Biology subjects

Khan, S.

Publications and source records attributed to Khan, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Lipid lowering alone fails to limit atherosclerosis progression and neutrophilic inflammation in middle-aged mice

While cholesterol lowering remains the cornerstone therapy for atherosclerosis and prevention of major adverse cardiovascular events, 33-50% of individuals on lipid-lowering therapy continue to exhibit elevated inflammation. Middle age (MA) represents a critical window for disease acceleration, underscoring a need to better understand non-resolving inflammation in this time frame. We rendered young (2 months) and MA mice (10 months) hypercholesterolemic with an AAV8-PCSK9 virus and fed a western diet (WD). Following 20 weeks on a WD, mice were switched to chow for 6 weeks to mimic lipid-lowering therapy. We found that MA atherosclerotic mice had increased plaque necrosis with increased circulating and bone marrow PMN compared to young mice. Upon lipid lowering, unlike in young mice, MA mice had increased necrosis, and reduced remodeling as well as increased circulating white blood cells and bone marrow hematopoietic stem cell progenitors (HSPCs). Circulating neutrophils correlated with necrosis in MA mice whereas young mice exhibited no correlation. Lipid lowered MA atherosclerotic mice had bone marrow HSPCs and neutrophils that exhibited a more activated phenotype relative to young. Elevated neutrophil-endothelial contacts were observed in the hippocampal vasculature of MA lipid lowered mice. While lipid lowering restrains atheroprogression in young, it is inadequate in MA, failing to reduce systemic inflammation and indicating the need for complementary therapies during this time frame.

immunology↗

bioRxiv2025

Staying in the loop to make ends meet: roles and regulation of GlmR in Bacillus subtilis

The metabolic networks of most life forms integrate cost-benefit analysis to properly budget carbon and other essential nutrients through continuous assessment of nutrient availability and environmental threats. Bacillus subtilis is a Gram-positive model bacterium found in diverse ecological niches such as soil, marine environments, and the human gut. As such, B. subtilis cells finetune metabolic pathways by monitoring signals indicating the presence of nutrients and stressors. A highly conserved protein, GlmR, is a key player in rationing carbon for the production of cell envelope precursors. This function of GlmR can be attributed to its role in cell shape regulation and antibiotic resistance. Given its central position in carbon utilization, GlmR is under post-translational regulation by phosphorylation and UDP-N-acetylglucosamine (UDP-GlcNAc) binding. GlmR is also linked to cyclic-di-AMP (c-di-AMP), a nucleotide second messenger involved in stress response. In this study, we probed the importance of GlmR in cell morphogenesis, c-di-AMP signaling, and investigated the physiological significance of post-translational regulation. Our results reveal that cells lacking glmR exhibit: (i) increased susceptibility to tunicamycin, a cell envelope targeting antibiotic; (ii) impaired division site positioning; and (iii) elevated intracellular c-di-AMP concentration. Furthermore, we show that the function of GlmR is finetuned by UDP-GlcNAc binding, phosphorylation, and acetylation. Additionally, we provide evidence showing that the recently discovered enzymatic activity of GlmR is integral for its function. We show that GlmR is a cell width determinant and propose a model suggesting close cooperation with an actin-like protein, MreB. Overall, our studies highlight that GlmR is at the crux of carbon flux with an important role in maintaining cell envelope integrity. IMPORTANCEBacteria must integrate feedback from multiple metabolic processes to efficiently allocate carbon to produce essential building blocks such as nucleotides, amino acids, and cell wall precursors to support life. GlmR is a critical metabolic factor involved in the making of cell envelope precursors in diverse bacterial phyla. In Bacillus subtilis, cells lacking GlmR are deformed and hypersensitive to cell wall targeting antibiotics. As siphoning off too much carbon from other essential processes is detrimental to cell viability, GlmR activity is tightly regulated. Here we report that absence of GlmR leads to aberrant placement of cytokinetic machinery and an increase in the levels of cyclic-di-AMP, a nucleotide second messenger that assists in the cell wall stress response. We also show that GlmR function is post-translationally finetuned by phosphorylation and acetylation. Furthermore, our data reveals that the catalytic activity of GlmR is required for its function. Thus, the activity of GlmR is tightly calibrated through multiple means for efficient carbon utilization.

