Biology subjects

Singh, N.

Publications and source records attributed to Singh, N..

At least 19 recordsLinked to original sources

bioRxiv2026

The RNA m6A reader YTHDF2 depletion phenocopies high glucose induced-cellular stress response in diabetic kidney disease

RNA modification, especially the m6A RNA methylation, has been increasingly associated with several physiological and pathophysiological processes. The proteins that help in establishment of the modification and its removal along with the reader proteins that facilitate the functional outcome, together play an important regulatory role in disease manifestation. Diabetic kidney disease (DKD) is one such example, which is categorized as a multifactorial disorder that involves complex metabolic, oxidative, inflammatory, and fibrotic alterations, mediated through genetic, epigenetic and signalling cascades. However, the contribution of m6A-associated RNA regulators in DKD manifestation is relatively less explored. In this study, using HFD+STZ-induced diabetic mice and high glucose-treated renal cell models, we identified the m6A reader protein, Ythdf2 as a consistently downregulated candidate. YTHDF2 depletion in the renal cell line altered cellular viability, proliferation and increased oxidative stress under high-glucose conditions. Click-IT EU labeling further revealed altered RNA processing effects following YTHDF2 depletion and glucose stress. In corroboration with the above observations in animal and cell line models, independent human DKD transcriptomic datasets demonstrated conserved inflammatory and fibrotic signatures as well as reduced YTHDF2 expression in DKD glomeruli. The YTHDF2 downregulation was found to be inversely correlated with several disease-associated inflammatory and fibrotic genes. Together, these findings identify YTHDF2 dysregulation as a potential post-transcriptional mechanism associated with cellular stress and disease-associated molecular programs in DKD.

physiology↗

bioRxiv2026

Beyond the Axenic Culture Filtrate Secretome: BONCAT and conventional proteomics together reveal the Infection-associated secreted protein repertoire of Acinetobacter baumannii in the human lung Epithelial cell line A549

Acinetobacter baumannii (Ab) is an important opportunistic pathogen responsible for pneumonia, bloodstream infections, and sepsis. Although several virulence determinants have been characterized, the bacterial secreted protein repertoire associated with host cell infection remains incompletely understood. Conventional proteomic analyses of axenic cultures provide valuable information but cannot distinguish proteins synthesized before infection from those produced during host pathogen interactions. This study combined bioorthogonal noncanonical amino acid tagging (BONCAT) with conventional proteomics to characterize the infection associated Ab secreted protein repertoire recovered from human lung epithelial A549 cells. To achieve pathogen selective metabolic labelling, an Ab clinical isolate that had caused neonatal sepsis (Ab7) was engineered to express a methionyl tRNA synthetase variant, MetRSNLL (L10N, Y258L, H299L), capable of incorporating azidonorleucine (ANL) into newly synthesised bacterial proteins. Labelling conditions were optimized using methionine biosynthesis inhibitory amino acids, which reduced endogenous methionine availability without appreciably affecting bacterial growth. Efficient ANL incorporation was confirmed by flow cytometry, confocal microscopy, biotin streptavidin assays, and in gel fluorescence. Metabolic labelling also remained compatible with bacterial intracellular survival. Sequential labelling with azidohomoalanine (AHA) before infection and ANL during A549 infection enabled the temporal assignment of bacterial protein synthesis. Subsequent SDS PAGE fractionation and LC MS/MS identified 152 infection-associated proteins, including 23 containing experimentally detected amino acid analogue modifications. Among these, 13 were AHA only, nine were ANL only, and one contained both analogues. Notably, 117 proteins were not detected in either axenic reference proteome. The infection associated fraction exhibited enrichment of predicted signal peptide containing and outer membrane proteins, while functional annotation identified enrichment of bacterial secretion system components among infection-only proteins. These findings demonstrate the complementary strengths of BONCAT and conventional proteomics: BONCAT provides evidence of bacterial protein synthesis during infection, whereas conventional proteomics expands detection beyond proteins containing identifiable analogue modified peptides. Although extracellular recovery does not establish active secretion, the combined approach reveals a host associated bacterial protein repertoire extending beyond axenic observations. This framework supports future investigations into bacterial adaptation, host pathogen interactions, and the prioritization of candidate diagnostic biomarkers, subject to functional characterization and clinical validation. The study establishes a methodological foundation for investigating infection-associated protein synthesis and release in complex host pathogen environments.

