Biology subjects

Agarwal, S.

Publications and source records attributed to Agarwal, S..

At least 19 recordsLinked to original sources

bioRxiv2026

The scaffold nucleoporin Nup188 undergoes liquid-liquid phase separation

Components of nuclear pore complexes are broadly classified into two categories: intrinsically disordered FG-repeat nucleoporins such as Nup98, which can undergo liquid-liquid phase separation, and scaffold nucleoporins, which form the NPC's structural framework. The basis for crosstalk of these nucleoporins within NPC and beyond is poorly understood. Here we show that human Nup188, a helical inner-ring scaffold protein, forms condensates both in purified form as well as at endogenous level. This condensate exhibits fusion, dissolved in 1,6-hexanediol and recovered after photobleaching, with dynamics and material properties comparable to those of hNup98 condensates. Moreover, these condensates are not colocalized to annulate lamellae. Our analysis also revealed that hNup188CTD is sufficient to form condensates and MD simulations revealed the key roles of IDR1-3 for condensation. Notably, hNup93 expression along with hNup188 suppressed the condensate formation. Together, these findings indicate that a structured scaffold nucleoporin can adopt a condensate-forming state via its CTD and Nup93 binding can suppress this phenomenon.

biochemistry↗

bioRxiv2026

Environment-driven active transport of influenza A virus

Biological media such as airway mucus and extracellular matrix are usually viewed as transport barriers that particles cross by passive diffusion or with internal engines. We show instead that a particle can move actively by modifying the landscape it traverses, creating environmental memory and directional cues in two and three dimensions. Influenza A virus (IAV) realizes this principle through its envelope proteins hemagglutinin (HA) and neuraminidase (NA), which bind and cleave sialylated glycan receptors, respectively. Combining theory, simulations and single-virus tracking, we connect bind--cleave kinetics and HA--NA organization to macroscopic transport. Cleavage dissipates chemical free energy, biases rebinding to the edited landscape and leaves a trail that shapes future encounters. In heterogeneous receptor landscapes, multivalent binding biases motion toward higher receptor density, while receptor destruction by NA can amplify this bias by sharpening the contrast sampled by HA. Experiments on reconstituted glycan membranes show that IAV steps are biased up local receptor gradients, as predicted. The theory suggests that virion-to-virion variability can distribute transport functions across a population, providing a physical hedge against complex receptor environments. Together, these results establish environment-driven active matter as a mechanism for motorless transport powered and guided by chemical modification of the environment.

biophysics↗

bioRxiv2026

Biomineralization from platelet δ-granules as the origin of cardiovascular calcification in humans and other animals.

Cardiovascular calcification is present in practically all cardiac diseases, which are the top killers in the world today1, and is particularly associated with atherosclerosis2, aortic stenosis3 and rheumatic fever4. If not the direct cause of death, calcification contributes considerably to complications that can lead to heart failure5. Nonetheless, the origins and mechanisms of cardiovascular calcification are still strongly debated3,6-12. Just over a decade ago, it has been reported that nano and micron-sized calcified spherical particles, formed from a single crystal of magnesium-containing calcium phosphate, were the first calcified structure that could be detected in cardiovascular tissue13. These particles were found even before any sign of cardiac disease was present and were present in all stages of cardiac diseases13. The ubiquity of these particles suggests their importance for the origins and development of cardiovascular calcific diseases. Here, we show that these particles originate from platelet {delta}-granules and are present in mammals, birds and lizards. Based on our results, we suggest a new mechanism for the origins of these particles, complementing existing models of cardiovascular calcification7,14, and bringing a new, early, and hitherto unaccounted key event in the process of cardiovascular calcification. This new mechanism model, along with a better understanding of the early stages of cardiovascular calcification, could open the path for the development of pharmacological prevention and treatment solutions for several cardiac diseases.

pathology↗

bioRxiv2026

Efficient Isolation and Phenotypic Characterization of Primary Rat Pulmonary Pericytes

