Biology subjects

Das, D.

Publications and source records attributed to Das, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Electrostatics Govern Protein Orientation on Citrate-Capped Gold Nanoparticles

Conjugation of proteins with gold nanoparticles (GNPs) is crucial in developing nanoparticle-based drugs, which are used in making targeted delivery systems for therapeutic and diagnostic use. This conjugation is often mediated by covalent gold-thiol (Au-S) bonds involving cysteine residues. However, the influence of other interactions, like electrostatics, on protein-GNP interactions remains poorly understood. While the independent roles of cysteine (thiol interactions) and lysine (electrostatics) are studied, how these two distinct mechanisms couple and mutually influence one another remains unknown. It is particularly unclear how the position and the electrostatics around cysteine residues within the protein structure modulate their interaction with GNPs. Hence, to isolate the electrostatic contribution from Au-S interaction, we systematically investigated the role of positively charged lysine in protein interaction with citrate-capped GNPs using protein L, a cysteine-free antibody-binding protein. Protein L has seven lysine residues, positively charged at pH 7.4. Each lysine was individually mutated to cysteine to create single-cysteine variants. The capping strength of these mutants on citrate-capped GNPs was assessed using various biophysical techniques. Surprisingly, not all cysteine mutants formed stable covalent conjugates. The K7C mutant, in particular, showed strong and irreversible GNP capping, not solely due to the introduced cysteine, but also because of nearby positively charged residues that promoted electrostatic attraction to the negatively charged citrate-capped GNP surface. This critical role of electrostatics was further confirmed using lysine acrylation, which effectively capped lysines and prevented their interaction. Our experimental plan was thus able to successfully decouple the contributions of Au-S bonding from lysine-driven electrostatics. These sequential and cooperative studies underscore that electrostatic and covalent factors are not independent but part of an interconnected process, thereby underscoring the need to consider local charge environments in protein engineering for nanoparticle conjugation.

biophysics↗

bioRxiv2025

Modeling collective cell migration in a heterogeneous environment: Drosophila border cell migration as a model paradigm

Collective cell migration underpins development and disease progression, yet the influence of heterogeneous microenvironments on intercellular forces and migration dynamics remains elusive. Focusing on the chemotactic migration of border cell clusters in Drosophila egg chambers, we present a computational framework that integrates intercellular force dynamics and cell shape changes during large-scale migration, validated with live imaging data. Our model replicates in vivo observations, such as rotations of the cell cluster and its tendency to follow a central path in the egg chamber. Using the model, we discover instantaneous acceleration/deceleration phases as the cell cluster migrates through junctions of other cell types. We also predict how environmental tissue topology and perturbations in chemoattractant gradients and adhesion impact cluster speed and rotation. By addressing the limitations of prior models of border cell migration, our approach offers broad applicability to other migration contexts involving interactions among different cell types in complex environments.

biophysics↗

bioRxiv2025

Circular permutants of azurin exhibit molten globule intermediates not observed in WT azurin

Molten globule (MG) states of proteins have been described as special types of intermediate states within the energy landscape diagram of proteins. In a cell, the MG state has functional significance compared to the native or unfolded states owing to its enhanced side-chain dynamics. In this study, we re-examined the acid-denatured state of the metalloprotein azurin and also investigated whether circular permutation (CP) of azurin can lead to modulation of the energy landscape to yield intermediate or MG states. CP is a protein engineering tool that allows the rearrangement of the secondary structural elements without perturbing the overall three-dimensional protein structure. We carried out comprehensive thermal, chemical, and pH denaturation studies to examine possible MG states in azurin WT. We additionally examined two CPs of azurin, whose energy landscapes in the metal-free and metal-bound forms have been previously characterized. One of the positions of CP is near the metal-binding loop (cpN42) and the other is within the active site of the protein (cpF114). We used thermal denaturation monitored by far-UV and near-UV CD, time-resolved fluorescence anisotropy of a buried Trp, and solvent accessibility of the same Trp residue to evaluate the presence of MG states in these proteins. WT azurin shows binding with ANS at a low pH due to ionic interaction with the fluorophore at this pH. We find that though WT azurin does not adopt an MG state, CP of azurin(s) exhibited an MG state. Our findings highlight the potential of CP to modulate protein energy landscapes and consequently the physiological functions of proteins.

