Biology subjects

Banerjee, P.

Publications and source records attributed to Banerjee, P..

At least 19 recordsLinked to original sources

bioRxiv2026

PINPOINT enables non-destructive nanoscopic quality control of individual lipid nanoparticles and their mRNA cargo

Quality control of lipid nanoparticles (LNPs) is central to development of mRNA therapeutics, yet ensemble measurements obscure critical heterogeneity in particle morphology and mRNA cargo loading. Here we introduce PINPOINT (Profiling Individual Nanoparticles with Precise Observation of INternalized Therapeutics), a single-particle nanoscopy platform for non-destructive, multiparametric profiling of preformulated LNP samples. Super-resolution imaging with Nile Red resolves size and morphology, while an optimized permeabilization strategy provides selective access to encapsulated mRNA without compromising LNP integrity. Single-particle fluorescence measurements, including RiboGreen and sequence-specific probes, enable quantification and validation of mRNA loading and identity. Comparative analyses of bare and encapsulated mRNA reveal kinetic and structural features, exposing heterogeneity masked by bulk assays. By integrating structural and molecular readouts at the single-particle level, PINPOINT establishes a quantitative framework for assessing of LNP composition and payload distribution, offering actionable insights for improving batch consistency and guiding the rational design of next-generation mRNA delivery systems.

biophysics↗

bioRxiv2026

HIV-1 Assembly is Assisted by IRSp53 Through an Interaction that Extends Gag Conformation

Budding of the immature virion is an essential part of the HIV-1 life cycle. However, understanding and inhibiting this process has been challenging, as both simulations and theoretical studies suggest that the energy required to bend the membrane is too high for the complete assembly of a spherical viral particle at the plasma membrane without the assistance of cellular host factors. Recent results suggest that the I-BAR protein IRSp53 may be one such host factor, but the molecular mechanism by which the Gag-IRSp53 interaction promotes HIV-1 assembly and budding remains poorly understood. We employ molecular dynamics simulations, starting from AlphaFold models, to show that the SH3 domain of IRSp53 interacts directly with the MACANTD domain of Gag and we experimentally validate this interaction. Our simulations show that the specificity of the interaction with IRSp53 arises from its ability to extend the MACANTD Gag conformation in a manner similar to RNA binding, which is known to activate Gag assembly. We then computationally design HIV-1 Gag mutants that, as confirmed by simulations, do not undergo conformational changes upon interaction with theIRSp53 SH3 domain. Experimentally, these Gag mutants are tested and shown to accumulate in cells. In addition, for these mutants we observe large quantities of Gag assembling at the plasma membrane but exhibiting large budding defects. These results validate the proposed mechanism by which the Gag-IRSp53 interaction promotes completion of HIV-1 budding and suggest new routes for inhibiting viral assembly or budding.

molecular biology↗

bioRxiv2026

Chemical proteomics reveals post-translational remodeling of endosomal pathways during macrophage activation.

Macrophages are central regulators of innate immunity, yet the post-translational mechanisms that enable rapid inflammatory responses remain incompletely understood. Here, we integrated transcriptomics, unenriched proteomics, phosphoproteomics, metabolomics, and cysteine reactivity profiling to comprehensively characterize primary human macrophages following Toll-like receptor and Stimulator of Interferon Genes (STING) activation. This approach revealed substantial discordance between transcript- and protein-level changes, as well as extensive post-translational remodeling. Differentially reactive cysteines were enriched in proteins involved in small-GTPase signaling, vesicular trafficking, and the endo-lysosomal system. Reactivity changes in two subunits of the endosomal mRNA-transport FERRY complex nominated it as a candidate target of inflammation-associated post-translational regulation. Biochemical and imaging analyses established FERRY complex assembly and early endosomal localization in primary human macrophages and identified DISC1 as a previously unrecognized FERRY-associated protein. This work provides a comprehensive resource of biochemical and post-translational remodeling during macrophage activation and implicates endosome-associated mRNA transport in innate immune regulation.