microbiology↗

bioRxiv2025

AutoSpectral improves spectral flow cytometry accuracy through optimised spectral unmixing and autofluorescence-matching at the cellular level

The advent of spectral flow cytometry has seen a rapid rise in the complexity of flow cytometry experiments, allowing the construction of assays with at least 50 fluorescent parameters. To correctly determine the contributions of each fluorophores signal to the high parameter data an accurate unmixing matrix needs to be generated. Even with single-stained controls, however, these matrixes include errors such as spillover spread, which compounds with each additional parameter, functionally limiting panel design. An additional source of errors is heterogeneity of cellular autofluorescence, which can affect both the unmixing matrix and misalign signals when the matrix is applied to individual cells in complex cell mixtures. Here we developed AutoSpectral, a statistical approach to automate the production of minimal-residual error unmixing matrixes and pair multiple distinct multifluorescent spectra to individual cells within a mixed sample, via an R-based software tool. AutoSpectral improves unmixing accuracy, improving incorrectly assigned cell positions by up to 9000-fold, reduces spread, particularly in samples with variable autofluorescence, and allows multi-lineage analysis of mixed populations, providing superior data for spectral flow cytometry experiments.

immunology↗

bioRxiv2025

Harnessing cGAS-STING signaling to counteract the genotoxic-immune nexus in tauopathy

Tauopathies are progressive neurodegenerative disorders characterized by aberrant tau aggregation, cognitive decline, and persistent neuroinflammation, yet the mechanisms driving neuroinflammation and disease progression remain incompletely understood. Here, utilizing human postmortem AD brains and a mouse model of tauopathy, we report that genotoxic stress-induced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) immune pathway form a self-amplifying loop that fuels neuropathology and cognitive deficits. Targeted disruption of this cycle through either genetic deletion of cGAS or pharmacological inhibition of STING restores immune homeostasis and attenuates tau pathology and cognitive deficits. Our results showed a significant accumulation of DNA double-strand breaks (DDSBs) and impaired DNA repair function, alongside elevated cGAS-STING signaling and type I interferon (IFN-I) responses in human AD brains compared to non-AD. In the PS19 transgenic (PS19Tg) mouse model of tauopathy, we found significantly elevated levels of DDSBs and altered expression of DNA repair proteins during early stages of disease, which preceded the dysregulation of cGAS-STING signaling and emergence of significant neuropathology in the later stage. Interestingly, genetic deletion of cGAS shifted microglial polarization from a pro-inflammatory M1 phenotype toward an anti-inflammatory M2 state, accompanied by a reduction in IFN-I signaling and improved cognitive performance in PS19Tg mice. Pharmacological STING inhibition reshaped the transcriptomic landscape, revealing selective regulation of pathways governing synaptic plasticity, and immune responses. This transcriptional reprogramming was accompanied by suppression of inflammatory responses, reduction in synaptic pathology, and attenuation of tau pathology in PS19Tg mice, underscoring STING as a therapeutic target for tauopathy. In conclusion, our findings reveal that genotoxic-immune crosstalk drives neuroinflammation and tau pathology and identify a conserved, druggable cGAS-STING axis that can be targeted to impede or slow disease progression in tauopathies. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/678980v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1339c89org.highwire.dtl.DTLVardef@16a1d5borg.highwire.dtl.DTLVardef@1ad4631org.highwire.dtl.DTLVardef@db4615_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Whole-genome DNA methylation profiling in COVID-19 positive patients reveals alterations in pathways linked to neurological dysfunction