biophysics↗

bioRxiv2025

Genetically encoded nAChR upregulation is neuroprotective in female parkinsonian mice

Parkinsons disease is projected to rise to pandemic proportions by 2050, which has resulted in an urgent need for disease-modifying treatments. In this regard, we previously showed that in a mouse model of parkinsonism with unilateral 6-hydroxydopamine (6-OHDA) injection into the dorsolateral striatum (DLS), low doses of the neuronal nicotinic acetylcholine receptor (nAChR) partial agonist and smoking cessation drug, cytisine exerts sex-specific neuroprotection in substantia nigra pars compacta (SNc) dopaminergic (DA) neurons of only female mice by reducing apoptotic endoplasmic reticulum (ER) stress. Although these data suggest that neuroprotection might occur via cytisine-mediated upregulation of {beta}2 subunit-containing ({beta}2*) nAChRs in SNc DA neurons, there is no direct evidence to support this idea. Therefore, this study asks the critical question of whether upregulation of {beta}2* nAChRs in SNc DA neurons alone is sufficient to reduce apoptotic ER stress and exert neuroprotection in a preclinical unilateral DLS mouse model of 6-OHDA-induced parkinsonism. To address this question, we generate and characterize a novel {beta}2-upregulated transgenic mouse line. These transgenic mice possess mutations in the M3-M4 intracytoplasmic loop of {beta}2 subunits that cause constitutive upregulation of {beta}2* nAChRs without nicotinic ligands. Surprisingly, when compared to wild-type littermates, only female {beta}2-upregulated transgenic mice demonstrate upregulation of {beta}2* nAChRs in SNc DA neurons as assessed by significant increases in Sec24D-containing ER exit sites (Sec24D-ERES). Using the optogenetic calcium and dopamine sensors, GCaMP6f and GRABDA respectively, we found significant increases in dihydro-beta-erythroidine (Dh{beta}E)-sensitive {beta}2* nAChR-mediated calcium influx in SNc DA neuron dendrites and Dh{beta}E-sensitive acetylcholine (ACh)-evoked dopamine release at SNc DA neuron terminals of the DLS in female transgenic mice. We then used four independent readouts to assess neuroprotection of SNc DA neurons following unilateral 6-OHDA injection into the DLS, viz., contralateral apomorphine-induced rotations, preservation of SNc DA neurons, inhibition of a major proapoptotic ER stress protein, C/EBP homologous protein (CHOP) and glial fibrillary acid protein (GFAP) expression in SNc astrocytes. In all four readouts, female {beta}2-upregulated transgenic mice showed significant neuroprotection. From a clinical perspective, this study shows that upregulation without nicotinic ligand-mediated activation of {beta}2* nAChRs in SNc DA neurons can be a translationally viable disease-modifying strategy for Parkinsons disease. In addition, we envision that the novel transgenic {beta}2-upregulated mice created in this study will provide a valuable tool for understanding the role of nAChR upregulation in major neurological disorders such as addiction, anxiety, depression and dementia.

neuroscience↗

bioRxiv2025

Cyclo-stationary distributions of mRNA and Protein counts for random cell division times

There is a long history of using experimental and computational approaches to study noise in single-cell levels of mRNA and proteins. The noise originates from a myriad of factors: intrinsic processes of gene expression, partitioning errors during division, and extrinsic effects, such as, random cell-cycle times. Although theoretical methods are well developed to analytically understand full statistics of copy numbers for fixed or Erlang distributed cell cycle times, the general problem of random division times is still open. For any random (but uncorrelated) division time distribution, we present a method to address this challenging problem and obtain exact series representations of the copy number distributions in the cyclo-stationary state. We provide explicit cell age-specific and age-averaged results, and analyze the relative contribution to noise from intrinsic and extrinsic sources. Our analytical approach will aid the analysis of single-cell expression data and help in disentangling the impact of variability in division times.