microbiology↗

bioRxiv2026

An AI-powered cloud biofoundry for autonomous biological research

Autonomous experimentation, where an automated system designs, performs, analyzes, and iteratively improves experiments with minimal human intervention represents a transformative scientific research paradigm. However, it remains hampered by the disconnect between computational design and experimental execution. Here we present iCloudBiofoundry, a cloud-accessible, scalable, and self-evolving platform that bridges this divide. A scientific multi-agent system, iBioGenie, plans research workflows by converting natural language research objectives into testable hypotheses and experimentally grounded protocols using literature, databases, and computational tools. A physical AI layer, BiofoundryAI, translates them into executable workflows that run autonomously on distributed robotic biofoundries, and collects the data for iterative design. We demonstrate the end-to-end capabilities of iCloudBiofoundry across three representative case studies spanning enzyme retrieval, protein engineering, and metabolic engineering. By integrating agentic AI with physical AI, iCloudBiofoundry provides a cloud accessible platform that unifies computational design and robotic execution, enabling autonomous biological research.

synthetic biology↗

bioRxiv2026

Collateral Immune Cell Signaling Compromises the Efficacy of T-cell Engaging Therapies for Cardiac Fibrosis

Cardiac fibrosis is causally linked to heart failure progression and survival. Currently, there are no approved treatments that directly target cardiac fibrosis. Recent studies have identified a subset of activated cardiac fibroblasts distinct from myofibroblasts that are marked by fibroblast activation protein (FAP) expression, emerge in the injured and diseased heart through inflammatory signaling, and contribute to fibrosis. Using a genetic mouse model, we demonstrate the potential benefits of FAP+ fibroblast depletion following myocardial infarction. Unexpectedly, while FAP targeted bispecific T-cell engaging antibodies (BiTE(R) molecules) effectively eliminate FAP+ fibroblasts from the heart, they surprisingly lead to accelerated deterioration of cardiac function, enhanced remodeling, and increased scar size. FAP BiTE(R) molecules elicit a robust cytokine response within the heart with prominent activation of interferon gamma (IFNg) and CD40 ligand pathways. Target cell killing was independent of IFNg; and CD40L signaling and blockade of these pathways was sufficient to unmask the protective therapeutic effects of FAP+ fibroblast depletion. Mechanistically, we reveal that IFNg; signaling to fibroblasts drives the differentiation of an independent lineage of activated fibroblasts not typically found in the infarcted heart, which are responsible for the harmful effects of FAP BiTE(R) molecules. Collectively, these findings highlight a previously unrecognized cardiac liability of BiTE(R) molecules and inform the design of the next generation of therapeutics.

immunology↗

bioRxiv2026

CD55-CD319-CX3CR1 flow cytometry gating strategy recapitulates scRNA-seq-defined memory CD8 T cell subpopulations

