Biology subjects

Zhang, X.

Publications and source records attributed to Zhang, X..

At least 109 records · Page 6Linked to original sources

bioRxiv2026

TRACER navigates rearrangement-driven sesterterpene chemical space via multimodal enzyme-product representation learning

Skeletal rearrangement drives the immense structural complexity of terpene, yet predicting it remains a formidable challenge due to sequence-function decoupling in terpene synthases. Here, we established TRACER (terpene rearrangement annotation via co-attentive enzyme-product representation), a multimodal framework mapping the latent associations between sequence-derived enzyme representations and product chemotypes. Retrospective validation proved TRACERs exceptional precision in predicting compound classes and discriminating skeletal rearrangement (SR) from non-skeletal rearrangement (NSR) pathways. TRACER-guided genome mining characterized two bifunctional synthases, FsPS and AcPS, uncovering four unprecedented carbon skeletons. Density functional theory calculations deciphered these cyclization cascades, pinpointing a critical 5/6/11 tricyclic intermediate as the key branching node for scaffold diversification. Mutagenesis and molecular dynamics simulations suggested that E305 in FsPS enables rearrangement by maintaining active-site water exclusion, whereas its alanine mutation causes premature carbocation quenching. Collectively, this work establishes a predictive paradigm for the rational discovery and mechanistic elucidation of complex terpene architectures.

synthetic biology↗

bioRxiv2026

Single Cell Mapping Identifies CD14+ Macrophages as Central Orchestrators of CD8+ T Cell Driven Immune Niches in clinical Type 1 diabetes

The authors have withdrawn this manuscript after identifying an error in the annotation of donor disease status in one or more datasets used in the study. The error arose from inadequate or ambiguous disease-status labeling in the corresponding GEO dataset at NCBI. The authors personally communicated with the dataset depositor to verify the disease status of the relevant samples. Subsequent verification indicated that the deposited metadata required correction to accurately reflect the disease status of these samples, which may affect the results and interpretation of the study. The authors therefore took the initiative to work with the GEO/NCBI database administrators to correct the relevant sample annotations and are currently reassessing the analyses using the corrected metadata. The authors believe that withdrawal of the current version is appropriate until this reassessment is complete and a revised version can be prepared. This withdrawn version should not be cited or relied upon as a reference until a revised version is made available. Any questions regarding the withdrawal or ongoing reassessment should be directed to the corresponding author.

pathology↗

bioRxiv2026

Photometallobiocatalytic Asymmetric Radical-Mediated Cross-Coupling of Organotrifluoroborate Salts and Pyridotriazoles

The cooperative integration of photoredox catalysis and metalloenzyme catalysis has emerged as a powerful strategy for enabling stereoselective radical transformations beyond the capabilities of either catalytic mode alone. Herein, we report a photometallobiocatalytic enantioselective intermolecular C-C cross-coupling of pyridotriazoles and secondary alkyltrifluoroborate salts through cooperative catalysis between an organic photosensitizer and an engineered protoglobin. By combining visible-light-mediated radical generation with enzymatic activation of pyridotriazoles to form reactive Fe carbenoid intermediates, this transformation enabled highly enantioselective radical C-C bond formation through a proposed outer-sphere coupling mechanism. Through biocatalyst mining and directed evolution, engineered Aeropyrum pernix protoglobin catalysts were developed that catalyzed this radical C-C coupling with excellent efficiency and stereocontrol. The photobiocatalytic platform exhibited a broad substrate scope with respect to both secondary alkyltrifluoroborate salts and pyridotriazoles, affording a range of valuable N-heterocyclic products in excellent yields and enantioselectivities. Mechanistic studies supported the involvement of radical intermediates and revealed spontaneous binding between the photocatalyst eosin B and the engineered metalloenzyme. By leveraging cooperative photometallobiocatalysis, this work established an underexplored strategy for asymmetric intermolecular radical cross-coupling via an outer-sphere mechanism, further expanding the catalytic repertoire of transition-metal carbenoid chemistry. Entry for the Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/744224v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@39f55borg.highwire.dtl.DTLVardef@11b3dfeorg.highwire.dtl.DTLVardef@1832ac2org.highwire.dtl.DTLVardef@6c4f56_HPS_FORMAT_FIGEXP M_FIG An enantioselective photometallobiocatalytic cross-coupling of pyridotriazoles and secondary alkyltrifluoroborate salts is developed. Cooperative catalysis using eosin B and an engineered protoglobin combines visible-light-mediated radical generation with enzymatic metal carbenoid activation, affording valuable N-heterocyclic products in excellent yield and enantioselectivity through an outer-sphere radical coupling pathway. C_FIG

biochemistry↗

bioRxiv2026

Stomatal Complex Ionomes Explain Divergent Gas Exchange Responses to Salinity in Maize and Faba Bean

