Biology subjects

Zheng, Y.

Publications and source records attributed to Zheng, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

siProGenA: Generative siRNA Candidate Construction via Position Proposal and Guide Generation

Small interfering RNAs (siRNAs) are short guide RNAs that recruit the RNA-induced silencing complex (RISC) to complementary target sites on messenger RNAs (mRNAs), triggering Ago2-mediated cleavage and gene silencing. siRNA design requires compact candidate sets that cover a target while preserving efficacy, specificity, and practical sequence constraints. Existing pipelines usually enumerate candidate windows, assign a canonical guide to each window, and then rank preconstructed siRNA--mRNA pairs. This has produced strong pairwise efficacy predictors, but leaves a candidate-construction gap: candidate positions and guide sequences are fixed before the model begins to rank them. We address this gap by decomposing siRNA candidate construction into two generative decisions: where to place candidates within an mRNA segment, and what constrained guide variants to consider at a candidate position. We instantiate this framework as siProGenA, using a Discrete Denoising Diffusion Probabilistic Model (D3PM) for mRNA-conditioned position proposal and a Bayesian Flow Network (BFN) for temperature-controlled guide generation. On 62 positive test segments, the diversity-aware final library reaches Hit@1 = 0.790 and Hit@5 = 0.903. In a measured-site controlled Stage~2 evaluation, seed- and cleavage-preserving variants outscore the canonical complement for 89.8% of measured sites, with supporting gains across additional computational scorers, random-mismatch controls, and biophysical diagnostics. Together, the results support a modular proposal--generation view of siRNA candidate construction for prioritizing compact candidate sets.

bioinformatics↗

bioRxiv2026

Cross-species analysis links cell-cell communication rewiring to NOTCH2 during serous endometrial carcinogenesis

Cell-cell interactions shape the fate of mutant cells during cancer initiation but how these interactions evolve during progression to pathologically recognizable lesions remain poorly understood. Here, we investigated cell-cell communication during serous endometrial carcinoma (SEC; also known as uterine serous carcinoma) development using a lineage-traceable mouse model and cross-species analyses of the mouse and human neoplastic endometrium. In mice, the early, pre-dysplastic stage was marked by a global decrease in inferred cell-cell interactions, followed by extensive communication network rewiring during neoplastic progression. Pathway-specific analysis revealed a similar pattern for NOTCH signaling, with NOTCH2 emerging as the dominant NOTCH receptor in Trp53/Rb1-mutant immature epithelial cells. Functionally, NOTCH2 promoted the outgrowth of more proliferative mutant organoids. Cross-species transcriptomic analysis identified conserved immature epithelial states in mouse and human neoplastic endometrial epithelium. In human tissues, NOTCH2 was overexpressed in serous endometrial intraepithelial carcinoma, a precursor of SEC, and in overt SEC. Furthermore, elevated NOTCH2 expression was associated with poor patient survival. These findings link cell-cell communication rewiring during experimental SEC development to conserved neoplastic epithelial states and identify NOTCH2 as an early marker and a potential target of disease interception.

cancer biology↗

bioRxiv2026

Microbial valerate is associated with CAR T dysbiosis and its supplementation enhances CAR T function in B-cell lymphoma