Human CD8 T cells have traditionally been classified into naive, central memory, effector memory, and terminal effector subsets using CCR7 and CD45RA expression, a framework that has guided immunological research and clinical immune monitoring for nearly three decades. However, recent single-cell studies have revealed transcriptionally distinct CD8 T cell populations, including GZMK-, GZMB-, and central memory-like states, raising important questions regarding their relationship to canonical flow cytometric subsets. Here, we systematically integrated transcriptomic, epigenetic, and phenotypic analyses to evaluate the correspondence between these classification schemes. We demonstrate that conventional CCR7-CD45RA gating generates heterogeneous populations containing extensive mixtures of transcriptionally and epigenetically distinct CD8 T cell states, resulting in poor resolution of biologically meaningful subsets. To address this limitation, we developed a surface-marker framework based on CD55, CD319, and CX3CR1 that accurately identifies transcriptionally defined human CD8 T cell populations using standard flow cytometry. This strategy enables direct isolation of viable cells, including GZMK-expressing cells increasingly implicated in aging, chronic inflammation, autoimmunity, and cancer, which previously could only be identified using intracellular staining or single-cell sequencing. Functional characterization of purified subsets revealed marked differences in proliferative capacity, cytokine production, and cytotoxic activity, demonstrating that transcriptionally defined states possess distinct immune functions. Together, these findings establish a biologically grounded framework for CD8 T cell classification and provide a practical platform for mechanistic studies, biomarker discovery, and cellular immunotherapy applications.

immunology↗

bioRxiv2026

Independent benchmark of H&E-based gene expression prediction in skin

Given the widespread availability of H&E slides, there is considerable interest in determining whether molecular information can be inferred directly from tissue morphology, potentially reducing the need for costly spatial transcriptomic profiling. We assessed three state-of-the-art methods for predicting single-cell gene expression from H&E images across three skin disease contexts and two Xenium panels. As controls, we included simple linear regression models trained on embeddings from multiple foundation models, totalling 16 models evaluated in this study. We show that all models performed poorly: for most genes, prediction accuracy was near zero, and reliable predictions were largely restricted to keratinocyte-associated genes. Predicted expression failed to preserve cell-type identity and spatial organisation, with only keratinocytes forming coherent clusters, while immune, fibroblast, and other dermal populations were extensively mixed. Notably, simple ridge regression on pretrained embeddings matched or outperformed the more complex published architectures, indicating that the predictive signal originates primarily from image representations rather than model design. Our results demonstrate that current H&E-based gene expression prediction methods are not yet suitable for single-cell-level interpretation of spatial transcriptomics in skin tissue.

bioinformatics↗

bioRxiv2026

P. aeruginosa liquid-based pathogenesis triggers HLH-30-dependent metabolic rewiring in C. elegans

Innate immunity is the first line of defense against invading pathogens and is essential for maintaining host survival. While the majority of innate immunity studies have focused on pathogen recognition and antimicrobial responses, increasing evidence suggests that lipid metabolism plays a fundamental role in shaping immune function. Understanding how these metabolic pathways contribute to immunity is crucial in the context of bacterial infections caused by opportunistic pathogens such as Pseudomonas aeruginosa. Host defense against P. aeruginosa requires the coordination of innate immune and metabolic responses; however, the mechanisms linking lipid metabolism to pathogen resistance remain poorly understood. Research into the relationship between lipid homeostasis and innate immunity may reveal factors governing host-pathogen interactions and identify novel strategies to enhance resistance to infection. Here, we demonstrate that P. aeruginosa liquid-based pathogenesis (LK-Pa) triggers a shift in host metabolism which differs from the one observed in response to agar-based pathogenesis. Our bioinformatic analyses revealed a highly similar metabolic profile (enrichment of lipid metabolism) in worms exposed to LK-Pa or the iron chelator phenanthroline, suggesting a shared host response to iron deprivation. We further characterized the host genetic factors driving this metabolic shift and established their importance for host defense against LK-Pa as well as liquid-based pathogenesis by Gram-positive pathogens Enterococcus faecalis and Staphylococcus aureus. Notably, our results indicate that LK-Pa triggers host lipid droplet depletion, an upstream component that leads to increased {beta}-oxidation. Finally, we demonstrate that LK-Pa triggers repression of MXL-3 which results in HLH-30-dependent metabolic rewiring.

cell biology↗

bioRxiv2026

Centrosome maintains the integrity of and repairs mature olfactory cilia in adult Drosophila

