Biology subjects

Nguyen, C.

Publications and source records attributed to Nguyen, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Low CAR affinity enhances T-cell synapse dynamics and activation

Low affinity chimeric antigen receptors (CARs) can clinically outperform high-affinity variants, yet the molecular mechanisms underlying affinity-dependent activity remain unclear. Here, we show that low CAR affinity enables faster CAR cluster motion, which is central to T-cell activation. We compared low-affinity CAT and high-affinity FMC63 CARs signaling at different stages of activation on supported lipid bilayers functionalized with defined CD19 densities. At low CAR expression on dense CD19 bilayers, low-affinity CAT CARs formed more mature and dynamic synapses, recruited ZAP70 more efficiently, and showed faster microcluster motion. These proximal signaling changes were accompanied by stronger calcium flux and increased NFAT nuclear translocation. Our data support a model in which weaker, lower-affinity CAR-antigen interactions promote receptor turnover, microcluster mobility, and signaling-adaptor recruitment, thereby enhancing downstream signal propagation. Our work provides a mechanistic basis for the superior functional performance reported for affinity-tuned CD19 CAR-T cells.

biophysics↗

bioRxiv2026

A stromal metabolic program suppresses NK-cell immunity to drive tumor progression in HER2-low breast cancer

Cancer-associated fibroblasts (CAFs) are major regulators of the tumor microenvironment, yet how distinct CAF states suppress innate immunity in HER2-low breast cancer remains poorly understood. Here, we identify an S100A4-enriched CAF population that expands during HER2-low breast tumor progression and establishes a metabolically immunosuppressive niche. Spatial transcriptomics and multiplex imaging of human HER2-low tumors reveal progressive CAF accumulation and an inverse spatial association between S100A4-enriched CAFs and immune infiltration, including natural killer (NK) cells. Using an immunocompetent HER2-low mammary tumor model, we show that S100A4-enriched CAFs promote tumor initiation and progression while suppressing NK-cell cytotoxicity, IFN-{gamma} production, perforin, and granzyme B. Fractionation of CAF-conditioned media and metabolic profiling identify a low-molecular-weight immunosuppressive program characterized by enhanced branched-chain amino acid catabolism and accumulation of branched-chain -keto acids (BCKAs). Mechanistically, BCKAs directly suppress NK-cell IFN-{gamma} production, whereas inhibition of the branched-chain aminotransferase BCAT1 reduces CAF-mediated NK-cell suppression and restores antitumor cytotoxicity. BCAT1 inhibition also suppresses HER2-low tumor growth in vivo, an effect attenuated by NK-cell depletion, establishing NK-cell restoration as a functional component of its antitumor activity. Together, these findings uncover a CAF-driven metabolic immune checkpoint in which S100A4-enriched CAFs exploit BCAT1-dependent BCKA production to suppress NK-cell surveillance and promote HER2-low breast tumor progression. Targeting stromal BCAT1 therefore represents a potential strategy to dismantle CAF-mediated immune suppression and restore innate antitumor immunity.

cancer biology↗

bioRxiv2026

Cyclic immunofluorescence platform using photocleavable linkers for direct antibody labeling enables cancer phenotyping

Advances in spatial proteomics through the development of multiplexed immunostaining platforms have facilitated analyses with increasing cellular and molecular granularity. However, currently available approaches are limited by harsh conditions for signal removal, restricting the number of antigens that can be probed in a single specimen without significant alterations to sample quality and structure. Here we present an approach for direct labeling of primary antibodies with fluorophores using a photocleavable linker (PCL) with a polyethylene glycol spacer (PEG) to enable cyclic immunofluorescence (cyCIF) with gentle signal removal conditions. Our innovative approach uses directly labeled primary antibodies to enhance staining specificity and cyclic immunostaining efficiency, while minimizing nonspecific background signal. Additionally, through integration of the PCL, this approach facilitates gentle cleavage of antibody conjugated fluorophore, preserving sample integrity over multiple rounds of staining. Direct PEG-PCL antibody labeling will promote greater multiplexing by minimizing specimen damage and allow for quantitative analyses of cyCIF spatial data. We demonstrate that cyCIF with PEG-PCL conjugated antibodies can be applied across a variety of cancer subtypes to identify and characterize rare neoplastic cell populations in both tumor tissue and fragile peripheral blood specimens.