Pericytes are essential regulators of pulmonary vascular homeostasis, but their isolation from rat lung tissue is challenging because no single marker uniquely identifies them and contaminating fibroblasts, endothelial cells, hematopoietic cells, and vascular smooth muscle cells can persist during isolation and culture. Here, we describe a rapid and efficient protocol for the isolation of primary rat pulmonary pericytes using sequential magnetic depletion of CD45-positive hematopoietic cells and CD31-positive endothelial cells, followed by positive selection for NG2-positive cells. The isolated cells were expanded in culture and characterized by immunofluorescence and functional co-culture assays. Cultured cells displayed typical pericyte morphology and expressed the pericyte-associated markers NG2, PDGFR{beta}, and 3G5, with minimal expression of CD31, CD45, PDGFR and MYH11. In endothelial cell-pericyte ECM gel co-culture assays, isolated pericytes associated with endothelial cords and were frequently observed near network branch points and junctions, further supporting their pericyte identity. This method yields an enriched population of primary pulmonary pericytes suitable for downstream applications, including cell culture and functional studies. Overall, this streamlined protocol provides a practical platform for studying pulmonary pericyte biology in rat models of health and vascular disease.

cell biology↗

bioRxiv2026

Bead-based RNA Detection with Cas13a: Multi-scale Design and Validation

CRISPR-based assays offer sensitive and specific nucleic acid detection for a broad range of diagnostic and surveillance applications. While standard solution-based assays are simple, they typically lack the ability to detect multiple targets individually without the need for more complex instrumentation and multi-step workflows. Surface-based strategies offer greater potential for multiplexing since the identity of a target can be associated with a location on a slide or a specific bead, as in the case of DNA arrays or Luminex bead assays. However, the design principles for optimizing surface-based CRISPR assays have yet to be developed. Here we present a multi-scale analytical framework integrating nanoscale enzyme kinetics, microscale diffusion constraints, and macroscale assay parameters that we use to develop a bead-based Cas13a assay where the bead becomes fluorescent when the target RNA for that bead is detected. The assay, which we call SurfCas, uses surface-bound guide RNAs and reporter RNAs to detect one or more soluble target RNAs in a one-pot assay. We show that SurfCas has a sensitivity approaching bulk reactions, and we demonstrate multiplexed detection of target RNAs in a complex biological matrix. The design principles we establish for surface-based CRISPR assays offer guidance on how to further improve SurfCas sensitivity and scale multiplexed detection of target RNAs in a single reaction.

bioengineering↗

bioRxiv2026

Programming the Internal Architecture of Synthetic Compartments by Coassembling Filamentous and Liquid DNA Phases

Living cells rely on filaments and condensates as key organizers of their interior. Developing these structural primitives together inside synthetic compartments using programmable components is a step toward building functional synthetic cells, composite biomaterials, and synthetic tissues. Here, we demonstrate that DNA nanotubes and DNA condensates can be co-assembled within cell-sized compartments, including water-in-oil droplets and giant unilamellar vesicles (GUVs). The two nanostructures form as expected, producing a single condensate surrounded by nanotubes in diverse morphologies that depend on DNA and salt concentration, as well as compartment size. By incorporating photoactivatable DNA linkers, we can control the order of assembly and trigger reconfiguration of nanotube networks into ring-shaped bundles enclosing a condensate, architectures reminiscent of a cellular nucleus within a cytoskeletal ring. An isothermal assembly protocol based on monovalent salts further extends this approach to GUVs. Together, these results establish DNA filaments and condensates as programmable, composable organizers of synthetic cell interiors.

synthetic biology↗

bioRxiv2026

NEK1 autophosphorylation is disrupted by amyotrophic lateral sclerosis-associated missense variants: activity biomarkers and structural insights