Mechanisms of cilium assembly are well studied; however, how mature metazoan cilia maintain their structures and functions in vivo remains unknown. It is also unclear whether the centrosome-derived basal body (BB) directly contributes to ciliary homeostasis. We combined biochemistry, genetics, high-resolution subcellular imaging, and electrophysiology to investigate long-lived ciliated olfactory sensory neurons (OSNs) and their olfactory behaviour in adult Drosophila. Several centrosome assembly proteins are absent, but another subset persists through dynamic protein exchange at the BBs of mature olfactory cilia. At this ciliary base, pericentriolar material (PCM) components, e.g., {gamma}-Tubulin23C, centrosomin, and pericentrin-like protein form an interconnected network required for ciliary maintenance. Adult- and OSN-specific depletions of these proteins, particularly in combination, disrupt accumulation of the heterotrimeric kinesin-2 at the ciliary base and, subsequently, in the shaft, causing loss of ciliary tubulin, EB1, and odorant receptor co-receptor. These dysregulations cause profound loss of ciliary shaft and impair olfactory function/behaviour. The centrosomal kinases PLK1/POLO and Aurora A also work with this PCM network and are required for mature ciliary homeostasis. Remarkably, these defects in ciliary structure, function, and adult olfactory behaviour are reversible, suggesting that this centrosome-derived BB is a dynamic homeostatic epicentre that regulates trafficking and ciliary structural and compositional integrity in vivo. These findings uncover an active, centrosome-dependent, cell-autonomous mechanism for maintaining and repairing mature metazoan cilia in fully grown organs in adulthood and also provide a possible explanation for how deregulation of conserved ciliary base components could lead to progressive, late-onset cilia-related disorders.

cell biology↗

bioRxiv2026

Bioactive Spermidine-Crosslinked DNA Hydrogel for rapid homeostasis and accelerated wound healing

Damage to the skin by trauma, burns, or surgical procedures often results in uncontrolled bleeding, which remains a leading cause of preventable death following injury, yet most conventional hemostatic materials are engineered solely to arrest bleeding and often adhere strongly to the wound bed, causing pain, rebleeding, and disruption of newly formed tissue upon removal. Here, we report a DNA hydrogel that structurally mimics neutrophil extracellular traps (NETs) and is crosslinked using a bioactive small molecule with potent autophagy-inducing, cardioprotective, anti-inflammatory, antioxidant, and mitochondria-protective properties, integrating rapid hemostasis with active support for tissue regeneration in a single biomaterial. The DNA network provides an intrinsically biocompatible, biodegradable scaffold capable of recruiting platelets and erythrocytes to achieve rapid clot formation, while the bioactive crosslinker is released as the network degrades, delivering a sustained cytoprotective and anti-inflammatory stimulus directly at the wound site. The hydrogel was characterised physiochemically and evaluated for cytocompatibility, hemolytic potential, hemostatic efficacy, and wound-healing performance in a murine model. Results demonstrate that the bioactive-crosslinked DNA hydrogel achieves rapid, effective hemostasis, while accelerating wound closure and supporting regenerative tissue remodelling. This dual-function platform offers a promising strategy for next-generation wound-care biomaterials that unite immediate bleeding control with accelerated, natural tissue healing.

bioengineering↗

bioRxiv2026

Multiomic analysis of non-glaucomatous human trabecular meshwork cells.

Glaucoma is an irreversible blinding disease that affects millions of individuals worldwide. Elevated intraocular pressure (IOP), regulated by the trabecular meshwork (TM) in the anterior eye, is the only modifiable risk factor. Human TM cells can be cultured from donor eyes, providing a precious resource for studying factors that induce or prevent glaucoma. The goal of this study was to produce datasets that define the molecular profile of human non-glaucomatous TM cells. Using 18 human TM cell strains cultured from non-glaucomatous individuals deposited from seven laboratories in the USA and UK, this study used transcriptomic, proteomic, lipidomic, and metabolomic analyses to characterize the molecular content of TM cells. The data herein provides the most comprehensive multiomic analyses of human TM cells to date and will be a useful resource for researchers and clinicians in the TM and glaucoma fields.