biochemistry↗

bioRxiv2026

CRISPR activation screens identify core protein-dependent regulation of heparan sulfate sulfation and ligand specificity

Heparan sulfate proteoglycans (HSPGs) are essential cell surface and extracellular matrix glycoconjugates that mediate diverse biological processes through interactions between their heparan sulfate (HS) chains and extracellular ligands. While HS sulfation patterning is known to dictate ligand specificity, how cells control HS assembly to regulate these interactions remains incompletely understood. To systematically identify genetic modifiers of HS-protein interactions, we performed genome-wide CRISPR activation (CRISPRa) screens in HEK293T cells using binding of antithrombin (AT), which selectively recognizes 3-O-sulfated HS motifs, or the N-sulfation-specific antibody 10E4 as functional readouts. Strikingly, the screens revealed proteoglycan core proteins as key modulators of HS function. In particular, syndecan-1 (SDC1) emerged as a preferential enhancer of AT binding compared to other syndecan family members. Targeted upregulation of syndecan family members increased total HS levels, but only SDC1 enhanced AT binding. Structural and enzymatic analyses demonstrated that SDC1-associated HS chains contain elevated 6-O-sulfation and serve as superior substrates for 3-O-sulfotransferases relative to SDC2-associated HS chains. Additionally, SDC1 exhibited slower cell surface recovery, which was blocked by cycloheximide treatment, consistent with extended trafficking and biosynthetic processing. Overall, these findings indicate that proteoglycan core protein identity influences HS sulfation patterning and ligand-binding specificity and trafficking kinetics may contribute to core protein-dependent regulation of HS modification.

Cell Biology↗

bioRxiv2026

Predicting Pain Using a Data-Driven Agent-Based Model of the Bilateral Central Amygdala

Sensory processing in the amygdala is a complex, dynamic process. Decades of surgical, electrical, pharmacological, optogenetic, and chemogenetic in vivo manipulations have revealed the nociceptive functions of anatomically- and genetically-restricted neuronal populations. In parallel, molecular and electrophysiological approaches have allowed for high-resolution, temporal examination of nociceptive-induced alterations in amygdala plasticity. Computational integration of this data is critical for future therapeutic development; in practice, these models would allow for in silico prediction of amygdala activity following injury, and in a reciprocal fashion, changes in pain-like behaviors following manipulation of discrete amygdala neuronal populations. To this end, we developed a three-dimensional computer model of the bilateral central nucleus of the amygdala (CeA). We employed agent-based modelling to integrate wet-lab data from two CeA cell populations: Calcitonin Gene-Related Peptide Receptor (CGRPR; Calcrl) expressing cells and Protein Kinase C delta (PKC{delta}; Prkcd) expressing cells. We integrated the spatial location, connectivity, neuronal activity, and electrophysiological properties of these neurons in our realistic bilateral model architecture. Our model captures properties of the amygdala that drive pain modulation, including hemisphere-specific physiological differences, and generates predictions of nociception related to bladder injury. Predictions from the model were compared retrospectively to pain outcomes during manipulation of CGRPR-expressing neurons in whole mice. These comparisons show strong alignment between our model and in vivo outcomes. Significance StatementNociceptive processing in the amygdala emerges from interactions among cell types. No framework has been developed that recapitulates this complexity. Here, we generated a 3-D agent-based computational model of the bilateral central amygdala (CeA) using data that accounts for pain-associated changes in neuronal physiology, cellular identity, anatomical positioning, and temporal specificity. The model revealed that hemispheric differences in intrinsic CeA neuronal excitability are dominant drivers of nociceptive output. Furthermore, when manipulated to mimic injury-associated plasticity, the model accurately predicted behavioral outcomes. This model is a publicly available computational tool that predicts hemisphere-targeted pain therapy efficacy. Moreover, the flexibility of this framework will allow future adaptations to other brain areas and disease contexts.

neuroscience↗

bioRxiv2026

Glio-SERS: Label-Free Molecular Profiling of Plasma Extracellular Vesicles in Brain Tumors Using SERS and Artificial Intelligence