systems biology↗

bioRxiv2025

Plastimos: a live cell imaging-based framework to study dynamics of EMT-mediated cellular plasticity in breast cancer

Cancer cell plasticity, primarily mediated by the process of epithelial-mesenchymal transition (EMT), plays a critical role in promoting therapeutic resistance, tumor heterogeneity, and metastasis. EMT-mediated plasticity enables cancer cells to undergo molecular and phenotypic changes, which alter their invasiveness and resistance to treatments. This study aims to develop a quantitative, high-throughput system to assess EMT-mediated plasticity, which can inform therapeutic strategies for metastatic cancers. To accomplish this, we developed Plastimos, a semi-automated imaging analysis pipeline. This framework utilizes time-series images to track live cells through deep learning-based segmentation and employs a greedy algorithm to map cell trajectories, enabling the extraction of cellular phenotypic features. These features are used to study the EMT-mediated plasticity state of cells in response to the well-known EMT-inducing factors EGF and TGF-{beta}1. We selected two breast cancer cell lines, MCF7 and MDA-MB-231, representing classical epithelial-like and mesenchymal-like cell. The pipeline assigns a Plasticity Index based on various parameters, including motility, morphology, and proliferation, thus providing a quantitative estimate for the plasticity of deviating from the epithelial state. Our results indicate that epithelial-like cells respond to EMT-inducing factors at both molecular and phenotypic levels, while mesenchymal-like cells responses are only seen phenotypically. The Plasticity Index classifies cells along the EMT spectrum, converting to a Plasticity Score that reflects mesenchymal proportions. Availability and implementationThe pipeline implementation and the source code of Plastimos can be found at: https://github.com/Durdam/Plastimos.git

bioinformatics↗

bioRxiv2025

Structural insights into the recruitment of viral Type 2 IRES to ribosomal preinitiation complex for protein synthesis

Picornaviruses employ internal ribosome entry sites (IRESs) in their genomic RNA to hijack the hosts translational machinery. The picornavirus, Encephalomyocarditis virus, employs a type 2 IRES present in its 5UTR and requires 43S ribosomal preinitiation complex (PIC), the central domain of eukaryotic initiation factor (eIF) 4G, eIF4A, and an essential ITAF (IRES trans-acting factor)-polypyrimidine tract binding protein 1 (PTB1) to form 48S PIC. In this study, we have used cryo-electron microscopy (cryo-EM) to determine the structure of EMCV IRES-bound mammalian 48S PIC in a scanning-arrested closed state at the start codon. The EMCV IRES domains contacts initiator tRNA (tRNAi) and 40S head at the inter-subunit interface, which reveals an altogether unique mechanism used by viruses to capture host translational machinery for its protein synthesis. The tRNAi is held away from the 40S body in contrast to canonical cap-dependent translation while the domain I apical region of EMCV IRES mimics 28S rRNA of 60S to interact with 40S ribosomal head proteins-uS13 and uS19. The structural analysis account for numerous biochemical studies on Type 2 IRES and shows how Type 2 IRES interacts with 43S PIC to form 48S PIC. This study provides mechanistic insights for understanding EMCV IRES-mediated translation initiation, which could be extrapolated to other IRESs sharing similar motifs and factor requirements including type 1 viral IRESs.

biophysics↗

bioRxiv2025

Hydrolysis-dependent severing tunes internal monomeric heterogeneity to shape actin length distributions