Anaerobe-depleting antibiotic exposure is associated with inferior progression-free survival after CD19 CAR T-cell therapy in large B-cell lymphoma, yet the cellular mechanisms linking gut dysbiosis to the CAR T-cell product and whether this imprint is reversible have remained undefined. In two independent CAR-T candidate cohorts, low stool valerate at the time of CAR-T eligibility identified a multi-metabolite-deficient dysbiotic gut microbiome state marked by depletion of fiber-fermenting commensals and loss of carbohydrate-fermentation, SCFA-biosynthesis, and amino-acid metabolism pathways. Reanalysis of single-cell RNA sequencing from 42 lymphoma patients stratified by piperacillin-tazobactam/imipenem/meropenem (PIM) exposure revealed that PIM-exposed CAR T-cell products were CD4-skewed, with significantly elevated AP-1/immediate-early gene (IEG) and cellular activation signatures that together predicted inferior progression-free survival. Ex vivo conditioning of CAR T-cells with valerate produced a chromatin and transcription factor program distinct from butyrate or propionate, characterized by KLF/SP/EGR family engagement, KLF4 promoter opening, and broad induction of AP-1/IEG and MHC class II transcripts, whereas butyrate drove broader chromatin remodeling with TBX21/EOMES/NF-{kappa}B gains and KLF2 promoter closure, and propionate induced an NFY-centered program with preferential commitment to low-mitochondrial-content states. Untargeted metabolomics confirmed valerate uptake and mitochondrial {beta}-oxidation in CAR T-cells, while dietary sodium valerate supplementation in meropenem-treated mice bearing A20 lymphoma significantly reduced tumor burden and extended survival compared with CAR T-cells alone. These findings identify stool valerate as a bedside-deployable biomarker of dysbiosis-imprinted CAR T-cell dysfunction and support ex vivo or dietary valerate supplementation as a clinically tractable strategy to improve CAR-T anti-tumor function in patients with disrupted gut microbiomes.

cancer biology↗

bioRxiv2026

Lamins promote trophoblast lineage-associated transcription and trophoblast giant cell development in placental organogenesis

Lamins are the major structural components of the nuclear lamina with a variety of roles in development and organogenesis. However, the function of lamins during trophoblast development, the first lineage to differentiate during mouse embryogenesis, remains unexplored. By utilizing an in vitro trophoblast stem cell differentiation model in a lamin null setting, we uncover that lamins maintain expression of genes related to trophoblast differentiation, while repressing genes involved in maintaining trophoblast stem cell stemness and off-lineage development. By deleting different combinations of lamins in mice, we show that both lamin triple-knockout and lamin-A and -B1 (lamin-A/B1) double-knockout result in placental defects, including reduced placenta size and disrupted placental organogenesis at embryonic day (E)9.5. At this stage, lamin-A/B1 are expressed in trophoblast giant cells of the placenta, and lamin-A/B1 loss leads to their impaired maturation in vivo. Lamin-A/B1 double knockout trophoblast giant cells exhibit reduced nuclear size along with a reduction of DNA damage signaling foci, suggesting a role for lamins in supporting trophoblast giant cell polyploidization. Similar to the transcriptional dysregulation observed during differentiation of lamin triple knockout trophoblast stem cells in vitro, lamin-A/B1 knockout in vivo results in downregulation of genes related to trophoblast giant cell function and upregulation of off-lineage genes. Our results suggest lamins are required for placental organogenesis by maintaining polyploidization and lineage-associated transcriptional programs in trophoblast giant cells.

developmental biology↗

bioRxiv2026

Vibrio parahaemolyticus metabolically adapts while tempering the host immune response during host cell invasion.

Vibrio parahaemolyticus (V. para) is an enteric pathogen that establishes a protected intracellular niche using its second type III secretion system. However, how this bacterium adapts to the host cytoplasm while overcoming cellular defenses has remained unclear. To define these mechanisms, we performed dual-transcriptomic profiling of both pathogen and host during invasion, intracellular replication, and late infection. Our analyses revealed extensive metabolic reprogramming by V. para, including induction of diverse nutrient transporters and metabolic pathways. We discovered that because mammalian cells are auxotrophic for aromatic amino acids, V. para must activate its own unique biosynthetic machinery, a requirement that proved essential for intracellular growth. Infected host cells mounted a sustained NF-kappaB response. Both heightened NF-kappaB activation and disruption of canonical NF-kappaB signaling restricted bacterial expansion, indicating that V. para exploits a finely tuned Goldilocks level of immune signaling to promote survival and replication. Together, these findings uncover fundamental metabolic and immune adaptations that drive pathogenesis.