Extracellular vesicles are increasingly recognized as important carriers of disease-associated molecular information, yet robust methods for their isolation and molecular characterization from limited clinical samples remain challenging. Here, we present an integrated approach combining standardized EV isolation, label-free Surface-Enhanced Raman Spectroscopy (SERS), and artificial intelligence (AI) for comprehensive molecular profiling of small extracellular vesicles (sEVs) from human plasma. Here, we show systematically isolated and characterized plasma sEVs using ExoTIC in accordance with MISEV2023 guidelines, with SERS analysis revealing quantifiable spectral differences across samples from patients with glioblastoma (n=20) and meningioma (n=23) compared to healthy controls (n=30). Among the evaluated AI models, the convolutional neural network most effectively captured group-level spectral differences in sEVs, achieving accuracies up to 88% in this pilot cohort. Further, an EGFR-based spectral regression model was explored to examine molecular variability across sEV samples. Parallel proteomic analysis presented statistically significant differences in several proteins elevated in glioblastoma or meningioma. This label-free, rapid approach provides a proof-of-concept framework for sEV molecular profiling establishing the basis for broad validation studies across diverse diseases.

bioengineering↗

bioRxiv2026

Bilayer acoustic force spectroscopy (BAFS) for quantifying receptor-antigen binding strength in immune synapses

Immune cell receptor - ligand interactions are key to cancer immunotherapy. However, receptor-ligand affinities often fail to predict T-cell mediated cancer killing, while immune-target cell binding strength measurements are limited by low precision and high non-specific binding. Here we present bilayer acoustic force spectroscopy (BAFS), a method to quantify the binding strength of receptors in immune synapses that virtually eliminates non-specific binding and increases the resolving power by up to 50-fold. By replacing target cells with a supported lipid bilayer functionalized with antigens, BAFS avoids antigen-independent interactions and target cell heterogeneity, while maintaining the spatial self-organization of receptors that typifies active immune synapses. We demonstrate the high sensitivity and control by showing how CAR T-cell synapse strength depends on CD19 antigen density, and by revealing that CD8 synergistically strengthens {beta}TCR-pMHC synapses independently of Lck recruitment to CD8. BAFS is a general method that can be used broadly in immunotherapy screening and to dissect the complex molecular interactions that underpin immune synapse activation.

biophysics↗

bioRxiv2026

S-SELeCT: A Human-Evolved Serine Integrase System for Efficient Large-Cargo Genome Integration

As a consequence of their sizes, many loss-of-function genetic mutations fall within large genes. A major gene-therapy tool that could be used to solve large swaths of the genetic diseases that result from these inherited mutations is large-fragment knock-in. I.e. instead of attempting to create separate treatments for each and every location that these mutations occur in, large groups of patients could be aided via a single safe-harbor integration of the full-length coding sequence. Towards this goal, we have created a set of early stage gene-editing enzymes that can help mediate large cargo integration at a safe harbor locus in human cells. When expressed in stable lines, our S-SELeCT (Site-Specific Large Cargo Targeting) integrase fusions can facilitate integration of a 10 kb plasmid at frequencies up to 32%, and when delivered transiently via plasmid transfection, we were able to achieve up to 13% knock-in. These are the first serine integrase enzymes that have been evolved fully in human cells, and the first to recognize an endogenous symmetric non-pseudosite - the first true human serine integrase attachment site. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/702954v2_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@1c22676org.highwire.dtl.DTLVardef@16b171corg.highwire.dtl.DTLVardef@1ba4796org.highwire.dtl.DTLVardef@b1d91f_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2026

Loss of nsp14-exonuclease activity impairs the replication, proofreading, fitness and pathogenesis of SARS-CoV-2