The coordination of actin-binding proteins (ABPs) is essential for forming a wide range of actin structures that drive vital cellular processes. Many of the ABPs, like the severing protein cofilins, exhibit affinities dependent on the nucleotide state of actin monomers. Experiments have shown that cofilins interact with actin filaments cooperatively in a concentration-dependent manner, where it severs less at both low and high concentrations. However, how this nucleotide state-dependent severing influences actin filament distributions is poorly understood. Here, we propose a computational model of actin filament dynamics that combines stochastic polymerization, phosphate release, hydrolysis-dependent cooperative binding of cofilins, and severing of actin monomers. This model simultaneously reproduces key experimentally observed features: The non-monotonic change of the filament length and the monotonic decay in actin cap size with the increasing cofilin concentration, as well as the linear increase of the cap size with the actin concentration. We elucidate how both these emerge from the tuning of the heterogeneity in the arrangement of cofilin decorated and undecorated monomers within a filament. We also predict that, with varying cofilin concentrations, steady-state length distributions can change from symmetric bell-shaped to long-tailed skewe d shapes. We show that, in some regimes, our model simplifies to a coarse-grained model enabling us to mathematically predict that the distribution of undecorated monomers is exponential with a decay exponent that depends on cofilin and actin concentration. This study highlights the importance of non equilibrium processes that can modulate structural heterogeneity within a filament to shape actin length variation.

biophysics↗

bioRxiv2025

Repurposing statins in combination therapy for effective ablation of metastatic breast cancer

Metastatic breast cancer (mBC) remains an incurable disease with limited treatment options, highlighting the need for novel therapeutic approaches. Combination therapy with chemotherapeutic agents along with targeted therapy are the most common methods of treatment used in the terminal stages of the disease. Eventual development of resistance to these approaches, leading to fatal outcomes suggests the presence or emergence of a heterogenous population of cells intrinsically resistant to commonly used regimens. Previous work identified a heterogeneous population of metastatic tumor cells with distinct molecular characteristics driven by Macc1 (Metastasis Associated in Colon Cancer 1) overexpression, which could be targeted by lovastatin (transcriptional inhibitor of Macc1). Building on this foundation, the efficacy of lovastatin in targeting metastatic cells with high Macc1 expression was evaluated in lung metastasis, and the regulatory pathways governing Macc1 expression and lovastatin treatment in mBC were investigated. The expression of Macc1, in breast cancer biopsies provides insights into the intra and inter tumor heterogeneity of the Macc1 gene and its correlation with disease outcomes. While some studies suggest synergistic effects between statins and chemotherapeutic agents, comprehensive evaluations of various combinations and their therapeutic outcomes are still needed. The therapeutic efficacy of lovastatin combined with chemotherapy to determine the most effective treatment regimen that maximizes tumor cell ablation demonstrated that lovastatin can effectively ablate the chemoresistant tumor cells in mBC. The translational implications of this research will identify patient subgroups that may benefit most from statin-chemotherapy combinations that could support the repurposing of statins as cost-effective adjuvant therapies for mBC.

cancer biology↗

bioRxiv2025

Automated high-throughput patch clamp electrophysiology of hiPSC-derived neuronal models

The advent of human induced pluripotent stem cells (hiPSCs) and their differentiation into neurons and brain organoids has revolutionized our ability to model brain disorders in a human context. However, current technologies to assay the electrophysiological properties of human neurons in these models remain limited by throughput, as single-cell manual patch clamp is laborious and resource intensive. Here, we provide methods to perform high-throughput automated patch-clamp (APC) on hiPSC-derived neurons. We describe how to dissociate and perform voltage-clamp recordings on human neurons from three well-established protocols - 2D directed differentiation of cortical neurons, NGN2-induced neurons, and 3D cortical organoids - using the Nanion Syncropatch 384, a commercially available high-throughput APC system. Using this approach, we investigated the biophysical properties of voltage-gated sodium channels (VGSCs) and provide direct comparisons between manual and APC recordings across all three hiPSC-derived model systems. We demonstrate the capability of this automated system for pharmacological analysis of native human VGSC isoforms, which will enable compound screening approaches. Lastly, we provide methods to sort specific cellular populations within these hiPSC models using fluorescence-activated cell sorting (FACS) followed by APC. These methods and results provide a transformative and novel high-throughput technique for quantifying passive and active membrane properties in cell-type specific and/or genetically modified hiPSC-derived neurons.

neuroscience↗

bioRxiv2025

Combined MEG and EEG suggest a limbic source network of the P3 in retrosplenial cortex, insula, and hippocampus

