertebrates. Significance StatementInterferons (IFNs) represent a hallmark of vertebrate antiviral immunity, yet their origin has remained elusive. Here, we reported bona fide IFN ligands and cognate receptors in both lamprey and lancelet, two key species placed at the transition from invertebrates to vertebrates. Using the VSV infection model, we further demonstrated the roles of lamprey IFN in antiviral defense and immune regulation. We also found that substitutions in residues essential for Dicer mediated dsRNA processing coincided with the emergence of the IFN system. Collectively, our findings provide key insights into the evolutionary origin of IFN-based immunity and its gradual replacement of RNAi as the dominant antiviral strategy in vertebrates.

evolutionary biology↗

bioRxiv2026

Human hindbrain organoids with segment-specific 5-HT neurons for motor modulation

The serotonergic system regulates human mood, reward, and motor control, and its impairment contributes to debilitating psychiatric disorders. Serotonergic neurons (SNs) arise predominantly in the hindbrain and acquire segment-specific identities and functions; however, the lack of human models remains a challenge to understanding the human serotonergic system. Here, we generate human serotonergic-enriched rostral and caudal hindbrain organoids (hsrHOs and hscHOs) that produce segment-specific SNs with regional identities corresponding to rhombomeres r2-r3 and r5-r8, respectively. hsrHOs and hscHOs produce SNs that recapitulate their in vivo counterparts in transcriptomic profiles, function, and ascending or descending projection preferences. Assembly with human neuromusculoskeletal organoids enables modeling of serotonergic modulation of motor control. This system further reveals that deficiency of the schizophrenia risk gene DISC1 disrupts human serotonergic development and impairs motor modulation. Together, our study establishes a human organoid platform for modeling development, circuit function and disease-associated dysfunction of the serotonergic modulatory system.

neuroscience↗

bioRxiv2026

A Comprehensive Computational Model of the Human head for Designing and Optimizing Visual Brain-Machine Interfaces

Brain-machine interfaces (BMIs) for vision restoration require models that accurately simulate the anatomy and electrical properties of visual pathways. However, current models focus only on isolated structures, such as the retina or brain, and overlook surrounding tissues. Here, we present a comprehensive computational model of the human head, incorporating the entire visual pathway--including the eye, optic nerve, and brain--along with critical neighboring tissues such as the orbit, paranasal sinuses, enabling precise simulations. Validation using human and large animal data demonstrated a strong correlation between the simulated and measured electrical potentials. Component elimination analysis revealed that the optimized comprehensive model outperformed simplified versions. The models utility was demonstrated through multiple applications: (1) comparative analysis of electrical neuromodulation technologies for optic neuropathy, revealing the filed intensity limitations of noninvasive approaches and the safety concerns of invasive intraorbital approach; (2) identification of optimal stimulation site, revealing that transnasal stimulation at the optic chiasm outperformed traditional approaches; and (3) in silico design of electrode arrays for optic nerve prosthetics, demonstrating theoretical advantages in invasiveness and visual field coverage compared to existing retinal and cortical prosthetics. This validated and versatile computational resource supports the development of neuromodulation strategies and visual BMI technologies.

bioengineering↗

bioRxiv2026

Uncovering the latent structure of interwoven population and temporal codes

Population analysis methods have become standard for navigating the complexity of neural data. However, these methods often assume a rate code, neglecting information encoded in the precise timing of spikes. Critically, additional information encoded in bursts of action potentials may be missed. Here, we develop a factor analysis method that disentangles the factors associated with bursts and individual spikes. This enables burst codes to be investigated directly from the structure of the data, without requiring external covariates. We demonstrate that analyzing firing rates alone obscures the latent structure and factors underlying bursts. Applying our method to simulated and experimental data, we show that it can infer the correct latent structure and be used to test for the presence of burst coding. By merging the population and burst coding perspectives, we provide a framework for linking changes in bursting to internal variables involved in attention, perception, and learning.

neuroscience↗

bioRxiv2026

Tripeptidyl peptidase II is essential for maintaining cerebrovascular homeostasis of female mice and represents a novel therapeutic target for vascular dementia