Coronaviruses (CoVs) replicate their RNA genomes with increased fidelity than other RNA viruses, a mechanism mediated by the proofreading and recombination activities of the exoribonuclease domain of replicase nonstructural protein 14 (nsp14-ExoN). Both murine hepatitis virus (MHV) and SARS-CoV tolerate nsp14-ExoN loss-of-function mutations (ExoN-) (D90A and E92A) but have impaired replication fidelity and pathogenesis yet identical substitutions in MERS-CoV and SARS-CoV-2 have been reported to be lethal. Here, we report a saturation mutagenesis approach facilitating the recovery and analysis of several constellations of SARS-CoV-2 nsp14 ExoN-inactivating, loss-of-function substitutions, including the canonical D90A and E92A. Biochemical assays with purified WT or ExoN- nsp10-14 fusion proteins confirmed that active site substitutions abolished ExoN activity (ExoN-). SARS-CoV-2 ExoN- viruses had impaired replication, RNA synthesis, and recombination, as well as decreased replication fidelity and loss of fitness in vitro. ExoN- viruses were significantly attenuated for replication in human primary airway epithelial cells and were attenuated for replication and pathogenesis in WT mice as well as the highly susceptible K18 transgenic mice. In the absence of interferon signaling in vivo, SARS-CoV and SARS-CoV-2 ExoN- viral replication could be nearly fully restored. These results demonstrate that SARS-CoV-2 ExoN- are viable but highly impaired for replication, fitness, and fidelity in vitro as well as innate immune antagonism and pathogenesis in vivo. Collectively, our results solidify the multiple critical roles for nsp14-ExoN across CoV genera and establish new approaches for rescue and analysis of loss-of-function substitutions for studies of CoV replication, pathogenesis, and evolution.

microbiology↗

bioRxiv2025

Discover Potential New Epitopes through Post Translational Modification in Sjogrens Disease

Sjogrens syndrome (SjD) is a chronic autoimmune disorder in which the immune system attacks the glands that produce tears and saliva, leading to symptoms such as dry eyes and dry mouth. If left untreated, SjD can also cause inflammation and damage to other parts of the body, including the skin, lungs, kidneys, and nervous system, and increase the risk of developing lymphoma. The human leukocyte antigen (HLA) class II molecule HLA-DR3 is strongly associated with SjD. To investigate how post-translational modifications (PTMs) influence the presentation of SjD-associated autoantigens by HLA-DR3, we employed a computational framework to analyze the binding of PTM-mimic peptides to HLA-DR3. Our analysis revealed that PTM-mimic substitutions at canonical anchor positions rarely improved predicted binding affinity using the Stabilized Matrix Method, with most modifications resulting in reduced affinity. However, a comprehensive analysis of full-length SjD-associated autoantigen sequences (Ro60, Ro52, La) identified discrete regions with high densities of PTM-eligible anchor sites, specifically, the Ro60 HEAT solenoid, Ro52 RING/B-box/PRY-SPRY modules, and the La motif-RRM1 region, suggesting that PTMs may alter epitope presentation in a sequence-dependent manner. Experimental validation of selected PTM-mimic peptides demonstrated enhanced T cell responses, which were associated with increased binding affinity to HLA-DR3. Structural modeling of a representative complex revealed that PTM-mimic peptides adopt a slightly shifted backbone orientation and altered side-chain positioning, leading to a larger peptide-DR3 interaction interface. These findings provide new insights into the role of PTMs in shaping the immunogenicity of SjD-associated autoantigens and highlight the potential for PTM-mimic peptides to modulate T cell responses in SjD.

immunology↗

bioRxiv2025

Evaluation of the safety and insecticidal efficacy of ivermectin-treated bird feed formulations in different avian species