cell biology↗

bioRxiv2026

EEG biomarkers of reinforcement learning and motivation: A multi-task battery

Serotonin and dopamine make dissociable contributions to reinforcement learning (RL) sub-components, yet we lack neural biomarkers capable of detecting their differential effects. Here, we report the development of a five-task EEG battery designed to probe these dissociable RL mechanisms. Using a model-free analytical approach in healthy volunteers, we identify distinct neural markers across probabilistic instrumental learning, motivational vigour, Pavlovian-instrumental transfer, reversal learning, and a working memory-RL task. A centroparietal P300 tracked incremental learning across three paradigms and showed cross-task convergent validity. Readiness potentials and beta suppression indexed value-based motor preparation, while frontal theta captured Pavlovian-instrumental conflict. The largely independent pattern across markers supports the batterys capacity to detect selective pharmacological effects on distinct neural systems.

neuroscience↗

bioRxiv2026

High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

T cells adopt diverse states in human disease, with tumor-specific CD8 T cells serving as the quintessential example. These cells are critical for immunotherapy response, yet acquire hypofunctional states of exhaustion that impair treatment efficacy. While significant heterogeneity is observed among exhausted CD8 T cells (TEX), the mechanisms directing their unique phenotypes remain unresolved, limiting our efforts to augment TEX cell function. Here, we profile TEX cells in human head and neck squamous cell carcinoma (HNSCC) tumors and define a major mechanism regulating their cell state. With single-cell RNA and T cell receptor (TCR) sequencing of 106,667 tumor-infiltrating CD8 T cells, we define and validate three fundamental TEX subsets, each harboring dominant clonotypes: a progenitor subset (TPEX) and two intra-tumoral populations marked by intermediate (TEX-int) or high expression of inhibitory receptors, including killer cell lectin-like receptors, such as KLRC1/NKG2A (TEX-KLR). Using an in vitro model, we show that TCR avidity-dependent NFAT signaling governs an axis between the TEX-int and TEX-KLR states across human cancers, with high-avidity signaling driving the TEX-KLR state. Additionally, we uncover that interleukin-12 signaling selectively antagonizes high-avidity TEX-KLR-specific genes, despite broadly potentiating activation and cytotoxicity. Finally, we find that TEX-int and TEX-KLR cells are associated with anti-PD-1 response in HNSCC, with both of these cell types enriched near viable cancer cells following treatment. Together, these findings reveal that TCR avidity orchestrates tumor-infiltrating TEX cell states through NFAT signaling, a tunable axis that may be targeted to improve TEX cell function.