Although cerebrovascular impairment is a known common driver of both Vascular dementia (VaD) and Alzheimers disease (AD), the underlying mechanisms remain poorly defined. Substantial evidence has demonstrated that brain-derived estrogen is essential for cerebrovascular health and neuroprotection. Consequently, therapeutic strategies that can replicate the beneficial effects of estrogen replacement therapy (ERT) in the brain while avoiding its peripheral risks are being actively pursued. Here, we demonstrate that tripeptidyl peptidase II (TPP2) is essential for cerebrovascular homeostasis in adult female mice by orchestrating intracellular Ca{superscript 2} distribution and local estrogen biosynthesis. Specifically, TPP2 deficiency triggers a vicious cycle of Ca{superscript 2} imbalance and estrogen deficiency, thereby disrupting the anticipatory unfolded protein response (UPR). This disruption consequently drives aberrant pexophagic flux and ultimately depletes ether-linked phosphatidylcholine (PC-O)--the essential building block of endothelial cell (EC) membranes. In addition, we identified PC-O as a key facilitator of choline uptake through FLVCR2 and therefore its deficiency causes subsequent choline depletion as well as significantly decreased acetylcholine (ACh). Consistent with the fact that choline is a building block of PC and that ACh is a primary driver of vascular dilation, TPP2 depletion leads to significant cerebrovascular degeneration characterized by narrowed lumens, decreased EC number, and abnormal aggregation of ECs within the blood vessel. Additionally, AAV-mediated specific expression of Far1 in ECs to promote ether-PC biosynthesis significantly increases cerebrovascular volume and diameter in hippocampi of adult female TPP2 knockout (T2KO) mice. Importantly, ectopic expression of Far1 in ECs not only significantly ameliorates memory impairment in global and conditional TPP2-depleted female mice, but also significantly improves memory performance of naturally aged female mice. In total, our findings establish TPP2 as a key determinant of cerebrovascular homeostasis in adult female mice, positioning it as a novel therapeutic candidate for the treatment of VaD and AD.

neuroscience↗

bioRxiv2026

Microglial pruning of extinction-ensemble synapses preserves fear memory

Fear extinction suppresses learned fear without erasing the original memory, yet how competing fear and extinction ensembles are selectively updated remains unknown. Here, we show that microglia preserve fear memory by selectively editing extinction-ensemble synapses during retrieval. In medial prefrontal cortex, microglial processes, but not somata, expand, ramify, and tighten their engagement with extinction-ensemble dendrites and spines, where they preferentially engulf excitatory postsynaptic material and bias spine remodeling toward elimination. This selectivity is instructed by local find-me, eat-me, and dont-eat-me cues: purinergic signaling recruits microglial processes, phosphatidylserine exposure licenses engulfment of extinction-ensemble synapses, and CD47-SIRP protects fear ensembles from removal. Weakening microglial recruitment or engulfment, or removing this protection, accelerates extinction without impairing fear acquisition. These findings identify microglial processes as active gatekeepers of ensemble competition and reveal a neuroimmune mechanism that preserves fear memory by limiting extinction. In BriefDuring memory updating, microglial processes selectively engage extinction-ensemble synapses rather than globally pruning active circuits. Local recruitment, engulfment, and protection cues determine this choice, allowing microglia to preserve fear memory by limiting extinction. HighlightsO_LIExtinction retrieval selectively recruits microglial processes to extinction ensembles. C_LIO_LIMicroglia preferentially engulf excitatory postsynaptic material from extinction ensembles. C_LIO_LILocal "find-me," "eat-me," and "dont-eat-me" cues determine synapse selection. C_LIO_LIDisrupting microglial pruning facilitates extinction without impairing fear learning. C_LI

neuroscience↗

bioRxiv2026

Optoretinography in R9AP-bradyopsia reveals the essential role of G-protein signaling in the human cone elongation response

Human cone and rod outer segments exhibit a rapid shrinkage followed by a slower elongation in response to light, forming the basis of the optoretinogram. The molecular basis of this optical assay of photoreceptor function remains incompletely understood. In mouse rods, the elongation response requires transducin, the G-protein -subunit activated in the initial amplifying step of the phototransduction cascade. Here, we measured human cone outer segment responses in subjects with bradyopsia arising from a triple-deletion mutation in R9AP, the anchor and transport protein for the GTPase-activating protein (GAP) RGS9. Immunoprecipitation showed that the mutant R9AP has greatly reduced affinity for RGS9, predictably reducing the level of RGS9 in the outer segment. The bradyopsia subjects elongation responses had normal activation kinetics, amplitude and photosensitivity, but markedly slowed recovery. These results indicate that normal levels of RGS9 are required for deactivation of the cone elongation response, situating the underlying molecular mechanism within the G-protein cascade at or prior to formation of the GAP complex. The recovery of the cone elongation response, measured in a paired-flash paradigm for stimuli isomerizing up to 75% of opsin, revealed that elongation requires a substrate that is depleted and recovers in a bleach-dependent manner. In particular, recovery from the highest bleaching exposure (75%) tracked the time course of cone opsin regeneration, implying that unregenerated cone opsin produces "dark light", known in rods to arise from constitutive activation of G-protein. Taken together, the results from controls and bradyopsia identify the inactive (GDP-bound) transducin complex as the essential substrate for the human cone elongation response. Suppression of the elongation response in the paired-flash paradigm further revealed the complete profile of the initial fast cone outer segment shrinkage response and reinforced its origin in opsin structural changes. Overall, these results identify key mechanistic elements of the optoretinogram and enable its use as a molecularly interpretable, non- invasive assay of cone function in disease and therapeutic response.

