Biology subjects

Tan, M.

Publications and source records attributed to Tan, M..

At least 19 recordsLinked to original sources

bioRxiv2026

scPRIME-seq uncovers synchronized multimodal molecular collapse facilitated by pre-neoplastic epigenetic burden in prostate cancer

Tumorigenesis involves alterations across the genome, epigenome and transcriptome, yet how these layers interact to drive malignant transformation remains unclear, largely due to the lack of scalable multimodal single-cell approaches. Here, we developed scPRIME-seq, a high-throughput platform for simultaneous single-cell profiling of transcriptome, genome and DNA methylome, and applied it to 436,908 cells from 29 prostate cancers and 15 benign tissues. This multimodal atlas resolved dynamic changes across 30 cell types and a unified tumorigenic trajectory. Integrated analysis revealed a cell-state transition tipping point, where the onset of aneuploidy is temporally coupled with global methylomic alterations and transcriptomic rewiring. Early focal epigenetic alterations, including TMEM106A promoter hypermethylation and the emergence of hypomethylated domains, were present in founder aneuploid clones and benign tissues, indicating a pre-neoplastic origin of epigenetic burden. These pre-neoplastic alterations coincide with transcriptomic changes that affect epithelial interactions with stromal and immune compartments, promoting a putative tumor-permissive microenvironment. scPRIME-seq provides a high-throughput multimodal framework for identifying conserved, cell-intrinsic molecular programs underlying tumorigenesis.

cancer biology↗

bioRxiv2026

PAR2 signaling shapes microbial and metabolic remodeling along the gut-lung axis

Introduction: Protease activated receptor (PAR2) is a prominent sensor of environmental and microbial proteases and may serve as an important interface between the host epithelium and mucosal microbes. However, whether PAR2 helps shape microbial community structure and function at barrier surfaces remains unclear. To address this, we examined how PAR2 deficiency or activation affects microbial composition and metabolic potential across the gut lumen, airway lumen, and lung tissue in the context of exposure to protease-rich house dust mite allergen (HDM) exposure. Methods: Wild type and PAR2 deficient littermates of both sexes received a single intranasal challenge with phosphate buffered saline, HDM extract, or a selective PAR2 agonist. Microbial communities from feces, bronchoalveolar lavage fluid (BALF), and lung tissue were profiled using 16S rRNA gene sequencing and PICRUSt2 based functional inference to assess compartment specific taxonomic and metabolic responses. Results: Alpha and beta diversity remained stable across all experimental groups, but distinct conditions resulted in compartment specific remodeling of the microbial population. At baseline, PAR2 deficiency altered multiple genera in the gut and lung and shifted predicted pathways linked to amino acid, lipid, and sulfur metabolism. HDM induced broad taxonomic and functional changes in the gut and lung tissue, including shifts in coenzyme A biosynthesis, reductive TCA activity, lysine fermentation, and nucleotide biosynthesis, while producing only limited taxonomic changes in BALF. PAR2 signaling accounted for a substantial portion of HDM driven remodeling, and direct PAR2 activation reproduced many compartment specific effects, including mucin derived sugar degradation in the gut and suppression of nucleotide biosynthesis in the lung. Sex moderately modified microbial and metabolic responses, with males and females exhibiting divergent, condition dependent functional biases across gut and lung. Conclusion: These findings identify PAR2 as a mucosal niche modifying receptor whose activation or loss reshapes microbial composition and metabolic potential along the gut-lung axis.

microbiology↗

bioRxiv2026

HnRNPAB-targeted antisense oligonucleotides ameliorate tau pathology and cognitive deficits by modulating Alzheimers disease-associated alternative splicing