BackgroundWest Nile virus (WNV) is maintained in an enzootic cycle between reservoir host birds and Culex (Cx.) spp. mosquitoes. This relationship presents a potential target for vector control strategies. Ivermectin (IVM), an endectocidal drug that selectively affects invertebrates while remaining safe at high concentrations in mammals and birds, can be delivered to Cx. tarsalis via blood meals from birds fed IVM-treated bird feed. In this study we evaluated the safety, efficacy, and utility of IVM-treated bird feed as a novel vector control strategy by assessing its impact on multiple bird species and mosquitoes. MethodsMosquitoes were collected during peak WNV transmission season in Northern Colorado and DNA extracted from blood meals to determine host species. Chickens, pigeons, zebra finches, and house sparrows were fed different formulations of IVM-treated bird feed and observed for clinical signs, and their sera were fed to Cx. tarsalis mosquitoes to evaluate mosquitocidal efficacy. Feeding rates and IVM serum concentrations in birds were analyzed by unpaired t-test and one-way ANOVA, and mosquito survivorship was analyzed by Kaplan-Meier curves and compared using paired log-rank tests. IVM serum concentration and mosquito survivorship were compared using Spearman correlation. ResultsSpeciation analyses conducted on blood meals from Cx. tarsalis collected during peak WNV transmission season in Northern Colorado determined that they feed primarily on songbird species that commonly visit bird feeders, with house sparrows representing the most frequent blood meal host. In laboratory experiments using multiple formulations and doses of IVM, chickens, pigeons, zebra finches, and house sparrows ate comparable amounts of IVM-treated bird feed compared to untreated feed, had similar weight gain, and exhibited no clinical signs of toxicity. Both colony-reared and locally captured Cx. tarsalis showed significant mortality after feeding on sera from IVM-treated birds compared to controls. ConclusionsThese results suggest that targeting songbirds with IVM-treated bird feed should be safe for wildlife and may elicit high rates of IVM-induced mortality by reaching a large proportion of WNV vector mosquitoes via their proclivity for feeding on passerine birds.

microbiology↗

bioRxiv2025

The impact of patient biology on racial disparities in breast cancer outcome

Hormone receptor positive (HR+) breast cancer is the most common subtype of breast cancer diagnosed globally. Despite effective targeted therapies, HR+ breast cancer remains a leading cause of cancer-related death in women. Long-standing epidemiological research identifies significantly worse outcomes for Black women diagnosed with HR+ breast cancer relative to White women. While structural factors such as access to healthcare and education level contribute to this outcome disparity, it persists even in analyses where these factors are controlled. In-depth analyses of the somatic molecular biology that may underlie these outcome disparities are hampered by a lack of datasets that represent Black patient populations. Here, we generate a HR+ breast cancer patient transcriptomic dataset that overrepresents Black women and controls for access to healthcare and education level. We find that signatures relating to the tumor microenvironment, i.e. collagen deposition and prognostically unfavorable T-cell landscapes are enriched in HR+ tumors from Black women. Importantly, we find, using experimental model systems in vitro and in vivo, that race-aligned collagen deposition patterns are at least partially attributable to tumor cell-intrinsic signaling and critical for Black breast cancer metastasis. We also find that unfavorable T-cell signatures in HR+ tumors from Black women, which have previously been attributed to race and ancestry, are more strongly poverty-aligned. Using multiple independent datasets, we identify STAT4 as a potential master regulator of this poverty-associated tumor immune signature. Together, these findings provide new evidence that somatic molecular biology of breast cancer patients can be modified by multiple structural factors such as self-identified race and poverty burden to promote poor patient outcomes. Integrating an understanding of structural factors into molecular cancer research is critical for implementing truly personalized, and maximally effective, oncology systems.

cancer biology↗

bioRxiv2025

Toll-like Receptor 4 Contributes to PCOS-like Metabolic and Reproductive Pathogenesis

Polycystic ovary syndrome (PCOS) is a reproductive disorder with heterogeneous symptoms and severity. Despite extensive research documenting chronic immune dysfunction as a hallmark of PCOS, the specific molecular mechanisms driving immune activation and its connection to the syndromes diverse symptoms remain poorly understood. Emerging evidence suggests that gut-derived bacterial endotoxins, particularly lipopolysaccharide (LPS), may breach the intestinal barriers in PCOS patients and trigger systemic inflammation through Toll-like receptor 4 (TLR4), a pattern recognition receptor of the innate immune system. This study investigated whether TLR4 serves as a critical mechanistic driver of PCOS pathogenesis by examining the effect of genetic TLR4 knockout (TLR4-/-) in a letrozole (LET)-induced mouse model of PCOS. Our results demonstrate that TLR4 deficiency reduces many PCOS-like symptoms, including elevated luteinizing hormone, anovulation, and metabolic dysfunction. TLR4 knockout also preserved estrous cycling and fertility, improved glucose tolerance, maintained gut barrier integrity, and reduced inflammatory markers in LET-treated females. These findings establish TLR4 as a key mediator orchestrating PCOSs multi-system pathology, positioning TLR4 as a critical convergence point rather than affecting individual symptoms in isolation. This novel work reveals that TLR4-mediated inflammation drives multiple PCOS pathologies, opening avenues for targeted anti-inflammatory treatments in women with this disorder. Significance StatementPolycystic ovary syndrome (PCOS) affects up to 15% of reproductive-age women worldwide. This study reveals that TLR4, an innate immune receptor, is key to the pathophysiology of PCOS-like symptoms in female mice. When TLR4 was genetically deleted, mice treated with letrozole to induce PCOS-like symptoms maintained normal weight, glucose regulation, estrous cycling, and fertility. The improvements coincided with preserved gut barrier breakdown and reduced inflammation. These findings identify TLR4 as a key mediator between gut health, immune activation, and PCOS pathophysiology, suggesting that targeting TLR4 could offer new therapeutic approaches for this common but poorly understood syndrome affecting millions of women.