The event-related P3 is evoked by most task-relevant or salient stimuli, but its neural sources remain controversial. Here we reevaluate the role of hippocampus, unambiguously demonstrated in intracranial recordings, and retro-splenial cortex as sources of the P3 in magneto- and electroencephalography. Observers detected rare visual targets of a deviant shape and ignored rare non-targets of a deviant color. Source analysis was based on noise-normalized minimum-norm estimates in an individual, MRI-based source space. Critically, the cortical source space was extended to include the cornu ammonis of the hippocampus. Source analysis at the P3 peak showed strong sources in retrosplenial cortex and a weaker source in hippocampus. Activity in left somatosensory cortex was related to the button presses. Subsequent activity was observed in insula and anterior mid-cingulate cortex. Further simulation studies explored the spread of these sources and the plausibility of their combination to explain the data, and demonstrate that the combination of magneto- and electroencephalography increased the specificity of the source estimate. Overall, these data support an interpretation of the P3 as physiological indicator of activity in the limbic system for target and distractor stimuli.

neuroscience↗

bioRxiv2025

Cilia dynamics creates a dynamic barrier to penetration of the periciliary layer in human airway epithelia

The ciliated epithelium of the human respiratory tract is covered by the airway surface liquid (ASL), a protective fluid consisting of two layers: the periciliary layer (PCL), where motile cilia reside and generate fluid flow, and an overlying mucus layer. The complex structure and stratified nature of the PCL complicate both the prediction and quantification of fluid flow at the scale of individual or small groups of cilia, making it difficult to connect microscopic flows to macroscopic clearance. To tackle this challenge, we developed a novel methodology that involves un-caging a fluorescent compound to trace the flow field within the PCL. Fluorescence is activated at micrometric spots within the cilia layer, and displacement vectors and diffusion are recorded using high-speed video. Our experiments reveal a complex fluid transport pattern, with displacement velocity along the epithelial surface varying due to a non-uniform vertical flow field. Additionally, we observed that cilia expel fluid at their tips, a mechanism likely aimed at preventing pathogen access to the epithelium. Simulations, where cilia are modeled as arrays of rigid rods with length asymmetry, support these findings and offer new insights into the dynamics of fluid transport in the respiratory tract and the critical role of cilia coordination. Significance StatementThis study introduces an experimental pipeline to investigate fluid velocity and diffusion within the PCL of the human respiratory tract. By integrating experimental data with simulations of ciliary motion, we offer a robust framework to understand how cilia, depending on their collective beating properties, propel periciliary fluid in this structurally and dynamically complex environment. Our findings significantly expand the understanding of ciliary function, revealing that when cilia are beating coherently near cilia tips fluid is actively driven away from the epithelial surface. This suggests that coordinated cilia movement not only plays a key role in maintaining respiratory health by clearing mucus, but may also provide a dynamic barrier against pathogen entry.

biophysics↗

bioRxiv2025

Benchmarking of variant pathogenicity prediction methods using a population genetics approach

MotivationVariant pathogenicity predictors are essential for identifying new associations between genetic variants and rare diseases. However, despite the availability of numerous predictors, there is no clear consensus on which methods provide the most reliable results. The common practice of training, testing, and benchmarking these predictors using known variant sets from disease or mutagenesis studies raises concerns about ascertainment bias and data circularity. ResultsWe benchmarked commonly used pathogenicity predictors using an orthogonal approach that does not rely on predefined "ground truth" datasets. By leveraging population-level genomic data from gnomAD and the Context-Adjusted Proportion of Singletons (CAPS) metric, we identified CADD and REVEL as the best-performing predictors for distinguishing extremely deleterious variants from moderately deleterious ones. REVEL demonstrated superior calibration. Additionally, we show that CAPS can serve as a meta-analysis tool for interpreting variant annotations and highlight biases in ClinVar-based predictor training. Availability and ImplementationCAPS analysis and benchmarking results are available at https://github.com/mgudVCCRI/PopGenVariantFiltering Contacte.giannoulatou@victorchang.edu.au

genomics↗

bioRxiv2025

NOR1 and Mitophagy: An Insight into Sertoli Cell Function Regulating Spermatogenesis