Aberrant alternative splicing alters multiple disease-associated splicing events, including MAPT exon 10 inclusion linked to tau pathology, and thereby impacts Alzheimers disease (AD) progression. Heterogeneous nuclear ribonucleoprotein AB (hnRNPAB) functions as a splicing regulator, yet its roles in AD remain poorly defined. Here, we identified AB332, the full-length isoform of hnRNPAB, as a novel repressor of MAPT exon 10 inclusion. AB332 binds a conserved AAUAU motif and recruits RBMX and RBMXL1 through its RRM and glycine-rich domains to assemble a complex. Beyond MAPT, AB332 modulates a network of splicing events in multiple AD-associated genes, including STAG2, ApoER2, MCL-1, PICALM, and zDHHC7. Importantly, the exon 7-skipping isoform AB285 lacks these activities. Transcriptomic analysis of post-mortem brain tissues from AD patients revealed reduced expression of hnRNPAB, while selective downregulation of AB332 was confirmed in the hippocampus of 3xTg AD mice. We performed a systematic antisense oligonucleotide-tiling screen targeting hnRNPAB exon 7 and its flanking intronic regions, identifying ASO30 that specifically upregulated AB332 expression. In 3xTg AD mice, ASO30 restored hippocampal AB332 levels, reduced pathological tau phosphorylation, and rescued cognitive deficits. These findings define a molecular mechanism by which AB332 regulates AD-associated splicing events, thereby providing a therapeutic strategy targeting hnRNPAB for AD. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/736008v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@621096org.highwire.dtl.DTLVardef@2f01b1org.highwire.dtl.DTLVardef@182e6forg.highwire.dtl.DTLVardef@190afd3_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Spatial analysis of Intraductal Papillary Mucinous Neoplasms reveals secretory cell-enriched neighborhoods

Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer-related deaths in the United States. Intraductal papillary mucinous neoplasms (IPMNs) are neoplastic lesions of ductal origin that seed 10-25% of PDAC. There are currently no markers that distinguish between IPMN that will remain benign and those that will progress to cancer. A heterogenous population of secretory cells, including chemosensory tuft cells and hormone-expressing enteroendocrine cells (EECs), form during metaplasia and neoplastic progression in the pancreas, but the relevance of these populations as it relates to IPMN progression is not well characterized. Here, we performed spatial transcriptomics as well as multiplex immunostaining and spatial statistics on surgically resected IPMN from 60 patients to characterize these populations in all subtypes (gastric foveolar, intestinal, pancreatobiliary) and grades (low-grade, high-grade, invasive). We found that POU2F3+ tuft-like cells, CHGA+ EECs, and a subset of pancreatic endocrine cells ([a] and {gamma} cells) were present in all types of IPMN. Further, serotonin-expressing enterochromaffin cells made up the bulk of EECs in low-grade disease. Enterochromaffin, tuft-like, and glucagon-expressing alpha cells were not evenly distributed and instead were significantly enriched in a spatial manner, which is overlooked using conventional whole tissue quantification approaches. Tuft-like cell clusters were enriched with monocytes and resident memory T cells and anti-correlated to activated fibroblasts (myCAFs, iCAFs). Overall, these secretory cell clusters may reflect clonal expansion resulting in formation of distinct stromal niches with unknown consequences for disease progression.

Cancer Biology↗

bioRxiv2026

Estimating chronological and brain age using risk-taking behavior under uncertainty

Risky decision-making under uncertainty reflects complex cognitive processes supported by distributed brain networks that are vulnerable to aging. However, it remains unclear whether risk-taking behavior can serve as a behavioral marker of brain aging. In the present study, we combined behavioral tasks, computational modeling, and structural magnetic resonance imaging to investigate the relationship between risky decision-making, chronological age, and brain age. A total of 55 young adults and 112 healthy older adults completed the Iowa Gambling Task (IGT) and the Balloon Analogue Risk Task (BART), along with neuropsychological assessments and neuroimaging scanning. Decision processes were quantified using computational models, including the Value-Plus-Perseveration model and Exponential-Weight Mean-Variance. Brain age was estimated from gray matter volume. The results showed significant age-related alterations in parameters reflecting feedback sensitivity, learning rate, and loss aversion in both tasks. Within older adults, several decision parameters were significantly associated with both chronological age and brain age. Regression analyses further showed that computational parameters significantly predicted chronological age and brain age, whereas traditional cognitive screening measures did not show significant predictive effect. Structural brain analyses indicated that IGT-related parameters were primarily associated with the basal ganglia, while BART-related parameters were linked to a broader network including prefrontal, cingulate, and temporal regions. These findings suggest that computational markers of risk-taking behavior capture subtle age-related changes in cognitive processes and brain deterioration. Therefore, risk-taking parameters may serve as reliable functional markers of brain aging, providing critical insights into the mechanisms underlying successful aging.