immunology↗

bioRxiv2025

Effects of fibroblast growth factor 2 on muscle precursor cells from mouse limb and extraocular muscle

Fibroblast growth factor 2 (FGF2) is known to play a role in skeletal muscle development and growth. We examined two populations of myogenic precursor cells for their responses to FGF2 in vitro using both extraocular and limb skeletal muscle. Fluorescence-activated cell sorting (FACS) was used to isolate two different populations of myogenic precursor cells, the EECD34 cells [positive for CD34, and negative for Sca1, CD31, and CD45] and PAX7-positive cells, from tibialis anterior and extraocular muscles of mice. These cells were cultured and treated with either proliferation or differentiation media in the absence or the presence of FGF2, followed by assays to determine its effects on proliferation and differentiation. These cells were also assessed for expression of fibroblast growth factor receptor (FGFR) 1, FGFR2, and FGFR4. Both the EECD34 cells and the PAX7-positive cells responded to FGF2 with significantly increased proliferation. Both myogenic precursor cell populations showed increased differentiation in the presence of FGF2, but also showed decreased rates of fusion into multinucleated myotubes in this in vitro system relative to control cells. FGF2 has pleiotropic effects on skeletal muscles. Contrary to the literature, FGF2 did not inhibit differentiation, but did appear to decrease fusion into multinucleated myofibers in vitro. These results provide a potential mechanism for reduction in myofiber number and size in the extraocular muscles in individuals with Apert syndrome, where FGF receptor 2 mutations maintain the receptor in an activated state.

cell biology↗

bioRxiv2025

A CRISPR-CAS9 high throughput machine-learning platform for modulation of genes involved in Parkinson's disease-associated PINK1-mitophagy in iPSC-derived dopaminergic neurons

Parkinsons disease (PD) is a progressive neurodegenerative disorder characterised by the loss of dopaminergic neurons, driven by complex molecular mechanisms that are not fully understood. To address this issue, we have developed a novel high-content phenotypic screening platform using human induced pluripotent stem cell-derived dopaminergic neurons to investigate the PINK1-PARKIN mitophagy pathway, a critical process in PD pathogenesis. Utilising high throughput, 384 well arrayed CRISPR-CAS9 genetic manipulation and high-content immunofluorescence imaging complemented with machine learning analysis, we examined ubiquitin (Ub) pSer65 levels. Ub pSer65, a potential PD clinical biomarker, is a key marker of mitophagy initiation in dopaminergic neurons upon mitophagy initiation using exogenous stimuli to mimic the disease relevant environment. The CRISPR-CAS9 knockout (KO) screen revealed two distinct phenotypic classes: essential genes causing cell death upon deletion, and genes modulating Ub pSer65 levels. Notably, KO of PINK1, PARKIN, and TOM7 genes decreased Ub pSer65 upregulation during mitophagy activation, confirming their established roles in the pathway and validating the suitability of the platform for target identification. This innovative platform provides a precise tool to further interrogate PD-associated genes, offering insights into mitophagy-related pathogenic mechanisms and identification of potential therapeutic targets. By bridging functional genomics with disease-specific neuronal models, this approach presents a promising strategy for advancing PD research and developing targeted interventions. To our knowledge, this is the first reported use of a human, translationally relevant cell model to study genetic perturbation within a disease relevant phenotype.

neuroscience↗

bioRxiv2025

obABPP-HT*: A Precision-Engineered Activity Proteomics Pipeline for the Streamlined Discovery of Deubiquitinase Inhibitors