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA betacoronavirus, causing coronavirus disease-19 (COVID-19). Infection with SARS-CoV-2 can result in a broad spectrum of clinical outcomes, ranging from asymptomatic or mild to a severe, deadly illness. Emerging evidence suggests SARS-CoV-2 affects host gene regulation through epigenetic mechanisms, such as DNA methylation, potentially contributing to immune dysregulation and post-acute sequelae, including neurological and psychiatric disorders. However, the extent and functional relevance of these epigenetic changes remain uncertain. Methods and resultsWe employed whole-genome bisulfite sequencing to profile DNA methylation in peripheral blood from SARS-CoV-2-positive patients across a spectrum of symptom severity, ranging from asymptomatic to severe (n=101), in comparison to SARS-CoV-2-negative individuals (n=105). We observed a widespread hypomethylation in the genomes of infected individuals, which was more pronounced in severe cases. Notably, we identified differentially methylated genes in patients with mild (19 genes), moderate (19 genes), and severe (35 genes) symptoms. These genes included those involved in canonical immune responses as well as known to be linked to neurodegenerative diseases. Subsequent pathway enrichment analysis further supported the significant association between the differentially methylated genes and those implicated in Alzheimers and Parkinsons disease, as well as neuropsychiatric conditions, suggesting potential epigenetic links between acute SARS-CoV-2 infection and long-term neurological outcomes. This is one of the first studies to comprehensively map severity-stratified genome-wide DNA methylation changes in COVID-19 patients. ConclusionOur findings underscore the potential importance of epigenetic regulation in the acute responses to SARS-CoV-2 infection and highlight an overlap with epigenetic mechanisms relevant for neuropsychiatric disease processes.

neuroscience↗

bioRxiv2025

High-temporal resolution of microbial food web dynamics and structure during phytoplankton blooms in the Baltic Sea

Heterotrophic nanoflagellates (HNF) are a key component of the microbial food webs, playing an essential role in nutrient recycling and energy transfer in aquatic ecosystems. They have been typically considered to be bacterivores, but they can be also omnivorous (feeding on prokaryotes and other eukaryotes) and predatory grazers (feeding on other eukaryotes). Here, we combine CARD-FISH with both short and long-amplicon sequencing to resolve dynamics of key HNF groups during two high-frequency sampling campaigns in spring (March-May) and autumn (September-November) phytoplankton blooms in the coastal waters of the Baltic Sea. This approach allowed us to resolve the microbial food web dynamics within HNF communities at the phylotype level at time scales relevant to HNF duplication times. Omnivorous katablepharids and predatory MAST-2 dominated the HNF community, especially in spring. Bacterivorous groups (e.g., MAST-1, CRY1) were less abundant. Long-read sequencing revealed distinct seasonal shifts in dominant phylotypes, with Katablepharis sp. and MAST-2D peaking in spring, while other lineages became more prominent in summer and autumn. The high abundance of omnivorous HNF, compared to bacterivores, highlights their key role both as grazers of bacteria and flagellates and as a food source for predatory and omnivorous ciliates. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/676335v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@c4ec55org.highwire.dtl.DTLVardef@77fd5dorg.highwire.dtl.DTLVardef@19b237borg.highwire.dtl.DTLVardef@368f39_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2025

Critical point drying of brain tissue for X-ray phase contrast imaging

X-ray phase contrast tomography is emerging as a powerful method for imaging large volumes of brain tissue at sub-cellular resolution. However, current sample preparation methods are largely inherited from visible light or electron microscopy workflows and hence are not optimised to exploit the full potential of X-ray contrast mechanisms. Here we propose to replace interstitial material by air to enhance X-ray phase contrast of the ultrastructural features. We used critical point drying (CPD) of heavy metal-stained mouse brain tissue to produce mechanically stable samples with preserved ultrastructure and enhanced refractive index boundaries, a nanofoam-like material that remains compatible with follow-up conventional resin embedding. Using two complementary synchrotron-based setups, a high-throughput microtomography beamline (P14, DESY) and a nanoscale holographic tomography beamline (ID16A, ESRF), we found that CPD samples consistently showed 2-4x stronger phase-shift signal than conventional resin-embedded tissue. The contrast gain remained consistent across samples, imaging conditions, and beamlines. Our results suggest that CPD offers a versatile route for preparing tissue for subcellular and ultrastructural-resolution X-ray imaging. It retains structural detail while improving signal, and is compatible with other processing procedures like femtosecond laser milling or electron microscopy, paving the path for biological tissue imaging beyond the mm3 scale.