Rare variants in NEK1, encoding a serine/threonine kinase, are amongst the most consistently implicated genetic contributors to amyotrophic lateral sclerosis (ALS), reported in approximately 2-3% of cases. Yet, whilst recent studies have characterised the cell biological consequences of NEK1 loss-of-function, the biochemical effects of ALS-associated missense variants on kinase activity have not been directly investigated. This distinction is mechanistically important, because missense alleles encode mutant proteins rather than simply reducing protein dosage. Here, we provide the most comprehensive cell-based phosphoproteomic map of NEK1 phosphorylation to date, identifying ten recurrent phosphorylation sites across independent expression and acquisition conditions. We experimentally assign pSer14, pThr156 and pSer418 as NEK1 autophosphorylation sites using kinase-dead controls, targeted extracted ion chromatogram analysis, phosphosite mutagenesis and phosphospecific antibodies. Leveraging activation-loop pThr156 as a readout of NEK1 activity, we assessed nine ALS-associated missense variants spanning the major functional regions of the protein. Amongst catalytic-domain variants, R261C produced the most robust reduction in pThr156 autophosphorylation, R232C produced a smaller reduction, and R232H increased pThr156; the basic-region variant, A313T, also showed a smaller reduction. Structural modelling provides a mechanistic framework for understanding these variant-specific effects. Our study establishes the first activity-based framework for functional classification of NEK1 missense variants, and provides direct evidence that ALS-associated missense variants can alter NEK1 autophosphorylation through a mechanism distinct from simple haploinsufficiency. The phosphospecific antibodies, isogenic cell lines and curated phosphoproteomic datasets generated here provide a community resource for future studies of NEK1 regulation, variant interpretation and therapeutic target validation.

neuroscience↗

bioRxiv2026

Sex-linked Lung Estrobolome May Contribute to Pulmonary Hypertension Penetrance of Bmpr2 R899X Mutation via an ET-1high Endoregulatory Macrophage Phenotype

Mutations in the bone morphogenetic protein receptor 2 (BMPR2) are a major genetic driver of pulmonary arterial hypertension (PAH), yet their penetrance is strikingly sex-biased: females are disproportionately affected, while males experience poorer outcomes. While hormonal and chromosomal factors have been implicated, the biological basis for this disparity remains not fully understood. Here, we investigated the role of the lung microbiome in sex-linked PAH pathogenesis. We hypothesized that increased BMPR2 mutation penetrance in females is partly driven by the accumulation of potent vasoactive molecules, such as endothelin-1 (ET-1), in response to lung microbiome dysbiosis. Using humanized Bmpr2+/R899X mice, we integrate lung metagenomics with basic functional immune profiling to show that females develop a distinct microbiome profile, characterized by increased microbial-derived lipopolysaccharide (LPS), potentially fueling the pathogenic effects of the estrogen metabolite 16-hydroxyestrone (16-OHE). These signals converge on macrophages, where co-exposure led to a hyperactivated state characterized by enhanced phagocytosis and ET-1 secretion. Tissue-level analyses confirmed immune cell infiltration and spatial association with elevated ET-1, providing evidence that these factors may contribute to the onset of sex-linked PAH. Taken together, these findings identify a previously unrecognized microbiome-estrogen-immune axis that amplifies BMPR2 dysfunction and provides a mechanistic basis for female-biased disease penetrance.

immunology↗

bioRxiv2026

Saturating variant analysis of TERC redefines the human telomerase reverse transcription mechanism

Genetic variation at the TERC locus, encoding the telomerase RNA component, is associated with human diseases and lifespan, but a comprehensive functional annotation of this critical non-coding RNA is lacking. Here, we performed saturating variant analysis of TERC in cells and unexpectedly redefine the human telomerase catalytic mechanism. Telomerase is understood to precisely reverse transcribe six TERC templating residues to generate GGTTAG repeats, however our screen identified dominant negative effects expected of templating bases inconsistent with this annotation. In vitro and in cells, we found human telomerase uses not six, but eight TERC residues flexibly for reverse transcription, most commonly yielding GGGTTA, but variably up to AGGGTTAG. An evolutionary change in TERC adjacent to the template explains the long-standing misannotation, reversion of which shifts the main templating register in TERC back to the one assumed for decades. Remarkably, this template-adjacent change also yields hyperactive TERC variants that rapidly lengthen telomeres when introduced into cells, including those from patients with genetic telomere diseases. By functional analysis of TERC variation in human cells, our work revises core tenets of telomerase reverse transcription and provides a new mechanistic model to inform the development of telomere-directed therapeutics.