microbiology↗

bioRxiv2026

Plasma titration provides a physical ruler for cross-platform proteomics

Plasma proteomics is expanding across platforms and cohorts, and integrating these data for AI demands comparability at the protein level, not merely concordant associations1,2. Affinity and MS platforms use distinct probes (antibodies, aptamers, or peptides) and signal readouts, yielding contradictory cross-platform results3,4. Without a known quantitative truth, we cannot distinguish biology from measurement distortion, leaving no gold standard for integration. Here we introduce Plasmix--a plasma reference suite with predefined male:female ratios (M, 1:0; Y, 3:1; P, 1:1; X, 1:3; F, 0:1)--and show that preserving this quantitative titration gradient, not just technical repeatability, predicts cross-platform concordance and identifies protein measurements suitable for integration. Profiling Plasmix across five platforms (Olink, SomaScan, NULISA, AAgAtlas, and MS-DIA) and 12 protocols across 17 batches, we found discordance is dominated by signal generation, not sample identity, and platforms distort signals in a protein-specific manner. Crucially, proteins retaining the titration response showed stronger agreement in an independent cohort; anchoring to the Plasmix midpoint (P) via sample-to-reference ratios reduced distortions, extending harmonizable coverage by 10-20%. Plasmix thus provides a physical ruler to benchmark accuracy, identifying genuinely integrable measurements before pooling datasets or training AI models--a critical bottleneck for plasma proteomics.

molecular biology↗

bioRxiv2026

Heart rate dynamics embed a shared representation of individual brain organization and cognitive function

Idiosyncratic brain functional organization shapes intricate cardiac dynamics through central-peripheral autonomic interactions, yet a comprehensive mapping between multi-scale heart rate dynamics and whole-brain functional architecture remains lacking. Here, combining highly comparative time-series analysis with multi-modal neuroimaging and intracranial electrophysiology, we establish heart rate dynamics as a physiological fingerprint that maps onto whole-brain functional architecture. Partial least squares analysis revealed a generalizable latent axis linking reduced heart rate temporal complexity and elevated micro-scale predictability to heightened resting-state functional connectivity across default mode, salience, and sensorimotor networks. This covariance aligns spatially with serotonergic, noradrenergic, and cholinergic neuromodulatory gradients, persists across physiological confound controls and cross-session validations, derives support from human intracranial electrophysiological recordings, and extends to active cognitive states. Furthermore, brain-covarying heart rate signatures underpin the predictive capacity of heart rate dynamics for individual fluid and crystallized intelligence, demonstrating a shared representational substrate. Our findings demonstrate a robust neurovisceral coupling architecture, establishing well-characterized heart rate dynamics as a scalable, neurobiologically anchored window into human brain functional organization and cognitive traits.

neuroscience↗

bioRxiv2026

Phagocytic activity of perivascular cell promotes CNS injury pathology

Injury, stroke, and neurological diseases cause persistent accumulation of cellular debris that deteriorates lesion microenvironment and impedes central nervous system (CNS) repair. Debris clearance in the injured CNS has long been attributed primarily to microglia and infiltrating macrophages. Here, we identify perivascular cells as previously unrecognized phagocytes that expand after injury and exhibit robust phagocytic activity. Perivascular cell phagocytosis is conserved across multiple mouse models of CNS injury and human stroke lesions. These cells exhibit key hallmarks of phagocytosis, including LC3-associated phagocytosis for efficient lysosomal degradation. Mechanistically, phosphatidylserine serves as the eat-me signal and Axl mediates myelin debris uptake. Myelin phagocytosis drives perivascular cell proliferation, fibrosis and lesion progression. Genetic deletion of Axl in perivascular cells or pharmacological inhibition with the FDA-approved Axl inhibitor Gilteritinib reduces pathology and improves functional recovery after spinal cord injury. Together, these findings establish Axl-dependent perivascular cell phagocytosis as a therapeutic target for CNS repair.

neuroscience↗

bioRxiv2026

StainX: GPU-accelerated batch stain normalization for computational pathology at scale