immunology↗

bioRxiv2026

E-cadherin-mediated neighborhood surveillance dictates pre-malignant outcomes

Stratified epithelia accumulate oncogenic mutations throughout life, yet overgrowths are rare. How epithelia detect and eliminate aberrant clones remains poorly understood. Using a mouse model of oncogenic clonal mosaicism in the skin, we find that pre-malignant epidermal cells redistribute E-cadherin to interfaces shared with wild-type neighbors, generating local tension heterogeneity that triggers elimination by cell competition. We show that gain or loss of E-cadherin can each drive competitive elimination, and although mechanical routes differ, both establish tension heterogeneity between neighbors, rather than any absolute adhesion state, as the critical determinant of epidermal fitness. This mechanism carries the seeds of its own failure: these tension differentials precipitate clonal sorting, depleting the wild-type contacts that surveillance requires. Pre-malignant cells then become supercompetitors, eliminating wild-type neighbors and expanding hyperplastically. Mechanical heterogeneity therefore endows tissues with an active, yet inherently fragile error-correction system whose collapse initiates a switch in competitive status, increasing tumorigenesis susceptibility.

cell biology↗

bioRxiv2026

Psi RNA-specific Binding Promotes HIV-1 Gag Conformational Change Critical for Immature Viral Particle Assembly

The immature HIV-1 virion is assembled by the Gag polyprotein using inositol hexakisphosphate (IP6) as an essential assembly co-factor. Gag binds the genomic RNA Psi packaging signal via the nucleocapsid (NC) domain and associates with the plasma membrane via the matrix (MA) domain. Previous studies revealed that Gag exists in both compact (C) and extended (E) conformational states in solution. Only E-Gag formed virus-like particles with the correct size and IP6 shifted the equilibrium of DNA-bound Gag to the E state. The influence of specific RNA elements on this conformational change is unknown. In this work, a dual dye-labeled Gag was prepared for probing the effect of RNA binding on Gag conformation using Forster resonance energy transfer (FRET). In low salt and in the absence of other factors, Gag was primarily in the C state. Psi RNA binding induced a more significant FRET decrease than binding to non-Psi RNAs, consistent with a shift to E-Gag. IP6 alone also promoted the E-Gag state in the absence and presence of RNA. Atomistic molecular dynamics simulations are consistent with and provide detail into the role of NC-Psi RNA binding in the conformational switch of C-Gag to assembly-competent E-Gag. Simulations also showed that this switch is driven by capsid (CA) linker domain orientational flexibility and MA-CA unbinding dynamics. Thus, the highly flexible multi-domain Gag polyprotein leverages both viral and host cell factors to sample and stabilize distinct conformations, thereby orchestrating the viral assembly process.

biophysics↗

bioRxiv2026

Extraction workflow determines marker-specific recovery andreproducibility in leaf-litter eDNA metabarcoding

Forest-floor leaf litter is a dynamic and structurally complex ecological transition zone and thus a promising substrate for terrestrial eDNA metabarcoding. Yet, extraction workflows for this heterogeneous matrix remain poorly standardized, especially in tropical systems, making it largely impossible to compare ecological functions across space, time, and taxa. To guide workflow selection across a series of selection criteria, including biological target, research question and practical considerations, we compared DNA extraction workflows for leaf-litter eDNA collected from 42 biological samples across seven different forest sites on Oahu, Hawaii. We evaluated four DNA extraction workflows: (1) Two low-volume approaches, with DNA extracted directly from 200 mg of homogenized litter using (i) CTAB or (ii) DNeasy PowerSoil(R); and (2) two high-volume approaches using PBS wash-based from 10 g of litter followed by (i) Centrifugation or (ii) Filtration. Taxonomic recovery from each workflow was evaluated with two COI primer sets targeting arthropods (ANML and shorter NoPlant), and one ITS marker targeting fungi. Results show that eDNA workflows tested here recovered site-level differences among forest-floor communities, but biodiversity recovery depended strongly on extraction workflow and marker. For low volumes, PowerSoil recovered the highest fungal richness (with ITS marker), and produced the most reproducible PCR-replicate profiles across markers, and required the least hands-on time, while CTAB was less expensive but required handling hazardous chemicals. For high volumes workflow, Centrifugation recovered higher arthropod diversity with ANML primer. Differences in community composition were nonetheless recovered by each method. At the same time, sampling sites explained more ASV-level compositional variation than extraction workflow across markers, showing that all workflows retained site-level ecological signals. Together, these results support a workflow framework in which extraction choice depends on target organism group, DNA state, reproducibility needs, and practical constraints.