neuroscience↗

bioRxiv2026

A Niclosamide Prodrug SSL-0024 with Enhanced Bioavailability Suppresses Hepatocellular Carcinoma via Multi-Pathway Signaling Inhibition

BackgroundHepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide, underscoring the urgent need for effective therapies. The FDA-approved anthelmintic, niclosamide, is a promising repurposed drug candidate for HCC; however, its clinical application in solid tumors is hampered by poor aqueous solubility and resulting low bioavailability. MethodsWe designed and screened eight novel niclosamide prodrug candidates for solubility, stability, and anti-proliferative activity in HCC cell lines. The lead compound, SSL-0024, was evaluated for pharmacokinetics and anti-tumor efficacy in an orthotopic patient-derived xenograft (PDX) model, and for cytotoxicity in patient-derived organoids (PDOs). Mechanistic studies assessed AKT-mTOR-STAT3 and other related signaling pathways. ResultsThe O-sulfate derivative, SSL-0024, demonstrated improved solubility and pH stability, and a sustained release of niclosamide over [~]48 h. Once-daily oral administration of SSL-0024 (100 mg/kg) achieved [~]60% tumor growth inhibition in PDX models at [~]46.8% of the niclosamide ethanolamine dose, with minimal toxicity. It also induced significantly greater cytotoxicity than sorafenib (p<0.05) in the PDO. Mechanistically, SSL-0024 suppressed major oncogenic pathways including AKT-mTOR-STAT3, RAF, and Wnt/{beta}-catenin. ConclusionsSSL-0024 overcomes key pharmacokinetic limitations of niclosamide while maintaining potent anti-tumor activity, supporting its further development as an orally bioavailable therapeutic candidate for HCC.

cancer biology↗

bioRxiv2026

Adaptive gene transcription in Escherichia coli under environmental stress

Escherichia coli is highly sensitive to acid and osmotic stress but adapts by modulating the expression of stress responsive genes. Nucleoid-associated proteins (NAPs) play key roles in DNA organization and sensing environmental changes. The histone-like nucleoid structuring protein H-NS is an NAP acting as a global regulator of stress genes. H-NS may alter local chromatin structure to modulate the expression of such genes in response to environmental stress. The H-NS homolog StpA co-regulates several target genes, but its precise role is poorly defined. To investigate the regulatory interplay between these two proteins, we examined transcription, DNA binding and chromatin structure at two regulated operons, hdeAB and proVWX, in E. coli following exposure to acid and salt shock. Our results show that H-NS senses pH and osmotic cues to remodel chromatin and relieve repression, while StpA compensates for H-NS loss, particularly at proVWX, highlighting a coordinated regulatory network.

microbiology↗

bioRxiv2026

Discovery and characterization of small molecule inhibitors of CBL-B that act as intramolecular glue to enhance T-cell anti-tumor activity

CBL-B is a RING-type E3 ubiquitin ligase that acts as a critical negative regulator of T-cell activation. It promotes T-cell anergy and suppresses immune responses through ubiquitin-mediated control of signaling proteins at the immunological synapse. T cells deficient in CBL-B activity lose their dependence on CD28 co-stimulation, exhibit heightened activation and increased cytokine production, and fail to re-establish anergy. In addition, mice deficient in CBL-B activity reject tumors. Together, this cellular mechanism and in vivo phenotype suggest inhibition of CBL-B may be a viable immuno-oncology therapeutic strategy. Here, we report the rational design and execution of a high-throughput screen (HTS) to identify small molecule inhibitors of CBL-B. This campaign led to the discovery of a scaffold that inhibits CBL-B E3 ligase activity with micromolar potency. Structural characterization revealed an intramolecular glue mechanism, in which the compound stabilizes the closed state of CBL-B, preventing phosphorylation of a tyrosine residue that is critical for activation and E2 binding. Iterative structure-activity optimization yielded compounds with nanomolar activity that enhanced T-cell activation and cytokine secretion in primary human T cells and suppressed tumor growth in a syngeneic colorectal mouse model. Together, these studies validate the biological rationale for pharmacological CBL-B inhibition and enabled the de novo discovery of intramolecular CBL-B glue inhibitors. This work culminated in the identification of NX-1607, a first-in-class oral CBL-B inhibitor now in clinical development for cancer immunotherapy.