neuroscience↗

bioRxiv2025

Mechano-activated chondroprogenitors (MACs) drive intrinsic cartilage repair; a process that is arrested in osteoarthritis

Articular cartilage, an avascular, matrix-rich tissue, is thought to have limited repair, thereby contributing to osteoarthritis (OA), the common degenerative disease of joints. Cartilage regeneration does occur, however, in OA joints that are mechanically off-loaded. Here we show that mechanical stress, through release of matrix-bound growth factors, reprogrammes chondrocytes, the primary cells of cartilage, into mechano-activated chondroprogenitors (MACs). Studying OA joint fluid before and after mechanical off-loading, reveals evidence of chronic MAC activity, which switches back to a chondrogenic one when mechanical stress is removed. Taken together, we conclude that OA is a disease of arrested repair in which mechanical stress signals need to be switched off before full repair can occur. This novel paradigm uncovers exciting new treatment opportunities.

cell biology↗

bioRxiv2025

Mono-allelic p.R37H Dehydrodolichyl Diphosphate Synthase variants lead to protein glycosylation defects, aberrant lipid profiles and interneuron scarcity in a novel mouse model of progressive epileptic encephalopathy.

Developmental delay and seizures with or without movement abnormalities (OMIM 617836) caused by heterozygous pathogenic variants in the DHDDS gene (DHDDS-CDG) is a rare genetic disease that belongs to the progressive encephalopathy spectrum. It results in cognitive delay in affected children, accompanied by myoclonus, seizures, ataxia and tremor, which worsens over time. DHDDS encodes a subunit of a DHDDS/NUS1 cis-prenyltransferase (cis-PTase), a branch point enzyme of the mevalonate pathway essential for N-linked glycosylation. We describe the first mouse model of this disease, DhddsR37H+/-strain, heterozygous for the human recurrent de novo c.110G>A:p.R37H pathogenic variant. DhddsR37H+/- mice present with seizures, myoclonus and memory deficits associated with reduced density or/and maturity of inhibitory interneurons in the cortex. Multiomics analyses of mouse CNS tissues, together with the enzymatic/structural characterization of the R37H DHDDS mutant protein, reveal that the variant produces a catalytically inactive enzyme and results in a brain dolichol deficit, aberrant glycosylation of brain glycoproteins, including those involved in synaptic transmission and major perturbations in the CNS proteome and lipidome. Acetazolamide, a carbonic anhydrase inhibitor clinically approved for treatment of glaucoma, epilepsy, and intracranial hypertension, and successfully used "off-label" to treat genetic movement disorders, reduces seizure susceptibility to pentylenetetrazol in DhddsR37H+/- mice, suggesting potential therapeutic value of using this drug in human DHDDS-CDG patients. Together, our results define cis-PTase as a master regulator of CNS development and function and establish that its monoallelic debilitating variants cause a novel congenital disorder of glycosylation associated with aberrant levels of neuronal proteins and lipids.

neuroscience↗

bioRxiv2025

Single cell decomposition of multicellular aging programs associated with impaired lung regeneration

Aging impairs the regenerative capacity of mammalian organs and is a major risk factor for organ fibrosis. Mechanisms underlying persistent fibrosis after lung injury in old individuals remain unclear. We used longitudinal single-cell RNA-seq after lung injury and dissected aging effects computationally and experimentally at baseline and during repair. In old mice, sustained fibroblast activation in the resolution phase of fibrosis was associated with prolonged epithelial senescence and persistent epithelial-mesenchymal crosstalk. Single-cell interpretable tensor decomposition analysis revealed that aging most strongly affected T/B-lymphocytes and macrophages. Notably, we identified a Granzyme K-high CD8+ T cell state that was unique to aged mice, co-localized with epithelial progenitors, and its co-culture or Gzmk treatments in lung organoids impaired progenitor function by inducing stem cell senescence. In summary, our study highlights the effects of immune aging on epithelial progenitor function and provides a time-resolved high resolution map of lung regeneration in the context of aging.