Salinity tolerance is commonly associated with whole leaf Na exclusion and maintenance of K homeostasis, but whether spatial ion partitioning among functional leaf compartments contributes to stress adaptation remains unclear. Here, we investigated the relationship between bulk leaf and stomatal complex ionomes and gas exchange performance under salinity using two contrasting genotypes in both maize and faba bean crops. Maize generally maintained higher photosynthesis and stomatal conductance than faba bean under salt stress, which was associated with lower Na accumulation, stronger K retention and distinct ion partitioning patterns between bulk leaf tissue and the stomatal complex. Enrichment analysis revealed that stomatal complex ion composition provided information beyond bulk leaf ion concentrations, with Na and Cl- showing distinct distribution patterns associated with photosynthetic performance. Integrating physiological and ionomic traits further demonstrated that stomatal-complex ion traits captured additional variation in salinity responses. These findings identify the stomatal complex as a functionally distinct ionomic compartment and reveal compartment-specific ion partitioning as an important mechanism underlying species-specific salinity tolerance.

plant biology↗

bioRxiv2026

Guard Cell Chloroplast Remodeling Under Salt Stress Correlates with Ion Enrichment in Faba Bean

Salinity stress alters cellular ion homeostasis and photosynthetic activity, yet how guard cell chloroplast architecture contributes to salt adaptation remains poorly understood. Here, we investigated salt-induced chloroplast remodeling in guard cells of two faba bean genotypes, Fuego and Scoop, by integrating 3D chloroplast imaging, ion enrichment analysis and photosynthetic measurements. Salt stress induced distinct genotype-dependent changes in chloroplast morphology, with Fuego exhibiting pronounced chloroplast enlargement and reduced surface area-to-volume ratios under Na2SO and high NaCl, whereas Scoop showed treatment-dependent remodeling with larger chloroplasts under low NaCl and higher surface area-to-volume ratios under Na2SO and CaCl2. These structural responses were associated with differential Na and Cl partitioning at the stomatal complex surface. In Fuego, chloroplast size was negatively associated with photosynthetic rate, whereas Scoop showed positive relationships between chloroplast size and photosynthetic performance. Multivariate analysis further revealed coordinated associations among chloroplast architecture, ion enrichment and photosynthesis that distinguished the two genotypes under salinity. Our findings demonstrate that guard cell chloroplast remodeling is closely associated with genotype-specific salt responses and local ion partitioning. Integrating organelle structural plasticity with local ion homeostasis provides a spatially resolved perspective on the cellular basis of genotype-dependent salinity adaptation.

plant biology↗

bioRxiv2026

A ligand-property-guided computational framework for prioritizing de novo protein binders for small molecules

Plant-derived small molecules possess highly diverse physicochemical properties, and the computational design of their protein recognition elements depends not only on the global structural quality of candidate backbones, but also on whether the local binding pocket, ligand-contact pattern, and predefined recognition conformation can be consistently retained after sequence design and structural back-prediction. To explore pocket-design strategies for different types of natural-product small molecules, this study selected capsaicin, (4R)-limonene, and quercetin as model ligands, representing a flexible amphipathic molecule, a compact hydrophobic monoterpene, and a rigid polyphenolic flavonoid scaffold, respectively, and covering the dimensions of pungent sensory flavor, volatile aroma, and flavonoid functional constituents. A ligand- physicochemical-property-guided computational design and multi-stage prioritization framework was established for candidate protein binders. The results showed that candidates with favorable initial global structural scores did not necessarily form reasonable local small-molecule binding pockets, indicating that evaluation of the local ligand environment is essential for candidate prioritization. After screening, 31 partial- pocket candidate backbones for capsaicin, 75 buried hydrophobic-pocket candidate backbones for (4R)-limonene, and 56 pocket-qualified candidate backbones for quercetin were obtained. Further sequence design and structural back-prediction analyses indicated that a subset of candidates could maintain the original pocket geometry and major ligand-contact patterns after sequence realization. Overall, these results suggest that the physicochemical properties of different plant-derived small molecules substantially influence the efficiency of de novo protein pocket formation, with compact hydrophobic ligands being more compatible with buried hydrophobic- pocket strategies, whereas flexible or multipolar ligands require a more refined balance between hydrophobic burial and polar exposure. This study provides a pre- experimental computational prioritization framework for natural-product small- molecule-recognizing proteins and offers candidate resources for subsequent protein expression, in vitro binding validation, active-constituent enrichment, and development of small-molecule biorecognition tools. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/743643v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@11297a9org.highwire.dtl.DTLVardef@1a303e1org.highwire.dtl.DTLVardef@153c550org.highwire.dtl.DTLVardef@bf1f76_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Synthetic Longitudinal Tabular Data Generation via Copula