Stain normalization reduces color variability in histopathology whole-slide images, but cohort-scale pipelines lack fused multi-image batch transforms for classical methods. We present StainX, a GPU-accelerated batch stain normalization framework built around a two-stage fit/transform interface. It implements histogram matching, Macenko, and Reinhard normalizers through a portable PyTorch backend and an optional CUDA backend that fuses per-pixel operations for batch throughput. On NVIDIA GPUs, the fused CUDA path outperforms the torch CPU backend by 168x, 70x, and 48x for Reinhard, histogram matching, and Macenko respectively, and exceeds the fastest GPU peers by 7-8x (Reinhard) and 2x (Macenko) at comparable accuracy. StainX also provides user-selectable precision modes, a documented Python API, continuous integration testing, and online documentation. Source code available at https://github.com/rendeirolab/stainx, and documentation at https://stainx.readthedocs.io. Implemented in Python. Runs on Linux, macOS, and Windows.

bioinformatics↗

bioRxiv2026

Pharmacological up-regulation of CALM1 restore endothelial cells in intracranial aneurysm pathogenesis

Subarachnoid hemorrhage (SAH) resulted from intracranial aneurysm (IA) rupture is an especially severe form of stroke. Endothelial dysfunction represents the initiating event of IA pathogenesis. Understanding the role of endothelial cells (ECs) underlying formation of IAs is helpful to seek for pharmaceutical treatment strategy. Based on single-cell RNA sequencing, proteomics, and metabolic analysis, we discovered a change in cell population in IA samples, majorly in ECs and macrophages (MPs). Abnormal ECs exhibit senescence and death in IA samples, which is absent in the control arterial samples. Cross-analysis of multi-omics revealed that CALM1, a calcium detector involved in mechanotransduction, is downregulated in the abnormal ECs. CALM1 knockdown leads to senescence and inhibits the proliferation and maturation of ECs under turbulent flow. Through high-throughput virtual screening, this work identified compound ZC04329651 as a potent CALM1 activator in maintaining the stability of endothelial cell junctions and attenuating cellular senescence. Thus, our findings showed compound ZC04329651 up-regulate the expression of CALM1 to restore ECs, which maybe a promising pharmacological treatment strategy for IAs.

cell biology↗

bioRxiv2026

Coevolution-driven reconstruction of multi-taxa siderophore interaction networks reveals topological diversity of microbial exploitation

Microbial communities are shaped by secreted metabolites that mediate ecological interactions, yet predicting these interactions from genomic sequences remains difficult, because the specific recognition between co-functional metabolites (CFMs), such as siderophores, and their receptor proteins (Rec) cannot be inferred from gene annotation alone. This difficulty arises from three factors: the prevalence of Rec-mediated exploitation, the lack of high-accuracy functional annotations, and the absence of genomic co-localization between functionally paired CFM-Rec in Gram-positive bacteria. Here we present the Coevolution-based Interaction Model (CIM), an automated framework that maps specific CFM-Rec pairings directly from uncurated genomic datasets. Using a dynamic joint optimization strategy that accounts for exploitation asymmetry and avoids combinatorial explosion, CIM identifies functional pairings solely through evolutionary covariation. We validated this approach by reconstructing macroscale iron scavenging networks across nine bacterial taxa. Experiments confirmed that CIM can bridge genomic distances exceeding 3 Mb in the Gram-positive genus Rhodococcus to identify unlinked cognate receptors, and can accurately predict cross-utilization by exploiter strains despite substantial receptor sequence heterogeneity in Burkholderiaceae and Rhizobiaceae. Finally, topological analysis of the reconstructed networks shows that siderophore exploitation acts as a universal topological glue, fusing fragmented microbial populations into highly connected communities, and that the exploitability of siderophore production reverses depending on network modularity. CIM thus offers a scalable, sequence-to-ecology approach for predicting interactions mediated by secondary metabolites in microbial communities.