neuroscience↗

bioRxiv2026

Generation and long-term expansion of human pancreatic islet organoids in vitro

The scarcity of expandable, functional human islet cells remains a major barrier to diabetes therapy. Here, we identify PROCR+ cells within adult human islets and establish a defined culture system to generate pancreatic islet organoids. These organoids self-organize into -, {beta}-, {delta}-, and PP cells at near-native ratios, exhibit regulated insulin and glucagon secretion, and support exponential in vitro expansion. Single-cell transcriptomics reveals a unique progenitor-like cell population that is transcriptionally primed for endocrine differentiation but shares molecular features with fetal trunk cells and endocrine progenitors. When transplanted, the organoids rapidly ameliorate hyperglycemia in diabetic mice. Importantly, in a non-human primate model, intraportal transplantation of these organoids reduced exogenous insulin requirements, restored glucose-stimulated C-peptide secretion, and achieved sustained glycemic control-representing a critical step toward clinical translation. This study provides a strategy for expanding human islet organoids, offering a scalable platform for diabetes treatment, disease modeling, and regenerative medicine.

cell biology↗

bioRxiv2026

Protocol-dependent cardiomyocyte states determine disease modelling capacity of human iPSCs

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are widely used to model cardiovascular disease, yet numerous differentiation protocols generate cardiomyocytes with heterogeneous molecular and functional properties, complicating experimental design. Here we systematically compare sixteen commonly used cardiomyocyte differentiation protocols and characterize their resulting cell states using single-nucleus RNA sequencing, functional phenotyping and computational integration with human genetic data. Despite similar cardiomyocyte yields, protocols produced distinct transcriptional programs, subtype compositions and physiological properties. By integrating protocol-specific gene expression signatures with genome-wide association studies of cardiovascular traits, we identify cardiomyocyte states enriched for genetic architectures underlying specific diseases. These analyses accurately predict protocols most suitable for modelling particular disease contexts, including electrophysiological defects associated with Brugada syndrome and metabolic vulnerability relevant to myocardial infarction. Our results demonstrate that differentiation protocols encode biologically distinct cardiomyocyte states with differential disease relevance and establish a framework for aligning stem-cell differentiation strategies with human complex trait genetics to guide model selection. This approach enables rational design of iPSC-based disease models and highlights how population-scale genetic data can inform experimental systems in stem cell biology.

systems biology↗

bioRxiv2026

Ryder: Epigenome normalization using a two-tier model and internal reference regions

Motivation: Sequencing-based epigenomic profiling methods are powerful but suffer from technical variability that complicates cross-sample comparisons and can obscure true biological signals. While existing normalization methods using spike-in controls or computational approaches have been proposed, they often rely on assumptions that may not hold across diverse experimental conditions or require additional data types. Results: We present Ryder, a flexible and robust Python package for the normalization of epigenomic signal tracks. Ryder leverages stable internal reference regions, such as invariant CTCF binding sites, to correct for technical artifacts genome-wide. Our results show that it effectively adjusts both background noise and signal intensity, ensuring accurate signal alignment across samples while preserving genuine biological differences. We demonstrate that Ryder performs robustly across diverse assays including DNase-seq, CUT&RUN, ATAC-seq, MNase-seq, and ChIP-seq, with or without spike-in controls. By reducing technical noise, Ryder improves the detection of genuine biological changes, such as quantitative reduction of chromatin accessibility at key enhancer elements by depletion of BRG1, a key subunit of the chromatin remodeling BAF complexes. Availability and Implementation: The Ryder source code, documentation and test data are freely available at: https://github.com/YaqiangCao/ryder . The software version used in this study is archived at Zenodo: https://zenodo.org/records/21267457 .

bioinformatics↗