ecology↗

bioRxiv2026

The CD19-4-1BBL antibody fusion protein unleashes the immune system against high-risk chronic lymphocytic leukemia

CD19-4-1BBL is a bispecific antibody fusion protein that targets CD19 and costimulates 4-1BB on T cells and other immune cells. Its antitumor activity has been reported in B-cell non-Hodgkin lymphoma with emphasis on its T-cell mediated cytotoxic activity. Its effect on other 4-1BB expressing immune cells is unexplored. Here, we investigated the molecular mechanisms and the antileukemic effect of CD19-4-1BBL in chronic lymphocytic leukemia (CLL), a B-cell malignancy profoundly marked by the immunosuppressive activity of myeloid-derived suppressor cells, tumor-associated macrophages and CD4+ regulatory T cells. We demonstrated that CD19-4-1BBL simultaneously mitigates the immunosuppressive phenotype and transcriptome machinery of these cells and promotes antitumor CD8+ T-cell immunity. Finally, in a preclinical, patient-derived xenograft model of CLL, we observed a favourable survival impact, especially in mice transplanted with immune cells from patients with high-risk/progressive leukemia. Our findings provide evidence that the CD19-4-1BBL treatment is a multifaceted, immune-based strategy that should be clinically explored in patients with chronic lymphocytic leukemia. KEY POINTSO_LICD19-4-1BBL sharpens the myeloid cell transcriptome and stimulates diverse memory CD8+ T cell clonotypic responses. C_LIO_LICD19-4-1BBL costimulation can be therapeutically exploited in high-risk chronic lymphocytic leukemia. C_LI

immunology↗

bioRxiv2026

FINDER converts zero-background kinetic fingerprinting into area-scalable attomolar biomarker detection

Background constrains analytical sensitivity: surveying larger sensor areas samples more analyte molecules but also accumulates false positives, limiting gains in detection performance. Here we introduce FINDER--Fluorogenic INstantaneous Digital Enumeration and Recognition--a single-molecule platform that combines kinetic fingerprinting with fluorogenic transient probes for rapid molecular classification under near-zero-background conditions. By suppressing both solution and surface-associated background at micromolar probe concentrations, FINDER classifies individual molecules within seconds-scale observation windows per field of view. This regime allows sensitivity to scale with surveyed sensor area, enabling amplification-free quantification of the miRNA cancer biomarker hsa-miR-16 with an 11 aM detection limit. FINDER further generalizes to HPV16 DNA biomarker detection, two-color RNA/DNA co-profiling, and rapid discrimination of clinically relevant EGFR single-nucleotide variants using multidimensional kinetic filtering. Rapid per-field classification permits tens of fields to be surveyed within minutes. By converting kinetic specificity into area-scalable sensitivity, FINDER enables semi-automated attomolar biomarker counting without amplification in practical workflows.

biophysics↗

bioRxiv2026

Topography structures of arthropod communities revealed by leaf-derived environmental DNA on Oahu, Hawaii