molecular biology↗

bioRxiv2026

From APOE Genetics to AI-Designed Drug Candidates: An Integrated Pipeline for Oral, Brain-Penetrant ACAT1 Inhibitors in Alzheimer's Disease

For more than 55 million people living with dementia worldwide, no oral disease-modifying treatment is currently available. Alzheimers disease (AD) remains one of the most urgent unmet needs in neurology, with recent genetic evidence estimating that 72-93% of AD burden is attributable to common APOE allelic variation. Mechanistically, the APOE {varepsilon}4 isoform impairs cholesterol transport in the brain, promoting cholesteryl ester accumulation in microglia; these lipid-laden cells lose phagocytic capacity for amyloid-{beta} clearance and autophagy-mediated degradation of phosphorylated tau, linking a single upstream metabolic disruption to both hallmark pathologies of AD. ACAT1/SOAT1, the brains predominant cholesterol-esterifying enzyme, is an attractive therapeutic target: its inhibition reduces cholesteryl ester formation, restoring microglial A{beta} clearance, reducing A{beta} production, and promoting tau degradation via autophagy, supporting a multimodal mechanism not addressed by currently approved therapies. However, prior ACAT inhibitor programs were limited by isoform non-selectivity, excessive lipophilicity, and lack of CNS optimization. Addressing these constraints, we present the CuraGenAI Drug Discovery Platform for the design of oral, brain-penetrant ACAT1-targeted small-molecule candidates. The pipeline integrates scaffold-constrained generative chemistry with multi-objective ADMET optimization across more than 30 criteria. From approximately 6 million generated molecules, multi-stage filtering yielded approximately 7,300 CNS-optimized candidates with predicted oral brain-penetrant profiles distinct from prior ACAT clinical compounds. Three nominated lead candidates showed predicted BBB probabilities >0.93, favorable CNS drug-like physicochemical profiles, and stable ACAT1 binding poses, and are prioritized for synthesis and in vitro validation.

neuroscience↗

bioRxiv2026

Microplastics Disrupt Predator-Induced Plasticity in Daphnia across Behavioral, Morphological and Molecular Levels

Microplastics (MP) are widespread in aquatic ecosystems and pose a threat to freshwater biodiversity. While numerous studies examine physiological effects on aquatic organisms, less is known about how MP alter chemically mediated interactions that regulate predator-prey dynamics. Predator-induced defenses in Daphnia depend on detecting kairomones and represent an important form of adaptive phenotypic plasticity. Whether MP interfere with these responses, and through which mechanisms, remains unclear. Here, we show that polystyrene MP impair predator-induced defenses across Daphnia species by disrupting predator-cue-mediated plasticity at the behavioral, morphological, and molecular levels. In D. longicephala, chronic exposure to PS fragments weakened Notonecta-induced morphological defenses, whereas additive-containing PS fragments nearly suppressed defense formation and reduced body size. Consistent with these phenotypic effects, proteomic analyses revealed alterations in pathways related to molting and chitin metabolism, linking MP exposure to impaired defense formation. In D. magna, PS particles attenuated fish kairomone-induced diel vertical migration, with stronger effects for larger particles, consistent with reduced effective availability or perception of predator cues. Natural limestone particles caused only minor effects, indicating particle-specific rather than general particle-driven responses. Our findings demonstrate that MP can disrupt adaptive predator-prey interactions with potential cascading consequences for freshwater food webs.