cell biology↗

bioRxiv2025

A Lung Tumor-on-a-Chip Model Recapitulates the Effect of Hypoxia on Radiotherapy Response and FDG-PET Imaging

Most solid tumors contain regions of hypoxia that pose a significant challenge to the efficacy of radiation therapy. This study introduces a novel 3D lung tumor-on-a-chip (ToC) model designed to replicate the hypoxic tumor microenvironment in vitro while also providing a platform for clinically relevant interventions such as radiotherapy and positron emission tomography (PET) imaging. To simulate the heterogeneous oxygen distribution found in tumors, the ToC model incorporates an oxygen gradient achieved through a straightforward chemical oxygen scavenging system. A unique innovation of this device is the integration of a thin scintillator plate, which enables high-resolution radioluminescence microscopy imaging of tumor metabolism under hypoxia and normoxia conditions using clinically approved PET tracers such as fluorodeoxyglucose (FDG). The response of this hypoxic model to radiation therapy (10 Gy X-ray) demonstrated [~]4-fold higher radioresistance compared to the normoxic ToC model, as assessed by colony formation potential. Additionally, DNA damage observed in the normoxic ToC model was [~]5-fold higher than that in the hypoxic model. Furthermore, the metabolic consumption of glucose was found to mirror the localization of hypoxia, validating the use of this biomarker for planning radiation therapy. The integration of high-resolution radionuclide imaging within ToC models enables on-chip PET imaging and facilitates oncology research and discovery, offering innovative capabilities for the preclinical testing of novel cancer therapies in a clinically relevant environment.

bioengineering↗

bioRxiv2025

Food-activated Microneedle Sensor for Real-time, Colorimetric Spoilage Monitoring of Pre-packaged Food

At a time of growing food insecurity, developing technologies to reduce food waste is critical. We report an inexpensive, colorimetric spoilage sensor for real-time food product assessment. The sensor is composed of dehydrated gelatin microneedles that exhibit high mechanical integrity in their base state. However, once exposed to fluid-rich food environments, they rapidly transition to a hydrogel sensing state. Food-derived anthocyanins embedded within these microneedles enable pH-based spoilage monitoring. When applied to sealed fish products, these microneedles non-destructively penetrate through packaging and are rehydrated by the underlying fish matrix. As the product ages, a defined colour shift occurs, demonstrating strong correlation with quantitative spoilage markers. When applied to unsealed fish products for rapid testing, the large microneedle sensing interface enables accelerated colorimetric sensing. Finally, successful fresh versus spoiled categorization of smartphone-acquired images of the sensor using machine learning removes readout ambiguity, empowering consumers with independent real-time product monitoring.

molecular biology↗

bioRxiv2025

Genetically-programmed Hypervesiculation of Lactiplantibacillus plantarum Increases Production of Bacterial Extracellular Vesicles with Therapeutic Efficacy in a Preclinical Inflammatory Bowel Disease Model

Inflammatory bowel diseases (IBD) affect over 6 million people globally and current treatments achieve only 10-20% rates of durable disease remission. Bacterial extracellular vesicles (BEVs) from probiotic lactic acid bacteria (LAB) are a promising novel therapeutic with mechanisms holding potential to drive increased rates of durable disease remission, including immunomodulation and intestinal epithelial tissue repair. However, translation of these cell-secreted nanovesicles is limited by long standing biomanufacturing hurdles, especially low production yields due to low biogenesis rates from cells. Here, our goal was to identify a candidate probiotic LAB that produces BEVs effective in a preclinical mouse model of IBD, and then genetically engineer the LAB for at least 10-fold increased production yields of BEVs, thereby passing a critical production threshold. We identified Lactiplantibacillus plantarum as a candidate LAB producing BEVs effective in treating acute dextran sulfate sodium (DSS)-induced murine colitis, and with greater efficacy than BEVs from probiotic Escherichia coli Nissle 1917. We then genetically engineered a hypervesiculating L. plantarum strain by inducible expression of a peptidoglycan-modifying enzyme, resulting in a 66-fold increase in BEV productivity. Finally, we confirmed hypervesiculating L. plantarum BEVs were therapeutically effective in the acute DSS mouse model of colitis and found these BEVs were superior in reducing mucosal tissue damage compared to live L. plantarum cells. These findings demonstrate that BEVs from genetically engineered hypervesiculating strain of L. plantarum are a promising preclinical therapeutic candidate for IBD that overcomes historical biomanufacturing limitations of BEV therapeutics.