systems biology↗

bioRxiv2026

Molecular principles for graded activation of dopamine D1 receptor

G protein-coupled receptors (GPCRs) signal across a continuum of activation states, yet how ligands encode distinct signaling efficacies remains poorly understood. Here, we define the molecular mechanism underlying graded activation of the dopamine D1 receptor (D1R), a major therapeutic target for neuropsychiatric disorders. Functional analyses reveal that two widely used pharmacological tools, LE300 and SCH23390, possess intrinsic efficacy as an inverse agonist and a weak partial agonist, respectively, rather than the efficacy-silent neutral antagonists. Structural, molecular dynamics and mutagenesis analyses capture previously unrecognized inactive and intermediate receptor activation states that bridge known active conformations, and reveal that ligand efficacy is encoded through the progressive engagement of a conserved activation pathway centered on the W6.48 toggle switch. Guided by this mechanism, a single chemical modification markedly increases the agonist efficacy of SCH23390. Comparison with dopamine D2 receptor structures further reveals a conserved mechanism of inverse agonism despite distinct subtype-specific recognition. Together, these findings establish a structural framework for graded agonism at D1R and provide general principles for the rational design of efficacy-tuned therapeutics at dopamine receptors and related GPCRs.

biochemistry↗

bioRxiv2026

Targeting NAT10 suppresses TFH responses and alleviates allergic asthma via an ac4C-TOX2 axis

Chemical modifications of RNA, such as N4-acetylcytidine (acC), fine-tune gene expression, but their roles in cell fate determination within the immune system remain poorly understood. Here, we identify the RNA cytidine acetyltransferase NAT10 as a pivotal regulator of T follicular helper (TFH) cell differentiation and the germinal centre response. T cell-specific ablation of Nat10 in mice severely impaired TFH cell development, germinal centre formation, and antibody production following viral infection. Integrated epitranscriptomic and transcriptional profiling revealed that NAT10 deposits acC modifications on a cohort of mRNAs critical for immune function. We pinpointed the transcription factor TOX2 as a key downstream target, showing that acC modification within its mRNA coding sequence enhances both transcript stability and translation efficiency, thereby promoting TOX2 protein expression. Enforced TOX2 expression fully rescued the TFH differentiation defect in NAT10-deficient cells. This NAT10-acC-TOX2 axis is conserved in humans, where its activity correlates with the magnitude of vaccine-elicited TFH responses. Furthermore, we identified R428 as a potent NAT10 inhibitor and demonstrated that its administration could suppress pathogenic TFH responses and alleviate symptoms in a mouse model of allergic asthma. Our work elucidates a central epitranscriptomic mechanism controlling T helper cell fate and humoral immunity, and nominates NAT10 as a potential therapeutic target for TFH-driven diseases.

immunology↗

bioRxiv2026

An Integrated Multi-omics Single Cell Atlas of the Human RPE and Choroid

The retinal pigment epithelium and choroid are critical for supporting the function and maintaining the homeostasis of the outer retina, and their dysfunction underlies a range of inherited and complex ocular diseases. To comprehensively characterize the cellular, transcriptomic, and epigenomic heterogeneity and dynamics within these tissues, we assembled an integrated multi-omics reference atlas comprising 719,813 single-cell/single-nucleus transcriptomes and 234,007 snATAC-seq profiles from 102 ancestrally diverse donors spanning 0 to 99 years of age, including cells from both the macula and periphery. This atlas resolves 48 distinct cell types or states and catalogs 448,567 open chromatin regions. Specifically, we resolved five distinct RPE subpopulations organized along a central-to-peripheral spatial axis, alongside two distinct stress/senescence states. We reconstructed the transcriptomic and epigenetic zonation of endothelial cells and expanded choroidal stromal heterogeneity by characterizing 11 fibroblast and two pericyte types. Age-associated compositional analysis revealed a significant fractional depletion of melanocytes, PI16+ fibroblasts, and venule endothelial cells with age, alongside a modest relative loss of central RPE and a corresponding increase in far-peripheral RPE. Cell-type-specific aging transcriptomics uncovered shared pathways related to inflammatory responses alongside distinct cell-type-specific signatures. Notably, significant age-associated epigenetic changes concentrated in the macula during the transition from early-to-middle adulthood and remained stable into old age, with transcription factors from the AP-1/bZIP family emerging as the dominant enriched motifs. Finally, integrating this atlas with AMD GWAS data provides novel variant-to-gene evidence implicating LIPG and COL4A3 in AMD pathogenesis. Together, this multi-omics atlas serves as both an invaluable community reference and a powerful discovery engine that translates genetic risk signals into localized target cells and candidate mechanisms, laying a foundation for understanding RPE/choroid biology in health and disease.