immunology↗

bioRxiv2026

Occurrence of Biased mTOR Signaling in Hepatocellular Carcinoma

BackgroundmTOR signaling promotes cell growth and anabolic processes in all eukaryotes. Hyperactivation of mTOR signaling is associated with various cancers along with hepatocellular carcinoma (HCC). HCC is a highly lethal malignancy with multiple aetiologies such as viral infection, alcohol abuse, and metabolic dysfunction. Therapeutic options for HCC remain limited due to an incomplete understanding of oncogenic drivers and poorly characterized mechanisms of disease progression. MethodsVarious regimens of DEN and CCl4 carcinogen dosage were investigated on C57BL/6J mice to induce HCC. The histological analysis for fibrosis and serum markers for liver function were performed. Molecular analyses of oncogenic drivers were performed in the HCC tissues obtained from mice and HCC patients. The impact of inhibition of mTOR signaling was assessed on HCC progression. ResultsWe established a rapid DEN+CCl4 induced (DCI) HCC model in C57BL/6 mice to study disease progression longitudinally. The molecular analysis revealed upregulation of MAPK and downregulation of mTORC1-S6K-S6 signaling in HCC. However, other branches of mTOR such as mTORC1-ULK1, mTORC1-4EBP1, and mTORC2-PKC were upregulated due to the increased expression. Similar observations were found in tissues derived from HCC patients. Furthermore, inhibition of mTORC1 alone by Rapamycin did not reduce HCC progression but Torin 1 mediated inhibition of both mTORC1 and mTORC2 significantly reduced HCC progression. ConclusionsWe propose this biased mTOR signaling modulates mTOR activity towards specific downstream processes that are crucial for cancer cell growth and targeting both the mTOR complexes has better therapeutic potential in HCC. Impact and ImplicationsThis study provides a rapid pre-clinical model for understanding HCC progression and to explore various intervention strategies. The study reports a novel phenomenon of biased mTOR signaling where deregulation of downstream substrate levels modulates the mTOR activity towards the specific branches, the master regulator of cell growth and metabolism. Furthermore, the study suggests that the clinical investigations exploring the rapalogs against HCC should be cautiously considered depending on the aetiology and signaling status of HCC. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/727188v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@ef3714org.highwire.dtl.DTLVardef@1091b25org.highwire.dtl.DTLVardef@d52f2forg.highwire.dtl.DTLVardef@cc158_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDEN and CCl4 treatment generated a well-established HCC within 4 months. C_LIO_LILiver fibrosis and serum markers correlated with HCC progression. C_LIO_LIUpregulation of mTOR pathway substrates create biased mTOR signaling. C_LIO_LIDual inhibition of mTORC1 and mTORC2 reduced HCC progression significantly. C_LI

cancer biology↗

bioRxiv2026

Identification of drug candidates for rescue of SOX17 gene targets in pulmonary arterial hypertension

BackgroundBoth rare and common variants in the SRY-Box Transcription Factor 17 (SOX17) locus are associated with pulmonary arterial hypertension (PAH). SOX17 dysregulation leads to pulmonary artery endothelial cell (PAEC) dysfunction and the obstructive remodelling that characterises PAH. HypothesisImpaired SOX17 expression contributes to the pathogenesis of PAH. Restoring the function of SOX17 or its downstream targets using compounds that mimic its transcriptomic signature will rescue PAEC dysfunction and prevent PAH development. Methods and ResultsWe defined thousands of genes with direct SOX17 genomic binding sites and identified important potential binding partners, including ETS-transcription factors such as ERG by ChIP-seq in PAECs. Through the integration of three PAEC RNA-seq datasets involving overexpression and silencing of SOX17, we defined a robust SOX17 transcriptomic signature. In PAH patients, circulating plasma protein levels of 10 SOX17 signature genes were associated with the SOX17 common risk variants. This included EFNB2 and UNC5B; knockdown of these genes altered the viability and apoptosis of PAECs in response to TNF treatment. The drug-transcriptome database Connectivity Map (CMap) was used to predict novel potential therapeutic compounds to correct the SOX17 transcriptomic signature. Five compounds were selected for in vitro testing and were able to partially reinstate SOX17 target gene expression in PAECs. One compound, BX-912, was selected for in vivo testing as it corrected the levels of multiple target genes, including suppressing Runt-related transcription factor-1 (RUNX1). BX-912 blocked the development of pulmonary hypertension in mice lacking the SOX17 enhancer associated with human disease. ConclusionWe have demonstrated the therapeutic potential of targeting SOX17 in PAH through correction of its gene targets, identifying BX-912 as a lead compound with in vivo efficacy.

pharmacology and toxicology↗

bioRxiv2026

The conserved coordination of acyl-homoserine lactone and PqsE signaling defines the RhlR-dependent quorum-sensing network in Pseudomonas aeruginosa clinical isolates