Synthetic data generation is increasingly used to enable data sharing and secondary analysis while protecting participant privacy, particularly for longitudinal tabular health data, where repeated measures per subject create within-subject dependence that most synthetic data methods are not designed to preserve. Existing generative methods, particularly generative adversarial network (GAN)-based approaches, can model complex distributions, but their estimated dependence structures are often difficult to interpret and their performance may be unstable or prone to overfitting in modestly sized datasets. Here we show that eCDF-copula, a statistically rooted approach using the empirical cumulative distribution function (eCDF) and copula modeling, preserves within- and between-visit dependence structure. To handle pervasive missing data, we propose a two-stage strategy combining multiple imputation with copula-based synthesis, enabling a variance decomposition that quantifies replication variability across methods. We benchmarked the proposed approach against four established methods on two longitudinal clinical datasets spanning markedly different sample sizes (n = 120 vs. n = 3, 612). eCDF-copula achieved resemblance and utility exceeding those of state-of-the-art synthetic data methods, while maintaining comparable privacy.

bioinformatics↗

bioRxiv2026

Pharmacologic eIF2B Activation Rescues Neuropathy in CMT2 Subtypes by Normalizing the Integrated Stress Response

Among the many subtypes of Charcot-Marie-Tooth (CMT) disease, several result from mutations in genes encoding aminoacyl-tRNA synthetases, enzymes required for tRNA charging during cytoplasmic and mitochondrial translation. We report that activation of the integrated stress response (ISR) pathway is a shared molecular feature of tRNA synthetase-associated and other axonal CMT subtypes. RTX-117, a CNS-penetrant small molecule currently in Phase 1 clinical trials, targets eukaryotic initiation factor 2B (eIF2B), a key modulator of protein synthesis and the ISR pathway. Using cryo-EM studies, we have characterized the binding mode of RTX-117 to the eIF2B decamer. In GarsP278KY/+ mice, which develop early onset motor defects and axonal pathology that recapitulate CMT2D symptoms from tRNA synthetase mutations, RTX-117 treatment started after disease onset reduced chronic ISR activation and produced significant functional and electrophysiological improvement. We further identify ISR targets, including secreted proteins such as GDF15 and FGF21 that may serve as translational biomarkers for treatment response to RTX-117 in CMT disease. Broader surveillance of the ISR pathway across models of neurodegeneration reveals strong activation in several diseases and a correlation with disease progression, particularly in models of Alzheimers disease. These findings identify chronic ISR activation as a recurrent, though not universal, pathological mechanism of neurodegenerative disease models. Overall, our study identifies candidate biomarkers for CMT disease subtypes associated with defects in translational homeostasis and supports eIF2[a]-ATF4 axis modulation as a promising therapeutic strategy for this disease class. One Sentence SummaryRTX-117, a clinical stage eIF2B activator, blunts chronic ISR activation and improves nerve and motor function in a mouse model of Charcot-Marie-Tooth Disease Type 2D.

cell biology↗

bioRxiv2026

Recombinant sialylated ApoE2 suppresses ApoE4 and sex-specifically strengthens brain metabolism and cognition in ApoE4 mice