The development and differentiation of germ cell (Gc) is supported by the Sertoli cel (Sc). We previously reported that a developmental switch in Sc around 12 days of age onwards in rats. During the process of differentiation of Sc, the differential expression of mitophagy-related genes and its role in male fertility is poorly understood. To address this gap, we evaluated the microarray dataset GSE48795 to identify 12 mitophagy-related hub genes - Egfr, Bcl2, Ccl2, Mmp2, Igf1, Fgf7, Apoe, Fos, Cxcl12, Ocln, Dcn, and Snca. We identify NOR1 as a potential mitophagy-related gene of interest due to its strong regulatory association with two hub genes Bcl2 and Fos. To determine the importance of our analysis of finding key mitophagy-related genes, we generated a transgenic rat model with Sc-specific knockdown of NOR1 during puberty. Adult male transgenic rats were found to be infertile due to impaired spermatogenesis, indicating a critical role for NOR1 in Sc function. These findings suggest that mitophagy-related genes may play an important role in the differentiation of Sc and thereby, regulate male fertility.

cell biology↗

bioRxiv2025

DeepPROTECTNeo: A Deep learning-based Personalized and RV-guided Optimization tool for TCR Epitope interaction using Context-aware Transformers

Background: The development of personalized cancer vaccines relies on accurately identifying neoepitopes capable of eliciting strong immune responses. T cell receptor (TCR)-epitope interactions are fundamental to cancer immunotherapy. Traditional computational approaches focus primarily on epitope-major histocompatibility complex (MHC) binding, often overlooking the critical contribution of TCR binding. Furthermore, the clinical applicability of existing methods is constrained by fragmented pipelines that require separate workflows for variant calling, HLA typing, and independent peptide-MHC (pMHC) or peptide-TCR (pTCR) evaluation stages. Results: We present DeepPROTECTNeo, a unified deep learning framework that integrates genomic variant detection, HLA typing, high-affinity pMHC binding prediction, variant-driven TCR repertoire mining, followed by a hybrid transformer-Convolutional Neural Network dual-branch feature extractor with an explicit cross-attention-based deep learning model for TCR-epitope binding prediction. Our reverse vaccinology-inspired biologically informed architecture integrates Bidirectional Long short-term memory (Bi-LSTM) sequence features, convolutional-attention physicochemical/evolutionary descriptors via gated fusion, and TCR numbered contextual embeddings to enable residue-level interpretable modelling. Under a strict TCR-split strategy, it achieved a mean AUROC of 0.7856 and AUPRC of 0.7932 outperforming six state-of-the-art predictors by 4-5% with tight inter-fold stability. The architecture maintains high robustness against structural hard negatives and imbalanced datasets, successfully recovering 18 of 34 validated high-affinity neoepitopes from a patient-specific cancer cohort. Conclusions: Experiments results demonstrate that DeepPROTECTNeo is a powerful, reliable end-to-end neoantigen prioritization framework that effectively models complex TCR-epitope interfaces directly from clinical sequencing data, providing a robust interpretable foundation to accelerate personalized cancer immunotherapy.

bioinformatics↗

bioRxiv2024

Threshold-dependent negative autoregulation of PIF4 gene expression optimizes growth and fitness in Arabidopsis