zoology↗

bioRxiv2026

Chemical toxicity of microplastics is stronger than particle effects in D. magna

Microplastics (MP) are ubiquitous environmental contaminants with diverse physicochemical characteristics. Many studies have shown that size, shape, and polymer type are responsible for their toxicity, but this also seems to differ among MP from the same plastic type. One parameter likely contributing to these differences is plastic chemicals, a broad class of compounds intentionally or unintentionally added to plastics during their production and manufacturing. However, knowledge on the composition of plastic chemicals and their effects remains scarce. Therefore, to elucidate the chemical aspect of MP toxicity, we exposed Daphnia magna individuals to MP (PET, PBS, and PDLLA), cellulose, extracted particles (eMP), and methanol-based extracts of these particles for 10 days. Chemicals within such extracts were analyzed via GC-MS. This study was conducted with reduced food availability to investigate plastic effects in an environmentally relevant scenario. The introduction of a high-food control suggests that a more realistic feeding regime might exacerbate the plastic effects of the selected treatments. Our results indicated that, depending on the polymer type, plastic chemicals determine MP toxicity, which varies according to the endpoint investigated (i.e., body length, reproduction, levels of ROS and LPO). Body length, in particular, was significantly impaired by PET and PDLLA extracts, whereas reproduction was affected by most treatments. The investigated biochemical parameters (ROS and LPO) were not affected by the exposure. These results suggest that MP toxicity strongly depends on their chemical composition, whereas adverse effects due to physical properties are present independently of chemical composition across all MP types. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/724551v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@b6ecc8org.highwire.dtl.DTLVardef@17366d7org.highwire.dtl.DTLVardef@164d3f4org.highwire.dtl.DTLVardef@973af0_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2026

A Perturb-seq screen guided by species divergence uncovers pathways for collateral artery formation

Collateral arteries are natural bypasses that can reroute blood flow around arterial blockages, limiting tissue injury during stroke and coronary artery disease. Despite their clinical effectiveness, therapeutic strategies to stimulate collateral artery growth remain unavailable due to our limited understanding of their developmental mechanisms. Remarkably, guinea pigs display exceptionally dense collateral artery networks across various organs, resulting in complete resistance to ischemic damage in the brain and heart. In this study, we compared single-cell RNA sequencing (scRNA-seq) from guinea pig and mouse tissues to identify endothelial cell (EC) gene expression patterns associated with extensive collateral artery development. We then developed an in vivo Perturb-seq platform in mice to test whether genes differentially expressed in guinea pigs influence artery EC specification. This pipeline identified artery repressors that were downregulated in guinea pigs and increased pial collateral abundance when inhibited in mice. Downstream analysis suggests that artery repressors, including WNT and hypoxia response genes, function in two capillary EC subsets--Esm1+ pre-artery and Apln+ angiogenic tip cells. Reduced activity of these repressors allows more ECs to acquire arterial identity, potentiating collateral artery formation. Collectively, our study establishes a strategy for discovering the genes underlying species-specific traits, suggests that guinea pigs have collaterals due to decreased activity of artery inhibitor pathways and hypoxia responses, and identifies novel targets for stimulating collateral artery formation (Graphical abstract). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/721711v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@18b36cdorg.highwire.dtl.DTLVardef@bafbaforg.highwire.dtl.DTLVardef@1e1a6bdorg.highwire.dtl.DTLVardef@189f585_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2026

Myeloid Gi signaling acts as a weight-independent immunometabolic switch controlling systemic insulin sensitivity