bioengineering↗

bioRxiv2025

Loss of CHMP2A implicates an ordered assembly of ESCRT-III proteins during cytokinetic abscission

The ESCRT machinery mediates membrane remodeling in fundamental cellular processes including cytokinesis, endosomal sorting, nuclear envelope reformation, and membrane repair. Membrane constriction and scission is driven by the filament-forming ESCRT-III complex and the AAA-ATPase VPS4. While ESCRT-III-driven membrane scission is generally established, the mechanisms governing the assembly and coordination of its twelve mammalian isoforms in cells remain poorly understood. Here, we examined the spatial organization and interdependence of ESCRT-III subunits during mammalian cytokinetic abscission by depleting CHMP2A, a core ESCRT-III component. Using live cell imaging, structured illumination microscopy (SIM) and correlative light-electron microscopy (CLEM), we show that CHMP2A knockout cells display a significant delay--but not failure--in abscission, accompanied by distinct mislocalization phenotypes across ESCRT-III subunits. While IST1 and CHMP2B were minimally disrupted, CHMP4B, CHMP3, and CHMP1B display progressively severe organization defects at the abscission site. Dual- protein imaging reveals disrupted coordination between ESCRT-III subunits in individual CHMP2A-deficient cells, supporting an ordered assembly of ESCRT-III subunits in cytokinetic abscission. Together, our findings provide the first in vivo evidence for hierarchical assembly of ESCRT-III subunits during ESCRT-mediated membrane remodeling and identify CHMP2A as a key organizer of ESCRT-III architecture essential for timely membrane abscission.

cell biology↗

bioRxiv2025

Focused ultrasound neuromodulation of mediodorsal thalamus disrupts decision flexibility during reward learning

When learning to find the most beneficial course of action, the prefrontal cortex guides decisions by comparing estimates of the relative value of the options available. Basic neuroscience studies in animals support the view that the thalamus can regulate this activity within and across the prefrontal cortex. We studied a group of patients (n=37) undergoing unilateral MR guided focused ultrasound for essential tremor, performing the restless bandit, a reward reinforcement learning task, immediately before and after thalamotomy. Thalamotomy significantly impaired the proportion of switch choices during the task without affecting overall performance. This effect was observed when the task was delivered to co-incide with maximal vasogenic thalamic oedema but not in a control group tested on the same day of their treatment. A reinforcement learning model fitted to the patients choices replicated the effect of thalamotomy when the model increased exploitation of the bandits learnt value estimate. This shift in the explore-exploit trade-off, manifesting as reduced choice flexibility, co-varied with the pattern of post-operative oedema extension into mediodorsal nucleus. These findings confirm a causal role of the thalamus and specifically the mediodorsal nucleus, in regulating the extent to which value estimates are used to guide decisions and learning from reward.

neuroscience↗

bioRxiv2025

Non-canonical enhancers control gene expression and cell fate in human pluripotent stem cells