PHYTOCHROME INTERACTING FACTOR 4 (PIF4) is a vital transcription factor that controls plant growth by integrating environmental signals like light and temperature. Though upstream regulators of PIF4 are known, transcriptional regulation of PIF4 is poorly understood. Here, we demonstrate that the PIF4 undergoes negative autoregulation. We show that PIF4 promoter activity is more in the pif4 mutant but significantly reduced in PIF4 overexpression transgenic lines. Moreover, CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), which enhances PIF4 protein stability, promotes PIF4 autoinhibition. However, Phytochrome B (phyB), a photoreceptor that decreases PIF4 stability, inhibits autoinhibition. We further develop a network-based mathematical model incorporating the PIF4 autoinhibition and other key interactions. Our modeling and data analysis reveals that PIF4 autoregulation depends on a threshold of cellular PIF4 concentration. Our model also successfully predicts the hypocotyl growth and PIF4 promoter activity in various light and temperature conditions. Moreover, we show that the transgenic lines with enhanced PIF4 function negatively influence biomass and yield, irrespective of photoperiod and temperature. Together, the negative feedback of PIF4 dampens its own function and restraints unregulated growth. Our study thus elucidates the mechanisms of how the phyB-COP1/DET1-PIF4 module controls PIF4 transcription in tune with the endogenous PIF4 level.

plant biology↗

bioRxiv2024

Dysregulated RNA splicing induces regeneration failure in alcohol-associated liver disease

Individuals with progressive liver failure are at a high risk of mortality without liver transplantation. However, our understanding of derailed regenerative responses in failing livers is limited. Here, we performed comprehensive multi-omic profiling of healthy and diseased human livers using bulk and single-nucleus RNA-plus ATAC-seq. We report that hepatic immune milieu alterations in alcohol-associated liver disease (ALD) prevent hepatocytes from transitioning to a proliferative progenitor-like state, trapping them into an unproductive intermediate state. We discovered striking changes in RNA binding protein (RBP) expression, particularly ESRP, PTBP, and SR families, that cause misregulation of developmentally controlled RNA splicing in ALD. Our data pinpoint ESRP2 as a pivotal disease-sensitive RBP and support a causal role of its deficiency in ALD pathogenesis. Notably, splicing defects in ESRP2-targets Tcf4 and Slk, amongst others, directly alter their nuclear localization and activities, disrupting WNT and Hippo signaling pathways, which are critical for normal liver regeneration. We demonstrate that changes in stromal cell populations enrich failing ALD livers with TGF-{beta}, which suppresses ESRP2-driven epithelial splicing program and replaces functional parenchyma with quasi-progenitor-like cells lacking liver-specific functions. This unprecedented account of transcriptional and post-transcriptional dysregulation in ALD suggests that targeting misspliced RNAs could improve recovery and serve as biomarkers for poor ALD outcomes.

molecular biology↗

bioRxiv2024

Single-Cell Transcriptomics Unveils Skin Cell Specific Antifungal Immune Responses and IL-1Ra- IL-1R Immune Evasion Strategies of Emerging Fungal Pathogen Candida auris

Candida auris is an emerging multidrug-resistant fungal pathogen that preferentially colonizes and persists in skin tissue, yet the host immune factors that regulate the skin colonization of C. auris in vivo are unknown. In this study, we employed unbiased single-cell transcriptomics of murine skin infected with C. auris to understand the cell type-specific immune response to C. auris. C. auris skin infection results in the accumulation of immune cells such as neutrophils, inflammatory monocytes, macrophages, dendritic cells, T cells, and NK cells at the site of infection. We identified fibroblasts as a major non-immune cell accumulated in the C. auris infected skin tissue. The comprehensive single-cell profiling revealed the transcriptomic signatures in cytokines, chemokines, host receptors (TLRs, C-type lectin receptors, NOD receptors), antimicrobial peptides, and immune signaling pathways in individual immune and non-immune cells during C. auris skin infection. Our analysis revealed that C. auris infection upregulates the expression of the IL-1RN gene (encoding IL-1R antagonist protein) in different cell types. We found IL-1Ra produced by macrophages during C. auris skin infection decreases the killing activity of neutrophils. Furthermore, C. auris uses a unique cell wall mannan outer layer to evade IL-1R-signaling mediated host defense. Collectively, our single-cell RNA seq profiling identified the transcriptomic signatures in immune and non-immune cells during C. auris skin infection. Our results demonstrate the IL-1Ra and IL-1R-mediated immune evasion mechanisms employed by C. auris to persist in the skin. These results enhance our understanding of host defense and immune evasion mechanisms during C. auris skin infection and identify potential targets for novel antifungal therapeutics.

microbiology↗