Metabolic dysfunction does not necessarily correlate with adiposity. Metabolically healthy obese individuals and insulin-resistant lean individuals represent a fundamental paradox that implicates immune cell intrinsic mechanisms in the pathogenesis of type 2 diabetes. Here, we identify myeloid Gi signaling as a previously unrecognized determinant of whole-body glucose homeostasis. Single-cell transcriptomic analysis of adipose tissue macrophages from obese mice and humans reveals marked alteration in Gnai isoform, suggesting that myeloid Gi signaling is functionally engaged during metabolic disease. Using complementary myeloid-specific rodent models of Gi inhibition (pertussis toxin) and chemogenetic Gi activation (DREADD), we demonstrate that inhibition of Gi signaling improves glucose tolerance and enhances insulin sensitivity under both regular chow and high-fat diet conditions, independent of body weight and energy expenditure. Whereas acute Gi activation in lean mice modestly enhances glucose disposal, the same intervention during diet-induced obesity markedly impairs systemic glucose homeostasis, revealing context-dependent pathway function. Mechanistically, Gi inhibition amplifies macrophage cAMP-CREB signaling to drive IL-6 production, engaging STAT3- and AMPK-dependent pathways in adipose tissue and skeletal muscle to support insulin action. Conversely, Gi activation engages a previously uncharacterized G{beta}{gamma}-mTOR/AKT-JNK cascade, driving IL-1{beta} secretion that directly impairs insulin signaling in adipocytes and myotubes. Pharmacological IL-6 receptor blockade abolishes the metabolic benefits of Gi inhibition, whereas IL-1 receptor antagonism fully rescues Gi activation-induced metabolic dysfunction, establishing these cytokines as obligate downstream effectors. This signaling architecture is conserved in human macrophages, and ATAC-seq profiling reveals chromatin remodeling at cAMP-CREB and IL-6 regulatory pathway loci, consistent with the observed transcriptional reprogramming. Together, these findings establish myeloid Gi signaling as a weight-independent immunometabolic switch that couples opposing cytokine programs to systemic insulin sensitivity and identify this pathway as a therapeutic target in obesity-associated metabolic disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/713834v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@15e4576org.highwire.dtl.DTLVardef@224cd5org.highwire.dtl.DTLVardef@1e24888org.highwire.dtl.DTLVardef@1080bf3_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

physiology↗

bioRxiv2026

Primer- and template-independent RNA polymerization by terminal nucleotidyltransferase TENT4B

RNA synthesis by eukaryotic polymerases requires existing polynucleotides to serve as templates or primers. Here, we describe primer- and template-free RNA generation by human terminal nucleotidyltransferase 4B (TENT4B) via de novo polymerization of free nucleotides. We observed that recombinant TENT4B (rTENT4B) consumes ATP to yield inorganic pyrophosphate in the absence of a primer or template, concurrent with the appearance of oligomeric poly-adenosine RNA products. Remarkably, 5 labels on {gamma}-phosphate-modified ATP or GTP are retained during polymerization in the presence of unlabeled nucleotide triphosphates (NTPs). These polymers are created at a similar efficiency irrespective of the inclusion of a primer, indicating robust RNA synthesis by rTENT4B from free NTPs. While canonical purine NTPs are favored, nucleotide diphosphates can also serve as substrates for rTENT4B-mediated de novo RNA polymerization. rTENT4B-mediated RNA synthesis using free adenosine nucleotides shows high processivity to generate 1000s-mers, whereas guanosine nucleotide polymerization is strongly and uniformly self-limited and yields a 3-exonuclease-resistant oligonucleotide. Interrogation of other RNA polymerases reveals potential capacity for de novo polymerization using free ATP, albeit at significantly higher substrate concentrations and lower efficiency compared to rTENT4B. Our data provide definitive evidence of efficient template-free de novo RNA synthesis by a eukaryotic polymerase.

molecular biology↗

bioRxiv2026

Activation of PPARγ redirects fibro-adipogenic progenitors to replace ectopic bone with fat in models of fibrodysplasia ossificans progressiva and trauma-induced heterotopic ossification

The pathologic, osteogenic differentiation of fibroadipogenic progenitor cells (FAPs) is the primary recognized contributor to ectopic bone formation in fibrodysplasia ossificans progressiva (FOP) and trauma-induced heterotopic ossification (HO). Both conditions are characterized by up-regulated BMP signaling - the former by a gene mutation rendering the BMP receptor ACVR1 susceptible to activation by inflammatory ligands (Activin A), and the latter by up-regulated presence of BMP2 ligand in the setting of unmutated BMP receptor. We performed an unbiased assessment of FDA-approved therapies which would optimally target the transcriptional aberrations observed in developing FOP and HO lesions based on publicly-available datasets. This analysis uncovered rosiglitazone, a peroxisome proliferator-activated receptor gamma (PPAR{gamma}) agonist as the highest scoring therapeutic option across three data sets for both conditions. Rosiglitazone treatment eliminated ectopic bone lesions in a mouse model of FOP, and replaced these lesions with ectopic adipose tissue; similarly, systemic and local rosiglitazone treatment eliminated ectopic bone lesions in a mouse model of trauma-induced HO and replaced these lesions with ectopic adipose tissue. Our findings were corroborated by a single case report from 2010 showing positive results with rosiglitazone in a non-diabetic patient with FOP, with no subsequent studies. Overall, our findings suggest that a previously FDA-approved therapeutic is likely to be a successful therapeutic agent for both FOP and trauma-induced HO, both conditions for which current therapeutic options remain inadequate. One Sentence SummaryWe show that a previously FDA-approved therapeutic known to induce adipogenesis reduces ectopic bone and induces ectopic fat formation in diseases of heterotopic ossification.