Arthropod communities on oceanic islands are shaped by spatial isolation, environmental gradients, and biological invasions, yet their structure remains difficult to resolve due to incomplete taxonomic coverage. In particular, it remains unclear (i) how non-native arthropods can influence community composition and (ii) how they interact with native and non-native plants. To answer the first question, we used leaf-derived environmental DNA (eDNA) to characterize arthropod communities across elevational gradients on five ridges on Oahu, using the native tree Metrosideros polymorpha as a standardized plant. To understand the second question, we compared leaf-derived eDNA from Metrosideros polymorpha (Native), Acacia koa (native), and Psidium cattleianum (invasive), co-occurring in two ridges on Oahu. Additionally, to overcome limitations of reference databases, we applied NIClassify to infer native versus introduced status without requiring species-level identification. Across 96 leaf samples (with 851 Arthropod ASVs), we found arthropod richness increased with elevation, while the proportion of introduced taxa declined significantly. Community composition was primarily structured by ridge, with strong distance-decay relationships indicating high spatial turnover in both native and non-native assemblages. In contrast, plant species effects were context dependent and did not show a consistent native versus invasive signal. Threshold analyses identified a community transition (native vs introduced) near 500 m elevation. These results show that plant-derived eDNA can resolve spatial and environmental structuring of arthropod communities while capturing invasion dynamics under incomplete taxonomic knowledge. Classifier-based inference enables community-level ecological interpretation beyond reference-limited taxa, providing a scalable framework for biodiversity monitoring in data-poor systems.

ecology↗

bioRxiv2026

Wnt stimulation and inhibition in the development and phenotype of patient-derived gallbladder organoids

Gallbladder cancer (GBC) is a highly lethal malignancy with limited experimental models to study disease biology or evaluate therapeutic responses. Although canonical Wnt activation is commonly used for patient-derived organoid (PDO) development and expansion, gallbladder PDOs has also been generated under Wnt-inhibitory conditions. No comparative assessment has determined how Wnt pathway modulation influences gallbladder PDO development, phenotype or drug response. This study systematically compared the impact of canonical Wnt activation (WNTAct medium containing CHIR99021) versus inhibition (WNTInh medium containing DKK1) on the establishment, propagation, molecular features and therapeutic responses of PDOs generated from malignant or non-malignant gallbladder tissues derived from the same patient. Both media supported successful PDO generation with comparable efficiency, preserving biliary epithelial functions and marker expression. Transcriptomic profiling confirmed selective enrichment of canonical Wnt target genes in PDOs generated in WNTAct cultures. WNTAct conditions enabled markedly superior long-term propagation, whereas WNTInh cultures more consistently retained the dysplastic features in malignant samples. Gemcitabine response assays demonstrated significantly greater drug sensitivity in PDOs grown in WNTAct medium, a phenotype reversible upon media switching but requiring extended adaptation, indicating a dynamic and context-dependent influence of Wnt signaling on chemotherapeutic vulnerability. Collectively, the findings reveal a trade-off between long-term propagation and histological fidelity in gallbladder PDOs and show that Wnt signaling modulates gemcitabine sensitivity in a reversible manner. This comparative framework provides practical guidance for selecting culture conditions for gallbladder PDO based disease modelling and precision oncology applications.

cell biology↗

bioRxiv2026

A PLUM Job: Peptide modeLs for Understanding and engineering antiMicrobial therapeutics

Motivation: Antibiotic-resistant infections in humans and animals are rising, creating an urgent need for new antimicrobial strategies. This challenge extends from human health to food production, where foodborne pathogens cause substantial animal and human illness annually. Antimicrobial peptides (AMPs) are promising alternatives because of their broad activity and potentially lower resistance risk. However, rational AMP design remains challenging, particularly when generating targeted peptides with desired function and length for downstream experimental testing. Results: We introduce Peptide modeLs for Understanding and engineering antiMicrobial therapeutics (PLUM), a structured conditional variational autoencoder for controlled AMP generation. PLUM organizes the latent space into components associated with function, length, and residual sequence context, enabling de novo and prototype-guided generation across the 5-35 amino acid range. In de novo generation, PLUM achieved the highest classifier-confirmed yields for both AMP and non-AMP targets. PLUM-generated AMPs were also substantially distinct from the training set and exhibited broad internal sequence diversity. They further showed favorable predicted safety, stability, and potency profiles, including low toxicity, the lowest median instability index among the evaluated models, and a large number of candidates predicted to have high antibacterial potency. In prototype-guided generation, PLUM yielded more AMP-classified variants than HydrAMP while also supporting non-AMP-targeted generation from existing prototypes. Together, these results support PLUM as a scalable framework for function- and length-controlled AMP design. Availability: https://github.com/priyamayur/PLUM

bioinformatics↗

bioRxiv2026

Comparison of environmental DNA and bulk DNA metabarcoding for assessing terrestrial arthropod diversity across three habitat types on Guam