immunology↗

bioRxiv2025

DINOcell: Learning Generalizable Perturbation Effects through Self-Distillation

AO_SCPLOWBSTRACTC_SCPLOWPredicting cellular responses to therapeutics is a promising approach for novel target discovery. However, state-of-the-art computational models designed to predict perturbation effects struggle to generalize and outperform simple baselines. We present DINOcell, a weakly supervised framework that adapts self-distillation to single-cell transcriptomics for predicting perturbation effects. We demonstrate that DINOcell outperforms baselines in predicting the effects of single gene perturbations. Furthermore, DINOcell accurately predicts non-additive effects of combination perturbations, indicating its capacity to model complex genetic interactions. Finally, we show that DINOcell learns representations that capture biological signals and is a promising, generalizable approach for in silico perturbation modeling, providing a valuable tool for accelerating therapeutic target discovery.

bioinformatics↗

bioRxiv2025

A comprehensive evaluation of ultraviolet irradiation at 254 nm and 222 nm in inactivating human noroviruses on surfaces

Human norovirus (hNoV) presents significant public health challenges due to its low infectious dose and environmental persistence. This study compared the inactivation efficacy of ultraviolet C irradiation at 254 nm (UV 254) and far-UVC radiation at 222 nm (UV 222) against four hNoV strains and two surrogate viruses, Tulane virus (TV) and bacteriophage MS2. A symptom scoring assay was developed to assess hNoV infectivity following microinjection into zebrafish embryos, being used in combination with reverse transcriptase quantitative PCR (RT-qPCR), long-range RT-qPCR, and RNase-treated RT-qPCR. With a general laboratory set-up of viruses being suspended in deionized water droplets in Petri dish, UV 222 was demonstrated with comparable, if not superior, performance in reducing hNoV infectivity and RNA integrity, and was significantly more effective than UV 254 in damaging viral capsids. MS2 exhibited inactivation patterns similar to hNoVs, whereas TV was markedly more resistant to UV 222. The performance of UV 222 was consistent in inactivating hydrated viruses on both stainless steel and porcine skin surfaces. However, the efficacy of UV-222 was substantially more reduced when virus inocula were dried or mixed with simulated vomitus containing high levels of organic matter, compared with UV-254. No evidence of viral adaptation or persistent genomic diversification was detected following repeated sublethal UV exposures. Taken together, UV 222 can be regarded as a promising technology in surface disinfection, especially for hNoV control, meantime keeping safe for human exposure. ImportanceHuman norovirus, the main cause of foodborne illness and non-bacterial gastroenteritis, can be transmitted through human-to-human contact. Indirectly, food or food related surfaces are readily contaminated by hNoV, completing the transmission route. So far, no standard cultivation tool is available for detecting viable hNoV, resulting in the challenges of evaluating inactivation effectiveness of various disinfection technologies, including UV 222 treatments. The significance of our study lays in attempts to quantify hNoV infectivity loss of four strains using zebrafish model during UV 222 and 254 treatments, together with the underlying antiviral mechanisms indicated by three different types of reverse transcription qPCR methods. In addition, the concerns over possible emergence of variant were subdued by genome wide sequencing results after consecutive UV exposures and passaging in vivo zebrafish model. SynopsisUV 222 is recommended to be applied after surface cleaning and ideally on moist surfaces.