Quorum sensing (QS) enables Pseudomonas aeruginosa to coordinate virulence and biofilm formation through cell density-dependent signaling. In clinical isolates from patients with cystic fibrosis (pwCF), mutations in canonical QS systems such as lasR and rhlI often lead to altered signaling hierarchies that complicate our understanding of QS regulation during chronic infection. Here, we dissect the relative contributions of the autoinducer N-butyryl-L-homoserine lactone (C4HSL) and the protein binding partner PqsE to RhlR-dependent transcription in CF clinical isolates. Using site-directed mutagenesis to generate RhlR and PqsE variants incapable of responding to C4HSL (RhlR A44M) or dimerizing to interact with RhlR (PqsENI), we show that both inputs are essential for the full expression of QS-regulated virulence factors, including pyocyanin and rhamnolipids. Transcriptomic analyses revealed that C4HSL and PqsE co-regulate a conserved set of 28 RhlR-dependent genes, encompassing canonical virulence loci as well as uncharacterized genes that are likely important for adaptation to the CF airway environment. These findings establish that clinical isolates maintain functional QS circuitry reliant on dual activation of RhlR by both C4HSL and PqsE, revealing a conserved regulatory module that underpins pathogenic behavior across genetically diverse isolates. AUTHOR SUMMARYUnderstanding quorum-sensing regulation in clinical isolates of Pseudomonas aeruginosa is essential to determine how the pathogen persists and adapts within the cystic fibrosis lung. While most studies have focused on laboratory strains, chronic isolates exhibit distinct genetic and regulatory adaptations that complicate our ability to generalize quorum sensing function. Our work defines the coordinated roles of C4HSL and PqsE in activating RhlR-dependent gene expression and virulence factor production in isolates from patients with cystic fibrosis. We identify a conserved core of quorum-sensing-regulated genes that remain dependent on both signals despite extensive genomic divergence. These findings highlight that, even within the evolutionary landscape of chronic infection, quorum-sensing signaling through RhlR remains a central and conserved determinant of virulence. By resolving the dual contributions of acyl-homoserine lactone and PqsE-mediated activation, this work provides a mechanistic foundation for future efforts to therapeutically target quorum-sensing pathways in clinical P. aeruginosa infections.

microbiology↗

bioRxiv2026

ReMind: A Retrospective Self-Report Paradigm for Studying Mind-Wandering Onset During Reading

Mind-wandering (MW) is a frequent and pervasive phenomenon, yet it is commonly assessed using self-reports or probe-based methods that offer limited temporal precision regarding its onset. In this study, we introduce a novel paradigm, ReMind, that estimates the onset and duration of MW episodes during natural reading by combining retrospective self-reports with eye-tracking. Participants indicated the words where they believed their mind started and stopped wandering, and these reports were aligned with gaze timestamps to estimate MW onset. Using data from 44 participants, we examined whether knowledge of MW onset improves the detection of MW from eye-tracking signals. To evaluate relevance for both self-report and thought-probe paradigms, we additionally simulated thought probes by randomly sampling time points during reading. Logistic regression classifiers trained on eye-tracking features extracted from time windows anchored to MW onset achieved AUROC scores of 0.659 and 0.621 under the self-report and simulated thought-probe paradigms, respectively, using leave-one-subject-out cross-validation. In both cases, onset-aligned windows outperformed classifiers trained using arbitrary MW windows. Sliding-window analyses further revealed systematic temporal changes around MW onset, with classification performance peaking at approximately 3 seconds after onset. Feature-level analyses showed reduced fixation rate and fixation dispersion, along with increased pupil size following MW onset. Together, these findings characterize the temporal progression from on-task reading to MW. Overall, ReMind provides a useful framework for studying the temporal dynamics of MW during naturalistic reading.