cell biology↗

bioRxiv2026

scAmp analyzes focal gene amplifications at single-cell resolution

Oncogene amplification on extrachromosomal DNA (ecDNA) is a common driver of tumor progression and is associated with acquired drug resistance and poor patient survival. While whole genome sequencing (WGS) studies have revealed the landscape of genes amplified on ecDNA in tumors, it remains challenging to study the subclonal heterogeneity and functional (e.g., transcriptomic) consequences of ecDNA on tumors. To address this, we introduce scAmp: a probabilistic algorithm for detecting and analyzing ecDNA from single-cell datasets. We demonstrate scAmps improved accuracy over WGS approaches on well-characterized cell-lines and its applicability to clinical histopathology. We further showcase scAmp by analyzing 73 patient tumors profiled with single-cell ATAC-seq, where we analyze the subclonal evolution of ecDNA+ subclones and identify the effect of ecDNA amplifications on the chromatin accessibility landscape of cancer cells. Together, we anticipate that scAmp will broadly enable further studies - both retrospective and prospective - that dissect critical questions of how ecDNA affect cancer cells and the tumors in which they reside.

cancer biology↗

bioRxiv2026

A closed-loop cell therapy engineered to autonomously secrete Activin A inhibitor protects from fibrodysplasia ossificans progressiva

Engineered cell therapies present an opportunity for endogenous, site-specific production of therapeutic agents. Here we describe a closed-loop cell therapy which secretes an inhibitor of Activin A, ActR2A-Fc, upon exposure to Activin A. We demonstrate in vivo therapeutic efficacy of this approach in a mouse model of fibrodysplasia ossificans progressiva (FOP), a morbid condition in which patients develop extensive heterotopic bony lesions in response to aberrant sensitivity to Activin A through a mutation in the type I BMP receptor ACVR1 (ACVR1 R206H). To blunt Activin A activity, we designed a transposon plasmid containing the transgene encoding ActR2A-Fc, with expression controlled by the BMP-responsive element (BRE). In cells containing the causative mutation, the BRE is pathologically activated upon exposure to Activin A. FOP-derived marrow cells modified with the BRE-ActR2AFc plasmid exhibited the desired closed-loop functionality, with increased ActR2A-Fc expression upon exposure to Activin A and reduced expression upon withdrawal of Activin A. Engineered marrow cells secreted bioactive ActR2A-Fc, and bone marrow transplantation of FOP marrow cells engineered with the BRE-ActR2AFc transposon into same-sex FOP mice resulted in absence of heterotopic bony lesions. Experiments with labeled, engineered FOP marrow cells verified trafficking of the therapeutic cells to sites at risk for FOP. These data provide proof-of-concept for the therapeutic utility of engineered cell therapy for the treatment of FOP. Significance statementIn this study, we describe our development of an autologous, closed-loop cell therapy which can migrate to sites of tissue injury and locally secreting an inhibitor of Activin A. Through our use of an Activin A-responsive promoter to drive expression of the recombinant Activin A inhibitor, this engineered cell therapy exhibits closed-loop behavior and effectively prevents heterotopic bone formation in a mouse model of fibrodysplasia ossificans progressiva (FOP). We believe that the findings in this manuscript impactful beyond FOP, and provide a blueprint for the development of marrow-derived cell therapies across the disease spectrum.

cell biology↗