Human APOE4 is the strongest genetic risk factor for late-onset Alzheimers disease (AD), whereas the relatively rare APOE2 confers exceptional protection. This study examines whether recombinant ApoE2 protein can therapeutically leverage this genetic advantage to strengthen aging brains at risk for AD. We produced recombinant human ApoE2 (rhApoE2) using the FreeStyle 293-F system, yielding rhApoE2 with extensive sialylation that closely resembles its natural form in the human brain. In ApoE4-knockin mouse primary neurons, exposure to rhApoE2 induced significant increases in hexokinase 2 expression and decreases in endogenous ApoE4 protein levels. rhApoE2 also protected ApoE4 neurons from oligomeric amyloid-{beta} toxicity and oxidative stress. We further developed a cadherin-derived peptide (ADTC5) that transiently increases paracellular porosity of the blood-brain barrier, enabling safe, non-invasive delivery of rhApoE2 into the mouse brain. When co-administered with ADTC5, weekly intravenous doses of rhApoE2 for 4-8 weeks in middle-aged and aged ApoE4 mice enhanced synaptosomal glycolytic and exocytotic activities, promoted cortical lipid metabolic dynamics and DHA utilization, and improved learning and memory, with sex differences observed in some outcomes. These findings offer preliminary evidence supporting the therapeutic potential of human brain-like rhApoE2 sialoprotein, which enhances metabolic robustness and cognitive strength in aging ApoE4 brains, possibly modulated by sex.

neuroscience↗

bioRxiv2026

Confinement considerations for CRISPR toxin-antidote gene drive systems

Gene drives are potentially powerful tools, able to spread throughout target populations. They could be used to modify or suppress disease vectors, invasive species, and agricultural pests. Yet, in many scenarios, confinement of the drive to only a target population is required. Several types of drives are capable of this, among the most promising of which are CRISPR toxin-antidote drives. Though showing good performance in simple models, such drives have not been thoroughly assessed in spatially structured populations. Here we evaluate three modification drive variants with varying levels of confinement. We find that Toxin-Antidote Recessive Embryo (TARE) drive and 2-locus TARE drives can usually spread in connected populations or from a single sufficiently large release. However, they can still be stopped by a migration corridor or by a sufficiently high population density gradient. 1-locus 2-drive TARE, on the other hand, will not be able to spread outside of release areas and indeed will often retreat in the face of wild-type alleles. When these drives arrive at a point source such as a port, only the standard TARE drive has a significant chance of establishing if releases occur at sufficiently high frequency and quantity. However, in models with parental care among a limited number of offspring, TARE drives become more invasive, with lower introduction thresholds. Overall, we find that spatial and other ecological factors can substantially affect the outcome of a confined CRISPR toxin-antidote drive release.

ecology↗

bioRxiv2026

A conserved vesicular circuit enables metabolic adaptation and maintains systemic heme homeostasis

Metabolic homeostasis depends on adaptive control of intracellular metabolite flux, yet how such control is reconfigured when canonical transport pathways fail is unknown. Here we define a conserved vesicular circuit that preserves systemic heme balance by rerouting intracellular heme flux. We show that loss of the intestinal heme exporter MRP-5 in Caenorhabditis elegans causes lethal heme sequestration within endolysosomal compartments. This defect is bypassed by disabling the vesicular adaptor AP-3, which stabilizes and reroutes the heme importer HRG-1, restoring heme export without increasing cytosolic heme. Unbiased genetics and transcriptomics identify two previously uncharacterized SLC49A family members, HRG-13 and HRG-14, as heme exporters with distinct affinities that engage in a vesicular importer-exporter handoff. Live imaging reveals heme-enhanced contacts between HRG-1 and SLC49A-containing vesicles, consistent with direct vesicular transfer. This circuitry extends to vertebrates as disruption of the SLC49A3 homolog impairs erythropoiesis in zebrafish and causes intracellular heme overload, premature hemoglobinization and apoptosis in differentiating human erythroid cells. Together, these findings establish SLC49A3 proteins as conserved heme exporters and uncover a general principle of metabolic adaptation in which reprogramming intracellular compartmentalization, rather than increasing nutrient supply, restores systemic homeostasis.

cell biology↗

bioRxiv2026

CD133+ progenitor cells promote pulmonary hypertension through CXCR4 signaling

BackgroundPulmonary hypertension (PH) is characterized by pulmonary vascular remodeling and smooth muscle cell accumulation, but the progenitor-like cells that contribute to this process remain incompletely defined. MethodsWe combined analyses of human pulmonary arterial hypertension lungs and experimental PH models with bulk and single-cell RNA sequencing, lineage tracing, inducible ablation of CD133+ cells, and conditional deletion of Cxcr4 in CD133+ cells. ResultsCD133 expression was markedly increased in human and experimental PH lungs. Transcriptomic analyses identified inflammatory, metabolic, chemokine-associated, and smooth muscle cell-like programs in CD133+ cells from PH lungs. Lineage tracing showed that CD133+ cells contributed to endothelial and smooth muscle cell compartments during experimental PH. Genetic ablation of CD133+ cells attenuated hypoxia-induced PH and pulmonary vascular remodeling, whereas Cxcr4 deletion in CD133+ cells reduced PH severity. ConclusionsCD133+ progenitor cells are functional contributors to pulmonary vascular remodeling, and CXCR4 signaling mediates their pathogenic activity. Targeting pathogenic CD133+ cell states or CXCL12/CXCR4 signaling may provide a strategy to limit vascular remodeling in PH.