genomics↗

bioRxiv2026

Peroxiredoxin 6 limits mitochondrial peroxidation to prevent mitochondrial ER contact site assembly and inflammatory signalling following adaptive stress

Peroxiredoxin 6 (PRDX6) is a multifunctional enzyme with peroxidase, calcium independent phospholipase A2 (aiPLA2) and lysophospatidylcholine acyltransterase (LPCAT) activities. Although PRDX6 can repair peroxidised phospholipids and help prevent ferroptosis, its role in the adaptive response to physiological oxidative stress remains unclear. In this study PRDX6 function was investigated using myoblasts exposed to an acute low concentration of H2O2 and Caenorhabditis elegans subjected to a swimming intervention. Mild oxidative stress promoted myogenesis and mitochondrial turnover in myoblasts, while exercise enhanced activity and longevity in C. elegans. The adaptive responses were associated with increased mitochondrial localisation of PRDX6. In contrast, loss of PRDX6 under mild stress conditions resulted in increased mitochondrial lipid peroxidation, enhanced mitochondrial ER contact sites (MERCS), mitochondrial calcium accumulation, release of mitochondrial DNA and activation of innate immune signalling pathways. Similar phenotypes were induced by the ferroptosis activator erastin and rescued by the lipid peroxyl scavenger Ferrostatin-1, indicating lipid peroxidation was the key triggering event. Furthermore, inhibition of mitochondrial calcium uptake in C. elegans prevented calcium overload and attenuated inflammatory signalling. Together, the results identify PRDX6 as a conserved regulator of mitochondrial adaptation to physiological oxidative stress, functioning to limit mitochondrial lipid peroxidation and prevent excessive MERCS assembly, mitochondrial calcium dysregulation and inflammatory activation.

cell biology↗

bioRxiv2026

Promoter architecture decodes response regulator phosphorylation into distinct transcriptional logics in Pseudomonas aeruginosa

Two-component systems convert environmental signals into transcriptional responses, but how one response-regulator phosphorylation state produces different outputs at different promoters remains unclear. Here we show that promoter architecture determines how the ParR response regulator in Pseudomonas aeruginosa interprets phosphorylation. Phenotypic, transcriptomic, biochemical and promoter-engineering analyses showed that phosphorylation of the conserved ParR receiver residue Asp57 lowered DNA-occupancy thresholds across target promoters. Individual promoters, however, converted this shared increase in binding into activation, repression or phosphorylation-dependent sign switching. Chromosomally D57 ParR variants reproduced these behaviours at endogenous loci and altered antibiotic susceptibility, biofilm formation and virulence-associated phenotypes. Mapping and engineering of representative promoters identified a two-tier cis-regulatory code: half-site sequence compatibility determines productive ParR engagement, whereas spacer length determines regulatory sign and magnitude. Spacer swaps were sufficient to reprogram promoter logic between regulatory modes. These findings separate regulator state from promoter decoding and show how bacterial promoter architecture can diversify the outputs of a shared phosphorylation signal. ImportancePseudomonas aeruginosa is an opportunistic pathogen that survives antimicrobial treatment and adapts to diverse host environments by rapidly changing gene expression. How a single sensory pathway generates different, and sometimes opposing, responses across many genes has remained unclear. We show that the regulatory protein ParR does not impose one fixed transcriptional program. Instead, the DNA architecture of each target promoter determines whether the same phosphorylation signal activates, represses, or reverses gene expression. The sequence and spacing of ParR recognition sites form a compact regulatory code, and changing spacer length alone can reprogram promoter behavior. Different ParR states were also associated with changes in antibiotic susceptibility, biofilm formation, and virulence-associated phenotypes. Our findings explain how one bacterial signaling pathway can generate diverse physiological outcomes without additional regulators and provide a framework for understanding regulatory flexibility in Pseudomonas aeruginosa and for engineering genetic circuits with multiple outputs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/740031v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@1b6ec28org.highwire.dtl.DTLVardef@4a3f2eorg.highwire.dtl.DTLVardef@79e528org.highwire.dtl.DTLVardef@b3aa77_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2026