DNA based methods offer a rapid and cost-effective way for detecting species occurrence and monitoring biodiversity; among them bulk DNA metabarcoding is well-established, and recently developed environmental DNA (eDNA)-based methods offer a non-destructive alternative. With a goal to develop suitable methods for assessing insect biodiversity in ecosystems for which DNA reference libraries are not well developed and incomplete, such as remote islands, we compared established bulk DNA metabarcoding methods with eDNA across three replicated terrestrial ecosystem types (limestone forest, degraded forest, and grassland) in Guam. Using two mitochondrial COI primer pairs, we performed bulk DNA metabarcoding of standard entomological collection methods (malaise traps, pan traps, vegetation beating), and compared the assessment of biodiversity with that from different eDNA sources (flowers, spider webs, leaves, tree trunks). In our samples, eDNA and bulk DNA metabarcoding both detected a large proportion of overall taxa (OTUs, 86.6% and 60.3%, respectively). Although DNA metabarcoding detected significantly more taxa, eDNA proved to be a reasonable non-destructive alternative. As expected, because of limitations in existing reference databases for remote habitats, species-level identification was achieved for only a few OTUs. Overall, the sampling approach was the dominant driver of arthropod diversity, explaining [~]17% of the observed variation, while habitat type accounted for [~]4%. Thus, each sampling approach captured some unique diversity signals and contributed to the complementary effect of maximizing detection. For rapid insect biodiversity surveys of terrestrial arthropods, we recommend an integrated metabarcoding approach, and in sensitive habitats where insect capture is undesirable, eDNA offers a powerful alternative to monitor diversity and community change.

ecology↗

bioRxiv2026

PLCβs are recruited to the plasma membrane in macrophages by both Gβγ and Gαq

PLC{beta} enzymes cleave PIP2 from the plasma membrane, producing IP3 and DAG, which regulate intracellular Ca2+ levels and protein kinase C activity, respectively. They are regulated by GPCR signaling through the G proteins G{beta}{gamma} and Gq and have been shown to function as coincidence detectors for dual stimulation of Gq and Gi-coupled receptors via these G proteins. PLC{beta}s are aqueous-soluble enzymes, but partition onto the membrane surface to access their lipid substrate. We previously demonstrated that membrane recruitment and orientation of the catalytic core on the membrane surface underlie G{beta}{gamma}-dependent regulation of PLC{beta} enzymes. Using macrophages as a model system, where PLC{beta} signaling is essential for responses to infection and tissue injury, we investigated the contribution of G{beta}{gamma}-dependent regulation and membrane recruitment of PLC{beta} in the context of endogenous signaling. By measuring Ca2+ mobilization, we demonstrate that both Gi and Gq-coupled receptors independently stimulate PLC{beta} activity. Using total internal reflection and stimulated emission depletion microscopy, we demonstrate that most of the PLC{beta}3 in the cell is localized away from the plasma membrane at rest but is rapidly recruited to the plasma membrane upon stimulation by both Gi and Gq-coupled receptors, illustrating that both G{beta}{gamma} and Gq recruit PLC{beta} to the plasma membrane. These results support an updated model for G protein-dependent regulation of PLC{beta} enzymes, where G{beta}{gamma}-induced regulation in the absence of Gq can occur and is apparently dictated by the local concentration of receptor, G proteins, and PLC{beta}. Significance StatementPLC{beta} enzymes are critical mediators of signal transduction with roles in neuronal, cardiac, and immunological signaling. Despite this importance, many aspects of their function and regulation remain poorly understood. PLC{beta}s are aqueous soluble but must partition onto the membrane surface to access their lipid substrate, which enables regulation at the partitioning step, the catalytic step, or both. We previously demonstrated that membrane recruitment and orientation of the catalytic core on the membrane surface underlie PLC{beta} regulation by G{beta}{gamma} in a reconstituted system. Using macrophages as a model system for physiological signaling, we demonstrate here that G{beta}{gamma} can activate PLC{beta} via membrane recruitment in cells.