Deubiquitinases (DUBs), and the dysregulation thereof, are implicated in human disease. The recent inclusion of selective DUB inhibitors in clinical trials has heightened interest in DUB-focused drug discovery. Current DUB screening methods remain constrained, however, as they often rely on recombinant proteins that are truncated or derived from non-human sources, typically necessitating extensive optimisation of initial hits. We introduce a high-throughput, endogenous human DUB-focused activity proteomics workflow designed for the simultaneous screening and profiling of small, targeted libraries of catalytic group-reactive compounds. In a proof-of-concept screen, this innovative platform expanded the repertoire of electrophilic groups targeting DUBs, leading to the discovery of potent and selective inhibitors for USP47, OTUD7B, and USP5. Remarkably, these inhibitors required minimal or no optimisation to confirm the previously reported biological roles of the three DUBs, underscoring the advantages of this methodology for drug discovery applications.

biochemistry↗

bioRxiv2025

Intracellular Delivery of Bone Nanoparticles to Mitigate Irradiation-Induced Damage in Bone Marrow Mesenchymal Stem Cells

Ionizing radiation (IR)-induced bone damage presents a major clinical challenge by impairing bone marrow function and disrupting normal bone remodeling. Bone regeneration depends on bone marrow-derived mesenchymal stem cells (BMSCs), which are highly sensitive to IR that causes DNA damage, oxidative stress, apoptosis, and a shift from osteogenesis to adipogenesis, ultimately leading to bone loss and impaired healing. This study evaluated the therapeutic potential of intracellularly delivered bone-derived nanoparticles (BPs) in mitigating IR-induced BMSCs damage. We found that IR exposure caused significant BMSCs dysfunction, including reduced proliferation, increased apoptosis, persistent DNA damage, and a shift toward adipogenic differentiation. Treatment with BPs led to efficient intracellular uptake, improved cell morphology, enhanced proliferation, reduced apoptosis, and preservation of balanced differentiation capacity. Transcriptomic analysis via RNA sequencing revealed that BPs restored key molecular pathways disrupted by IR, particularly those involved in cell cycle regulation, extracellular matrix (ECM) remodeling, and apoptosis. By reversing these transcriptional impairments, BPs supported genomic stability and the regenerat ive function of BMSCs. Overall, these findings suggest that BPs effectively counteract IR-induced cellular damage and enhance the regenerative capacity of BMSCs, offering a promising therapeutic strategy for radiation -induced skeletal injuries.

bioengineering↗

bioRxiv2025

siRNA-based Therapeutic Candidate Targeting PRDM2 for Inhibition of Lung Cancer Progression

Lung cancer is the leading cause of tumor-related fatalities worldwide. The current precision therapies are effective but target only those molecules that are impacted in a minority of lung cancer patients. Positive Regulatory Domain 2 (PRDM2), a member of the PRDM gene family, also known as Retinoblastoma Interacting Zinc finger (RIZ) protein. RIZ is found to be misregulated in more than 60% of lung cancer patients, representing a significantly affected population when compared to other mutation-prone oncogenic factors. RIZ expresses two contrasting variants, a tumor suppressor RIZ1, and an oncogenic RIZ2 protein. In lung cancer, the RIZ1/RIZ2 ratio alters, and the epigenetic silencing of RIZ1 promotes RIZ2 overexpression. In this study, we used siRNA (ARIZ-047) to knockdown and inhibit the effect of the oncogenic protein RIZ2. Inhibiting RIZ2 with ARIZ-047 treatment increased RIZ1 expression and decreased the viability of a lung cancer cell line (A549). The ARIZ-047 treatment also upregulated the negative regulators of the Wnt signaling pathway and restrained the tumor progression in the xenograft mice model. In conclusion, this study identifies ARIZ-047 as a targeted therapy in lung cancer, which specifically inhibits RIZ2 and suggests the role of the Wnt signaling pathway in RIZ2 overexpression. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/636957v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1a5eb34org.highwire.dtl.DTLVardef@1f4dc2forg.highwire.dtl.DTLVardef@a3ce29org.highwire.dtl.DTLVardef@33f576_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