pathology↗

bioRxiv2026

Diet-derived peptides mediate the effects of dietary protein source on gastrointestinal health

Plant-based diets support gastrointestinal (GI) health while animal-based diets can disrupt gut homeostasis. Although multiple aspects of these diet types are believed to confer their respective effects, the role of their protein component is less well understood. Here, we conducted a randomized crossover-controlled feeding trial wherein healthy subjects consumed 70% of their daily protein intake in the form of pea protein (PP) or egg white protein (EWP) isolate (NCT05619939). Individuals who consumed EWP reported increased GI symptoms and exhibited elevated intestinal permeability. In contrast, these endpoints did not change following PP consumption. Fecal analysis showed increased diet-derived peptides only following EWP consumption, which was associated with resistance of EWP isolate to degradation by digestive enzymes in vitro. Metagenomic, metaproteomic and metabolomic analyses of stool after the EWP-based diet showed reduced abundance of multiple gut-protective bacterial species and increased bacterial amino acid utilization compared to samples following the PP-based diet. Dietary peptides in the gut luminal content of EWP-fed subjects reduced metabolic function of intestinal epithelial cell in culture. Providing an amino acid-based diet mimicking EWP composition to mice prevented colonic accumulation of diet-derived proteins and GI dysfunction associated with EWP diet consumption. Collectively, these findings demonstrate that dietary protein source is a key mediator of GI function, revealing a modifiable lifestyle factor that impacts human health.

physiology↗

bioRxiv2026

Gram-scale one-pot enzymatic synthesis of CDP-ribitol by a designed bifunctional fusion enzyme

D-Ribitol-5-phosphate (Rbo5P) plays vital roles in bacterial and mammalian development. In mammals, Rbo5P is an essential component of the O-mannosyl glycan on -dystroglycan, and its defective biosynthesis causes a group of dystroglycanopathies. Supplementation with cytidine diphosphate ribitol (CDP-ribitol, CDP-Rbo) and its analogues has shown therapeutic potential for certain dystroglycanopathies. However, facile and scalable strategies to prepare CDP-Rbo remain unavailable. Here, we report a practical one-pot enzymatic strategy for gram-scale CDP-Rbo production. Starting from inexpensive ribitol, Rbo5P is first generated by L-ribulokinase (AraB) and then converted to CDP-Rbo by CDP-ribitol pyrophosphorylase (TarI) in 90% yield. To streamline the process, AraB and TarI were fused into a single bifunctional biocatalyst, AraB-TarI, which converts ribitol directly into CDP-Rbo on a gram-scale in a one-step reaction. Furthermore, a nucleotide recycling strategy was developed to lower the cost and increase the atom economy from 47% to 75%. This work provides a green and scalable route to CDP-Rbo and a reliable material supply for developing therapeutics against dystroglycanopathies.

biochemistry↗

bioRxiv2026

Off-the-Shelf Multilayer Vascular Grafts with Damage-Resistant Hydrogel Coatings Incorporating Integrin Targeting

Synthetic grafts remain ineffective for small-caliber vascular applications due to thrombosis and intimal hyperplasia. To address these limitations, our lab designed a multilayer graft consisting of a hydrogel coating that promotes post-implantation endothelialization and an electrospun mesh that matches arterial mechanical properties. Damage-resistant hydrogels were engineered using a double-network system composed of polyether urethane diacrylamide and N-acryloyl glycinamide to enhance fracture resistance through hydrogen bonding. In this study, we utilized redox initiation to apply conformal, durable hydrogels to electrospun grafts. Bioactivity wa introduced using streptococcal collagen-like proteins containing 1{beta}1 and 2{beta}1 integrin-binding motifs, enabling selective cell-material interactions that support endothelialization while preserving acute thromboresistance. To establish the feasibility of these grafts as off-the-shelf devices, we evaluated coating integrity and bioactivity retention following sterilization and dynamic physiological loading. Sterilized composites exhibited surgically-associated damage resistance, indicating that sterilization did not compromise hydrogel durability. Coating integrity and bioactivity were also preserved after six weeks of physiological loading. Acut thromboresistance was supported by both static platelet adhesion assays and dynamic whole-blood bioreactor studies using heparinized blood, with low platelet adhesion observed relative to ePTFE. Finally, a pilot ovine carotid model demonstrated successful surgical handling and sustained graft patency. Collectively, these results highlight the promise of multilayer vascular grafts as durable, thromboresistant conduits for small-diameter vascular applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/741222v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@14ec910org.highwire.dtl.DTLVardef@17259e8org.highwire.dtl.DTLVardef@6cb611org.highwire.dtl.DTLVardef@1251d7d_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