biophysics↗

bioRxiv2025

Mechanism of Phosphatidylserine Lipid Scrambling by Human SERINC3, an HIV-1Restriction Factor

The HIV-1 restriction factor, hSERINC3, functions as a lipid scramblase, translocating lipids across the bilayer in reconstituted proteoliposomes and the viral envelope. Phosphatidylserine(PS) scrambling and exposure at the outer leaflet are recognized to play important roles in several biological processes. To understand the mechanistic basis for hSERINC3-mediated PS lipid scrambling at atomistic resolution, we implemented the transition-tempered metadynamics (TTMetaD) enhanced sampling method. Our simulations sampled close-to-open hSERINC3 conformational transition during PS scrambling and demonstrated that while other non-ATP-dependent lipid transporters with similar architecture transport lipid following a "trap-and-flip" mechanism, hSERINC3 adopts a "credit card" mechanism of lipid scrambling and does not follow the classical "alternating access" mechanism. Notably, we observe unfolding of the H8 NTD, consistent with the cryo-EM density map of WT-hSERINC3, mediates PS scrambling. A cluster of hydrophilic residues in the hSERINC3 central cavity, forming central gates and interacting with the PS headgroup, stabilizes the intermediate state of inner-groove scrambling and is also observed in the AlphaFold2 model of hSERINC5 that exhibits the highest viral restriction activity. Surprisingly, our simulations reveal distinct pathways for lipid translocation and pathway-dependent alterations of hSERINC3 central cavity, providing direct evidence for a non-canonical, closed-state out-of-groove PS scrambling in a complex membrane environment.

biophysics↗

bioRxiv2025

Environmental DNA Metabarcoding Effectively Detects Invasive Species, Pests, and Community Changes in Taiwan Rice Fields

Rice fields represent man-made semi-aquatic wetlands primed for invasive pests. Monitoring rice field biodiversity using conventional methods, however, is time-consuming and laborious. Environmental DNA (eDNA) methods can provide a fast and effective means to monitor rice field communities and inform management decisions. Our study provides proof-of-concept of rice field eDNA biodiversity assessments, with a focus on native and non-native pests across cultivation phases. We collected eDNA samples from locations in southern Taiwan during planting and harvesting, employing eDNA metabarcoding (COI) to detect diverse taxonomic groups. We assigned 77 ASVs across all sites to animal taxa, 34 of which were identified to species. Overall, 18 species were designated as native or non-native (83.3% and 16.6%, respectively), including three major rice pests, Chilo suppressalis (native), Coptotermes formosanus (native), and Pomacea canaliculata (non-native). Cultivation status affected overall diversity, with higher species richness during planting compared to harvesting. No significant differences were observed between native and non-native taxa between cultivation phases. Altogether, we detected a complex environment across trophic levels comprised of both native and non-native agricultural pests using limited sampling effort, demonstrating eDNA analysis as an efficient biomonitoring approach in rice agroecosystems with direct applications for pest, invasive species, and vector surveillance within Taiwan.

ecology↗

bioRxiv2025

Redox regulation of neuroinflammatory pathways contributes to damage in Alzheimers disease brain