microbiology↗

bioRxiv2025

Inferring and Evaluating Network Medicine-Based Disease Modules with Nextflow

Most human diseases result from complex molecular interactions of genes and proteins. Various network-based computational methods characterize these mechanisms by expanding seed genes into disease-associated subnetworks, or disease modules. Evaluating these diverse methods is tedious due to unique installation and data preparation requirements. Moreover, the underlying algorithmic strategies differ, making it difficult to determine which of the created modules are most useful or biologically plausible. To address this challenge, we developed an all-in-one Nextflow pipeline that enables automated and reproducible analyses. It handles installation, input preparation, execution, and systematic evaluation of six widely used module detection tools, considering module topology, functional coherence, robustness, and the capacity to recover seeds. In addition, it annotates the resulting disease modules with biological context information, prioritizes potential drug candidates, and generates visualizations and a comprehensive summary report. To showcase the value of our pipeline and offer guidance to potential users, we performed a comprehensive evaluation across 50 different disease-network combinations, revealing substantial variability among the derived disease modules. We show that this variability is driven by differences in modeling approach, input network, and seed composition. While most methods are robust to minor perturbations, they struggle to recover omitted seeds, and none consistently outperforms others, underscoring the need for careful method selection. Our work enables the research community to systematically compare approaches for disease module discovery, promoting reproducible network medicine research. Integrated into the nf-core project (https://nf-co.re/diseasemodulediscovery), it is intended as an extendable, long-term resource for tracking progress in the field.

bioinformatics↗

bioRxiv2025

Microsynteny Analysis Clarifies the Early Diversification of Cypriniform Fishes

While genomic data are powerfully informative for phylogenetic inference, traditional sequence-based analyses can be confounded by issues like homoplasy and incomplete lineage sorting, leaving deep nodes unresolved. Synteny, the conservation of gene order along a chromosome, presents a promising alternative source of phylogenetic information that is potentially less prone to these issues. Here we compare sequence and synteny phylogenetic phylogenomic approaches to unravel the evolutionary history of Cypriniformes, one of the most species-rich lineages of freshwater fishes. This clade has been plagued by uncertainty, particularly concerning the phylogenetic relationships between its four major subclades: Gyrinocheilidae (algae eaters), Catostomidae (suckers), Cobitoidei (loaches), and Cyprinoidei (minnows and carps). Phylogenomic approaches require high-quality genomic resources, which have been lacking for the family Gyrinocheilidae, a key lineage for understanding cypriniform evolution. To address this, we generated the first chromosome-level genome assembly and annotation for the Chinese Algae Eater (Gyrinocheilus aymonieri). We utilized this new genome along with 42 other fish genomes to conduct comprehensive phylogenomic analyses and compare results from sequence-based methods with a microsynteny-based approach. Although chromosome-level macrosynteny is broadly conserved among cypriniforms, we demonstrate that microsynteny has impressive power to resolve deep phylogenetic nodes. Both sequence-based and microsynteny-based analyses find that Gyrinocheilidae is the sister lineage to all other cypriniforms, resolving a long-standing phylogenetic point of contention. However, while sequence-based analyses provided weak support for the relationships among the remaining subclades, our microsynteny analysis revealed a novel and strongly supported sister relationship between Catostomidae and Cyprinoidei. This conclusion is supported by a 3- to 12-fold greater number of shared microsynteny clusters (synapomorphies) compared to the alternative topologies. We also demonstrate that microsynteny synapomorphies often arise from gene family expansions. Conservation of synteny concentrated in specific genomic regions may harbor the genomic underpinnings of the unique aspects of these clades and have played an important role in the evolutionary history of these taxa. Together, these results provide a robust phylogenomic backbone for Cypriniformes. More broadly, our findings highlight the power of microsynteny to resolve recalcitrant phylogenetic nodes where sequence data is uninformative, while also underscoring the critical importance of high-quality reference genomes, as our work also shows that including fragmented assemblies can lead to spurious results. Beyond phylogenomics, our results also provide a functional genomics glimpse into how conserved, clade-specific blocks of collinear genes (synteny synapomorphies) might contribute to what makes this hyperdiverse clade so unique and evolutionarily successful.

evolutionary biology↗

bioRxiv2025

O-Acyltransferase Genes Involved in the Production of Volatile Sex Pheromones in Caenorhabditis elegans