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

Integrated ex vivo screening and transcriptomic profiling to prioritize drug combinations for rare cancers

Discovering effective drug combinations requires testing many dose combinations across a diverse panel of tumor models. This approach is limited in rare cancers by a scarcity of cell lines and representative high-throughput models that would make exhaustive screening feasible and predictive. Patient-derived xenograft (PDX) models are genomically representative but too low-throughput for this purpose. Culturing PDX cells ex vivo in three-dimensional (3D) matrices offers a genomically representative and clinically relevant platform for preclinical drug testing, capturing the microenvironmental cues that shape in vivo drug response while requiring only limited tissue per assay. Toward this end, we designed and validated an experimental-computational framework, "ex vivo assessment of combination therapies" (EXACT), to enable drug combination discovery in rare tumors. Using PDX models of malignant peripheral nerve sheath tumors (MPNST), we built a platform to culture PDX cells ex vivo over multiple days, monitoring drug sensitivity and measuring transcriptomic responses to treatment. Computational analysis of these transcriptomic responses then identifies which compensatory pathway creates a unique vulnerability to a second drug. EXACT thus offers a biologically informed, scalable approach for prioritizing drug combinations in rare tumors, nominating drugs alongside biological rationale. Using this methodology, we identified a MEK inhibitor plus HDAC inhibitor combination with enhanced activity in vitro and in vivo, forming the basis of an active clinical trial. This platform could be adapted for real-time use with primary patient specimens, enabling personalized therapeutic discovery. SignificanceEXACT integrates PDX-derived 3D drug screening with biologically informed computational analysis to identify and explain effective combinations, providing a scalable strategy for therapeutic discovery in rare cancers such as MPNST.

cancer biology↗

bioRxiv2026

Inhibition of Pseudomonas aeruginosa-secreted protease IV reduces lung

The opportunistic pathogen Pseudomonas aeruginosa secretes numerous proteases that disrupt host defenses. Among them, the lysyl endopeptidase PrpL has been implicated in virulence, but its role in inflammatory responses has remained unclear. This study shows that purified PrpL activates the AP-1 transcription factors following instillation into mouse lungs, and drives robust production of IL-1{beta}, IL-6, and TNF, as well as clinical symptoms. A proteolytically inactive mutant of PrpL fails to elicit these responses. Structural analysis of the complex of PrpL and its natural inhibitory propeptide (PrpLPP) using X-ray crystallography revealed the inhibitory mechanism of PrpLPP, which guided the identification of a pre-existing dipeptide inhibitor (LasBi) that blocked PrpL activity and restricted AP-1-driven cytokine induction and clinical symptoms in vivo. This study shows that the alkaline protease AprA degrades PrpLPP and functions redundantly with LasB to liberate mature, active PrpL. Our findings: 1) establish PrpL as a key virulence factor that triggers AP-1-mediated inflammatory signaling, 2) provide structural and functional insights into PrpL inhibition, and 3) identify AprA as a novel upstream PrpL activator. Together, these results highlight PrpL as a promising anti-virulence therapeutic target. Given the functional redundancy of inflammatory effectors produced by P. aeruginosa, strategies aimed at mitigating P. aeruginosa-induced lung inflammation through PrpL inhibition would likely need to be combined with approaches targeting additional pro-inflammatory bacterial factors. Author SummaryWe identify PrpL as a critical Pseudomonas aeruginosa protease that activates the inflammatory transcription factor AP-1 and drives potent lung inflammation. Structural analysis of a natural PrpL inhibitor guided development of an existing dipeptide inhibitor that significantly reduced the production of proinflammatory cytokines and clinical responses in vivo. Moreover, discovery of the AprA protease as an additional PrpL activator refines the activation model and suggests potential upstream therapeutic strategies. These findings highlight PrpL as both a driver of pathogenesis and a promising therapeutic target for treating P. aeruginosa infections.

microbiology↗