Enhancers are key gene regulatory elements that ensure the precise spatiotemporal execution of developmental gene expression programmes. However recent findings indicate that approaches to identify enhancers may not capture the full repertoire of active enhancers in mammalian genomes. Here, we combine massively parallel enhancer assays with chromatin structure and transcriptome profiling to functionally annotate enhancers genome-wide in human induced pluripotent stem cells. We find that a substantial fraction ([~]40%) of accessible chromatin regions with enhancer function lack key features associated with active enhancers, including the active enhancer mark histone H3 lysine 27 acetylation and enhancer-associated RNAs. Perturbation of this class of non-canonical enhancers by CRISPR-mediated epigenome editing results in decreased levels of target gene expression and, in one instance, loss of pluripotent stem cell characteristics. Collectively, our data demonstrate enhancer activity for a class of gene regulatory elements that had until now only been associated with a neutral or inactive status, challenging current models of enhancer function.

genomics↗

bioRxiv2025

A Plasma Membrane Vesicle Imaging-Based Platform for Studying Membrane Fusion

Membrane fusion is central to biological processes such as viral entry, fertilization and cell-to-cell fusion. Gaining a mechanistic understanding of fusion requires the ability to visualize and quantify the dynamic interaction between two membranes and their associated protein machineries at high temporal and spatial resolution. However, studying these processes in live cells remains challenging due to the complexity of the cellular environment. Here we demonstrate a versatile cell-free platform based on giant plasma membrane vesicles (GPMVs) that enables controlled, quantitative analysis of receptor binding and membrane fusion kinetics in a native membrane context. As proof of concept, we reconstitute the SARS-CoV-1 Spike-ACE2 interaction, capturing specific receptor engagement and accumulation at the membrane interface using confocal microscopy and micropipette aspiration. Fusion was induced by proteolytic activation and quantified using both high-resolution microscopy and high-throughput Imaging Flow Cytometry. The platform also reveals the influence of membrane composition on fusion efficiency, demonstrated by the impact of cholesterol depletion. This approach provides a broadly applicable system for dissecting membrane fusion and protein-protein interactions across membranes, with compatibility for biophysical, imaging and structural analysis. It offers new opportunities for mechanistic studies and inhibitor screening in a biologically relevant yet experimentally accessible context.

biophysics↗

bioRxiv2025

3D Biomimetic Liver Cancer Model: Diethylnitrosamine-Induced Proteomic Dysregulations in Stromal-Epithelial Milieu

Hydrogel-based three-dimensional (3D) co-culture systems are emerging as biomimetic platforms that more accurately recapitulate tissue architecture and microenvironmental interactions compared to conventional two-dimensional (2D) cultures. This study introduces an engineered 3D liver-like model to investigate compartment-specific responses to the potent hepatocarcinogen Diethylnitrosamine (DEN), with a focus on early events in carcinogenesis and tumor-stroma interactions. AML12 and 3T3 cell lines were treated with DEN or vehicle either in 2D culture or in 3D hydrogels in four experimental groups: (1) DEN-treated AML12 with vehicle-treated 3T3, (2) DEN-treated 3T3 with vehicle-treated AML12, (3) both cell types DEN-treated, and (4) both vehicle-treated. The cultured recombinants were subjected to proteomic profiling via mass spectrometry, followed by bioinformatics analysis and the results were validated through immunocytochemical staining (ICC). Gene ontology analysis revealed that cytoskeletal, RNA metabolism, and scaffold/adaptor proteins were among the most significantly enriched in 3D versus 2D models. Structural proteins emerged exclusively in mixed 3D co-cultures, reinforcing the organotypic nature of the system. Enriched pathways in 3D included intermediate filament organization, actin dynamics, and focal adhesion--pathways closely associated with liver carcinogenesis. Protein-protein interaction analysis demonstrated maximal network complexity in 3D cultures where both compartments were DEN-exposed. Survival analysis further identified poor-prognosis biomarkers (KRT20, KRT15, KRT14) uniquely enriched in this condition. ICC staining supported the proteomic findings. This organoid-like 3D co-culture model provides a physiologically relevant platform for investigating early-stage liver carcinogenesis and highlights the critical role of stromal-epithelial interactions. Its ability to replicate organ-level complexity and generate clinically relevant proteomic signatures supports its utility in translational cancer research and future drug discovery applications.

cancer biology↗