A multi-agent workflow converts CAR-T patient evidence into experimentally testable hypotheses

Clinical studies of chimeric antigen receptor (CAR)-T therapy generate diverse molecular and clinical evidence that remains fragmented across publications, public repositories and patient-derived datasets, limiting systematic therapeutic discovery. Here we present BioPathfinder, an evidence-guided multi-agent workflow for closed-loop biomedical discovery. BioPathfinder constructs a provenance-aware knowledge resource linking publications with patient single-cell RNA sequencing (scRNA-seq) datasets and clinical metadata, and uses role-specialized large language model agents to generate, review and prioritize diverse, falsifiable and dataset-aware mechanistic hypotheses for computational and experimental validation. Applied to a curated corpus of CAR-T-treated patient studies and matched scRNA-seq datasets, BioPathfinder identified candidate mechanisms underlying CAR-T persistence, dysfunction and therapeutic resistance. The workflow prioritized the hypothesis that genes associated with an NK-like transition programme could be targeted to reduce CAR-T exhaustion and improve persistence. Analysis of patient scRNA-seq datasets showed enrichment of this programme in exhausted post-infusion CAR-T cells. Virtual perturbation prioritized transition-associated receptor genes, including KLRC1, KLRD1 and KLRG1, and expert review selected KLRC1, encoding NKG2A, for experimental validation. In vitro and in vivo chronic-stimulation models showed that NKG2A marked activated, exhaustion-associated CD8 CAR-T cells, whereas NKG2A blockade enhanced antitumour activity and persistence-associated functional readouts in vivo. BioPathfinder establishes a generalizable framework that transforms fragmented clinical single-cell evidence into experimentally validated therapeutic hypotheses, providing a scalable strategy for AI-guided biomedical discovery. HIGHLIGHTSO_LIBioPathfinder integrates fragmented CAR-T clinical studies, patient scRNA-seq datasets and metadata into a provenance-aware evidence resource for AI-guided hypothesis discovery. C_LIO_LIA multi-agent workflow generates, critiques and prioritizes diverse, falsifiable, dataset-aware mechanisms underlying CAR-T persistence, dysfunction and therapeutic resistance. C_LIO_LIBioPathfinder prioritizes KLRC1/NKG2A as a therapeutic target, and experimental validation demonstrates that NKG2A blockade enhances CAR-T persistence and antitumour function. C_LI

bioengineering↗

bioRxiv2026

Promoter architecture diversifies ParR-dependent transcription in Pseudomonas aeruginosa

Two-component systems convert environmental signals into transcriptional responses, but how one response-regulator phosphorylation state produces different outputs at different promoters remains unclear. Here we show that promoter architecture determines how the ParR response regulator in Pseudomonas aeruginosa interprets phosphorylation. Phenotypic, transcriptomic, biochemical and promoter-engineering analyses showed that phosphorylation of the conserved ParR receiver residue Asp57 lowered DNA-occupancy thresholds across target promoters. Individual promoters, however, converted this shared increase in binding into activation, repression or phosphorylation-dependent sign switching. Chromosomally D57 ParR variants reproduced these behaviours at endogenous loci and altered antibiotic susceptibility, biofilm formation and virulence-associated phenotypes. Mapping and engineering of representative promoters identified a two-tier cis-regulatory code: half-site sequence compatibility determines productive ParR engagement, whereas spacer length determines regulatory sign and magnitude. Spacer swaps were sufficient to reprogram promoter logic between regulatory modes. These findings separate regulator state from promoter decoding and show how bacterial promoter architecture can diversify the outputs of a shared phosphorylation signal. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/738673v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1a0a26forg.highwire.dtl.DTLVardef@10836a8org.highwire.dtl.DTLVardef@3c6cd8org.highwire.dtl.DTLVardef@10dfefb_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsParR coordinates antibiotic resistance, biofilm formation and virulence-linked phenotypes in P. aeruginosa. Promoters convert ParR phosphorylation into activation, repression or sign switching. Half-site compatibility and spacer length form a cis-regulatory code for ParR output.

biochemistry↗