Gene family expansions are critical for functional diversification, yet paralog contributions to metabolic pathways are often unclear. In Caenorhabditis, the expanded O-acyltransferase (OAC) family--enzymes that transfer acyl groups to hydroxylated substrates--remains poorly characterized despite having been implicated in lipid metabolism. Using CRISPR-Cas9 mutagenesis, behavioral assays, gas chromatographic-mass spectral (GC-MS) analyses, and metabolomics, we systematically analyzed 59 OAC-family protein-coding genes to define their roles in regulating signaling molecules. We found that four adjacent paralogs (oac-13, oac-16, oac-25, and oac-28) on chromosome I are required for synthesizing volatile sex pheromones (VSPs)--airborne signals critical for male mate-searching. Specifically, oac-13 and oac-16 are necessary for producing both major pheromone components, while the identical tandem paralogs oac-25 and oac-28 regulate the production of the later-eluting component in gas chromatography. Disruption of these genes reduced production of key pheromone components and impaired male attraction. Metabolomics revealed that oac-16 and other OACs also modulate synthesis and secretion of non-volatile ascaroside pheromones, indicating dual roles in chemical signaling. This work uncovers functional specialization within an expanded gene family, illustrating how redundancy and divergence enable adaptive evolution of communication systems.

biochemistry↗

bioRxiv2025

Divergent ontogeny of Tissue Resident Memory and Tissue Resident Exhausted CD8+ T cells underlies distinct functional potential

Persistent antigen stimulation promotes differentiation of exhausted CD8+ T (TEX) cells. TEX cells are distinct from circulating memory T (TCIRCM) cells but share many features with tissue-resident memory (TRM) cells established following infection resolution. CD8+ T cells co-expressing residency- and exhaustion-associated molecules in chronic diseases often correlate with clinical outcomes. However, the relationship between these cells and conventional TRM or TEX cells remains unclear. Here, we show that chronic antigen stimulation drives development of tissue-resident TEX (TR-TEX) cells that are ontologically and functionally distinct from TRM cells generated after antigen clearance. TR-TEX phenotypically resembled TRM cells but were regulated by distinct transcriptional networks and were uniquely dependent on Tox for residency programming. Although TEX progenitor cells acquired residency features upon entering chronically infected tissues, they failed to generate conventional TRM cells after antigen withdrawal. Conversely, TRM cells were able to differentiate into TEX cells during chronic antigen stimulation. Deriving cell-state specific transcriptional signatures revealed a selective association of TR-TEX cells with patient responses to immune checkpoint blockade, and only TR-TEX but not TRM cells responded to PD-1 pathway inhibition in vivo. These data suggest that TR-TEX and TRM cells are developmentally distinct cell types that share a tissue-residency program but have distinct roles in disease control.

immunology↗

bioRxiv2025

Cytokine Regulation of Human Antibody Responses to Influenza Vaccines

Studies show that human vaccine responses widely vary. Here, we analyzed data measuring 66 cytokines from 4 different inactivated influenza vaccine (IIV) cohorts over 5 seasons (N=581) and identified a significant correlation between baseline/day 0 serum IL-18 and IFN-{beta} concentrations and the vaccine-specific antibody response on day 28, suggesting these factors may have an adjuvant-like effect. To investigate this further, we tested the impact of 19 cytokines on the development of anti-influenza antibody response when administered together with the IIV vaccine in human tonsil and spleen organoids. We found that Type I IFNs (IFN-{beta} and others), IL-21, IL-12, IL-10, but not IL-18 or IFN-{gamma}, enhanced the antibody response. The live attenuated influenza vaccine (LAIV) induced a stronger antibody response than the inactivated one in organoids. Adding a single cytokine, IFN-{beta}, to IIV stimulation recapitulated most of the live vaccine-specific cytokine activation program. It increased the antibody response of the inactivated vaccine to that of the LAIV. Two other antibody-boosting cytokines, IL-12 and IL-21, were induced by LAIV but not by Type I IFNs, indicating different cytokines can affect different pathways leading to a robust antibody response. We then generated cytokine mRNA lipid nanoparticles (LNPs) to test the effect of cytokines on IIV response in a mouse model of immunization. We found that IL-21-LNPs augmented the quantity and breadth of the antibody responses, while IFN-{beta} LNPs enhanced durability. These findings identified parallel cytokine pathways regulating human vaccine responses and provide a rationale for using cytokines as adjuvants to mimic the effectiveness of live-attenuated vaccines without the risk of viral replication.