The mechanism(s) whereby redox stress mediates aberrant immune signaling in age-related neurological disorders remains largely unknown. Normally, the innate immune system mounts a robust response to infectious stimuli. However, unintentional activation by host-derived factors, such as aggregated proteins associated with neurodegenerative disorders or by cytoplasmic genomic or mitochondrial DNA, can elicit aberrant immune responses. One such immune response is represented by the cytosolic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. Using redox chemical biology and mass spectrometry approaches, we identified S-nitrosylation of STING cysteine 148 as a novel posttranslational redox modification underlying aberrant type 1 interferon signaling in Alzheimers disease (AD). Critically, we observed S-nitrosylated STING (SNO-STING) in postmortem human AD brains, in hiPSC-derived microglia (hiMG) exposed to amyloid-{beta} (A{beta})/-synuclein (Syn) aggregates, and in 5xFAD transgenic mice. Mechanistically, our findings reveal that STING S-nitrosylation is critical in initiating signaling cascades by promoting the formation of disulfide-bonded STING oligomers. This leads to neuroinflammation early in the course of disease in vivo in 5xFAD mice with consequent synaptic loss. Collectively, our research supports the role of SNO-STING in neuroinflammation associated with AD, and points to a novel druggable cysteine residue in STING to prevent this S-nitrosylation reaction with its inherent inflammatory response. One Sentence SummaryS-Nitrosylation of STING triggers activation of cGAS-STING signaling in Alzheimers disease brain and subserves a novel link between excessive nitrosative stress and dysregulated innate immunity, thus contributing to disease progression.

neuroscience↗

bioRxiv2025

Insights into gallbladder cancer pathogenesis from a living organoid gallbladder cholangiocyte biorepository

Gallbladder cancer (GBC) while rare worldwide has a high prevalence in India. Pathogenesis is unclear and outcomes poor. Gallbladder cholangiocyte organoids (GCOs) or gallbladder carcinoma organoids (GBCOs) were developed and serially propagated from surgically resected gallbladder tissues with benign or malignant diseases, respectively. Patient derived organoids (PDOs) were derived from 15 normal; 58 inflamed; 12 xanthogranulomatous cholecystitis (XGC); 5 pre-invasive neoplasm and 13 invasive malignant gallbladder pathologies. Protocol optimisation achieved 58% (69/119) success in organoid generation and expansion. Organoids maintained tight junction integrity; P-gp pump and enzymatic activity; preserved tissue-specific gene and protein marker expression; histological features and genetic variations. Cryopreserved organoids from 62 patients with primary tissue and high-quality DNA, RNA and protein derivatives have been banked. In gene expression analyses of tissue, XGC samples clustered with malignant subtypes, separate from benign pathologies. Derived XGC organoids showed a similar clustering. Enriched hallmark pathways in XGC support neoplastic change through chronic inflammation. PDOs generated from different gallbladder pathologies are a promising model to investigate the pathogenesis of GBC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/651639v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@465f3dorg.highwire.dtl.DTLVardef@16c5f0org.highwire.dtl.DTLVardef@11d4807org.highwire.dtl.DTLVardef@163d072_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Spatial distribution of cytoskeleton-mediated feedback controls cell polarization: a computational study

In the social amoeba Dictyostelium, cell motility is regulated through a signal transduction excitable network that interfaces with the cytoskeleton to control actin polymerization patterns. In turn, the cytoskeleton influences the signaling machinery via several feedback loops, but the nature and function of this feedback remain poorly understood. In this study, we use computational models to discern the essential role of complementary positive and negative feedback loops in polarizing cells. We contrast two potential mechanisms for the negative feedback: local inhibition and global inhibition. Our results indicate that both mechanisms can stabilize the leading edge and inhibit actin polymerization in other sites, preventing multipolarity. While some experimental perturbations align more closely with the local inhibition model, statistical analyses reveal its limited polarization potential within a wide excitability range. Conversely, global inhibition more effectively suppresses secondary and tertiary leading-edge formation, making it a more robust polarization mechanism. This raises an intriguing question: if local inhibition better replicates experimental observations but is less effective for polarization than local excitation and global inhibition, could there be a supplementary mechanism enhancing its polarization potential? To address this, we propose a novel mechanism involving the dynamic partitioning of back molecules which enhances communication between the front and back of the cell and can be leveraged by local feedback interactions between the cytoskeleton and signal transduction to improve polarization efficiency.

cell biology↗