bioengineering↗

bioRxiv2026

Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

Genome-wide association studies have identified rare and common mutations associated with increased risk of pulmonary arterial hypertension (PAH), but the mechanism by which impaired SOX17 expression increases PAH risk is not known. Notably, SOX17 plays a critical role in endothelial identity during development by suppressing RUNX1 through binding to its promoter and directing stem and progenitor cells toward an endothelial rather than a hematopoietic cell fate. RUNX1 functions as a key regulator of myeloid differentiation, aberrant angiogenesis and adverse cardiac remodeling. Previously, we found that RUNX1 inhibition reverses pulmonary hypertension (PH) in multiple animal models. Here, we hypothesize that impaired expression of SOX17 in PAH leads to endothelial cell (EC) dysfunction by failing to suppress RUNX1. METHODSHuman pulmonary artery endothelial cells (HPAECs) with stable SOX17 CRISPR/Cas9 knockout or RUNX1 overexpression were generated and examined for endothelial and hematopoietic gene expression, proliferation, migration, apoptosis, and angiogenesis. Immortalized lymphoblastoid cell lines (LCLs) from PAH patients with SOX17 mutations and healthy controls were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into ECs. The effect of RUNX1 inhibition on Sugen/hypoxia-PH was examined in rats, SOX17 enhancer knockout (SOX17enhKO) mice, and Cdh5-CreERT2;Runx1(flox/flox);SOX17enhKO triple transgenic mice. SOX17 and RUNX1 expression were analyzed in peripheral blood samples from PAH patients (n=359). RESULTSHPAECs with SOX17 deletion or RUNX1 overexpression exhibited decreased expression of EC markers, enhanced proliferation and migration, defective angiogenesis, and decreased apoptosis. RUNX1 siRNA knockdown or RUNX1 inhibition by Ro5-3335 partially restored the endothelial properties in SOX17 KO HPAECs. ECs differentiated from SOX17 mutant PAH patient iPSCs exhibited upregulated RUNX1 expression and loss of endothelial identity, which was also partially restored by RUNX1 siRNA or Ro5-3335. In addition, SOX17enhKO mice had increased RUNX1 expression and susceptibility to Sugen/hypoxia-induced PH (SuHx-PH). Treatment with RUNX1 inhibitors or inducible endothelial-specific deletion of RUNX1 rescued SuHx-PH susceptibility in SOX17enhKO mice. RUNX1 inhibitors Ro5-3335 and Ro24-7429 also reversed SuHx-PH in wild-type rats. In addition, plasma RUNX1 expression was higher in PAH patients lacking detectable SOX17 expression than in patients with detectable SOX17 expression. CONCLUSIONSImpaired SOX17 expression increases the risk of PAH through insufficient suppression of RUNX1, leading to pulmonary endothelial dysfunction. RUNX1 inhibition mitigates PH associated with SOX17 deficiency and may represent a novel therapeutic strategy for PAH, especially those with rare or common SOX17 mutations.

cell biology↗

bioRxiv2026

Astrocyte immunosuppressive activity in glioblastoma depends on ZEB1 and is counteracted by CXCL14

Glioblastomas are incurable and lethal brain cancers. Immunotherapies offer new and promising treatment options for glioblastoma patients, but the highly immunosuppressive nature of these cancers presents a challenging clinical obstacle. Glioblastoma immune evasion is driven by cell-cell interactions in the tumor microenvironment and recent studies have identified astrocytes as important contributors to immune silencing [1, 2]. Cell plasticity is a key feature of reactive astrocytes that drives heterogeneous, pro- or anti-inflammatory states [3], but the molecular regulators of astrocyte-immune interactions remain incompletely understood. Here, we investigate whether cell plasticity of glioblastoma-associated astrocytes promotes or opposes tumor progression and show that loss of astrocyte plasticity results in T-cell recruitment and immune activation. We evaluate how astrocytic cell plasticity contributes to immune functions in the glioblastoma microenvironment using single cell sequencing from preclinical models, in vivo genetic perturbations and in vitro mouse and human experimental systems. We show that astrocytes surrounding glioblastoma express the stem cell-associated transcription factor, ZEB1, and that conditional-inducible astrocytic deletion of Zeb1 remarkably reduces glioblastoma growth and extends survival. Increased recruitment and activation of T cells in astrocytic Zeb1-deficient mouse models is linked to increased expression of the immunoattractant cytokine CXCL14, and viral delivery of CXCL14 in experimental glioblastoma models increases survival. Our data support that CXCL14 is a candidate therapeutic target for reprogramming the tumor microenvironment that can restrict and reduce glioblastoma growth and progression.

cancer biology↗