immunology↗

bioRxiv2025

Hydrogel viscoelasticity modulates cell nascent extracellular matrix deposition

Polymeric hydrogels are valuable platforms for determining how specific mechanical properties of native tissue extracellular matrix (ECM) regulate cell function. Recent research has focused on incorporating viscous and elastic properties into hydrogels to investigate cellular responses to time-dependent mechanical properties of the ECM. However, a critical aspect often overlooked is that cells continuously remodel their microenvironment in hydrogels, such as by the deposition of newly secreted (nascent) ECM. While this nascent ECM has been demonstrated to play a vital role in transmitting mechanical signals across various biological contexts, the mechanisms by which it regulates cellular function in response to time-dependent mechanical properties remain poorly understood. In this study, we developed an interpenetrating polymer network that enables independent control of viscous and elastic hydrogel properties. We show that cells cultured on high-viscosity hydrogels deposit increased nascent ECM which also correlates with enhanced hydrogel remodeling. Interestingly, higher nascent ECM deposition on high-viscosity hydrogels was decoupled from intracellular contractility. These results establish a relationship between hydrogel viscosity and nascent ECM deposition that may extend to diverse cell types and offer new insights into cell-hydrogel interactions.

bioengineering↗

bioRxiv2025

Deconvolution of multiplexed peptidoform mass spectra enables high-resolution profiling of complex protein modification patterns

Combinatorial patterns of post-translational modifications (PTMs), like the histone code, play critical roles in regulating protein functionality. However, in bottom-up proteomics, isobaric modification peptidoforms (IMPs) frequently co-elute and co-fragment during liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), resulting in multiplexed mass spectra that cannot be properly analyzed by conventional protein identification and quantification methods. Existing solutions are either limited to highly narrow scenarios or unavailable for public use. Here, we introduce PTMdecoder, a robust and versatile software tool for discriminating and quantifying IMPs by deconvoluting their multiplexed MS/MS spectra and ion chromatograms without requiring a spectral library of individual peptidoforms. Tested on 34 samples of synthesized IMPs, PTMdecoder achieved an overall sensitivity of 95.7% for IMP identification and root mean square errors mostly <0.05 for quantification. In human histone samples, it detected and quantified 36.7-44.8% more IMPs than conventional methods, providing a higher-resolution view of histone modification patterns.

bioinformatics↗

bioRxiv2025

Visible-Light-Controlled Residue-Selective Cross-Linking for Deciphering Protein Complexes and Dynamic Protein-Protein Interactions in Live Cells

Cross-linking mass spectrometry (XL-MS) has emerged as an attractive technology for investigating protein complexes and protein-protein interactions (PPIs). However, commonly used cross-linking strategies present significant challenges for precise analysis of protein complexes and dynamic PPIs in native biological environments. Here we present the visible-light-controlled lysine-selective cross-linking (VL-XL) strategy for in-depth analysis of protein complexes and dynamic PPIs both in vitro and in live cells, building on light-induced primary amines and o-nitrobenzyl alcohols cyclization (PANAC) chemistry. We demonstrate that the VL-XL strategy effectively explores the dynamic dimerization of PD-L1 stimulated by exogenous modulators. Moreover, the VL-XL strategy successfully profiles the time-resolved EGF-stimulated EGFR interactome, providing valuable insights into the regulatory mechanisms of EGFR signaling and intracellular trafficking. Importantly, the VL-XL strategy efficiently deciphers the molecular glue (MG) induced dynamic PPIs and substrate profile of MG degrader, opening an innovative avenue for identifying neo-substrates. By harnessing the advantages of temporal controllability, good biocompatibility, and lysine selectivity, the VL-XL method simplifies MS data analysis and facilitates the acquisition of accurate structural information of protein complexes and the elucidation of elusive PPIs in live cells. Overall, the VL-XL strategy expands the XL-MS toolbox, and realizes in-depth analysis of protein complexes and dynamic PPIs, which will inspire innovative solutions for protein interactomes research and structural systems biology.

biochemistry↗