Biology subjects

Zafar, A.

Publications and source records attributed to Zafar, A..

9 recordsLinked to original sources

bioRxiv2026

Cdc42 small GTPase is a novel regulator of the fibrogenic activation of human intestinal myofibroblasts

Cell division cycle 42 (Cdc42) is a member of the Rho family of small GTPases, which plays crucial roles in regulating cytoskeletal remodeling, and membrane trafficking. While previous studies implicated Cdc42 in controlling intestinal epithelial homeostasis, the involvement of this small GTPase in the process of intestinal fibrogenesis remains unexplored. Our study was designed to determine whether Cdc42 regulates the fibrogenic activation of intestinal myofibroblasts in vitro. The study was conducted using a CCD-18Co normal human colonic fibroblast cell line, and primary human intestinal myofibroblasts (HIMF) isolated from Crohns disease (CD) patients. CCD-18Co and HIMF cells were stimulated by transforming growth factor-{beta}1 (TGF-{beta}1). Cdc42 was inhibited either genetically, using siRNA-mediated knockdown, or pharmacologically using specific Cdc42 inhibitors, ML141 and CASIN. Genetic and pharmacologic inhibition of Cdc42 markedly reduced TGF-{beta}1 induced expression of the major contractile cytoskeletal proteins, -smooth muscle actin, calponin 1 and L-caldesmon. Furthermore, Cdc42 inhibition significantly attenuated expression of key extracellular matrix (ECM) proteins, fibronectin and collagen I, in activated CCD-18Co cells and HIMF. Interestingly, decreased expression of contractile and ECM proteins in Cdc42-depleted myofibroblasts was not due to downregulation of the TGF-{beta}1 signaling, decreased mRNA transcription or increased lysosomal or proteasomal degradation of these proteins. Such suppressed pro-fibrotic activation of Cdc42-deficient CCD-18Co cells and HIMF involved a selective inhibition of protein translation due to inactivation of the AKT-mammalian target of rapamycin (mTOR) signaling module. These findings highlight Cdc42 as a key regulator of intestinal fibrosis that controls mTOR activation to enhance ECM production and contractile actomyosin cytoskeleton in intestinal myofibroblasts. In briefZafar et al. unravel a novel role of Cdc42 small GTPase in fibrogenic activation of human intestinal myofibroblasts. Genetic and pharmacologic inhibition of Cdc42 markedly reduced TGF-{beta} induced expression of contractile cytoskeletal proteins and extracellular matrix proteins in activated myofibroblasts. The mechanisms underlying such profibrotic activity of Cdc42 involve regulation of de novo protein translation via activation of the AKT-mTOR signaling module. HighlightsO_LICdc42 regulates TGF-{beta} dependent activation of human intestinal myofibroblasts C_LIO_LIProfibrotic activity of Cdc42 involves regulation of de novo protein translation C_LIO_LICdc42 regulates myofibroblast activation via AKT-mTOR signaling pathway C_LIO_LITargeting Cdc42-Akt-mTOR signaling may facilitate the development of novel antifibrotic therapies C_LI

cell biology↗

bioRxiv2026

Approaching the limit: Modeling the physiological bounds of playable space in football

AO_SCPLOWBSTRACTC_SCPLOWPitch control models quantify spatial dominance in football by estimating which player can arrive first at each pitch location, but they treat all players as equivalently capable regardless of preceding effort. We introduce physiology-aware pitch control (Phys-PC), a model-agnostic time-to-arrive correction that imposes two physiological capacity channels calibrated from tracking data: a transient recoverable burden capturing incomplete recovery from recent high-intensity efforts, and a cumulative non-recoverable drain accumulating across match play. Both channels reduce a bounded access scale that modulates kinematic TTA before any downstream pitch-control computation. All parameters are anchored to exercise-physiology benchmarks; no laboratory measurements are assumed. Applied to a 64-match international tournament, Phys-PC reveals structure that kinematic models cannot detect. In head-to-head races, the dominant burden channel shifts from transient to cumulative over the course of a match, with a transient resurgence in the final 15 minutes. These physiological asymmetries predict match outcomes: relative reserve advantage is associated with higher odds of winning ground challenges (OR = 1.20, p = 0.006; +4.2 pp), completing over-the-top passes past recovering defenders (OR = 1.45, p = 0.034; +8.3 pp), and progressing possession sequences into the final third (OR = 1.31, p = 0.013; +5.1 pp). At the player-profile level, an acute-cumulative decomposition of contested space access separates roles and individuals whose territorial reach is maintained through sustained positioning from those whose access is rebuilt through repeated high-intensity actions, providing a physiological lens on team tactical structure.

bioengineering↗

bioRxiv2025

MYOSIN IIA MOTOR REGULATES ATTACHING-EFFACING BACTERIA INTERACTIONS WITH INTESTINAL EPITHELIUM

Attaching effacing (A/E) bacteria, such as Enteropathogenic E. coli (EPEC) and Citrobacter rodentium, colonize intestinal epithelial cells (IECs) by inducing remodeling of the epithelial cytoskeleton and formation of prominent actin pedestals at bacterial attachment sites. While non-muscle myosin II (NM II) is a key regulator of the actin cytoskeleton, whether it regulates IEC colonization by A/E pathogens is not known. To address this question, we targeted NM IIA and NM IIC, the NM II paralogs expressed in IECs. Our in vivo studies utilized mouse models with either intestinal epithelial-specific deletion of NM IIA (NM IIA cKO mice), expression of a NM IIA motor domain mutant, or total deletion of NM IIC (NM IIC tKO mice). In vitro experiments utilized IECs (HT-29cF8 and Caco-2BBE) with CRISPR-Cas9-mediated deletion of NM IIA or NM IIC. In addition, NM II activity in vitro was modulated pharmacologically, using either the pan-myosin inhibitor, blebbistatin, or a specific NM IIC activator, 4-hydroxyacetophenone (4-HAP). NM IIA cKO and NM IIA mutant mice demonstrated higher C. rodentium colonization along with more severe mucosal inflammation and colonic crypt hyperplasia as compared to their controls. By contrast, NM IIC tKO mice was indistinguishable from their control with regard to C. rodentium colonization. Blebbistatin treatment increased EPEC attachment to IECs monolayers, whereas 4-HAP did not affect bacterial attachment. Genetic knockout of NM IIA, but not NM IIC, increased EPEC adhesion to IEC monolayers. Importantly, the increase in EPEC attachment exhibited by NM IIA-deficient IECs required intact bacterial Type 3 secretion system and functional Tir effector, indicating that NM IIA functions in actin pedestal assembly. In summary, we describe a novel role for NM IIA in limiting intestinal epithelial colonization by A/E pathogens via inhibition of pathogen-induced remodeling of the actin cytoskeleton.

cell biology↗

bioRxiv2025

Molecular dynamics simulations of intrinsically disordered protein regions enable biophysical interpretation of variant effect predictors

Predictive models for missense variant pathogenicity offer little functional interpretation for intrinsically disordered regions, since they rely on conservation and coevolution across homologous sequences. To understand the extent to which biophysics modulates model performance compared to genomic conservation, we model biophysics of IDRs explicitly for improved interpretation of variant effects. We develop MDmis, a method that uses biophysical features extracted from molecular dynamics (MD) simulations of IDRs to predict pathogenicity. We find that pathogenic variants in Long IDRs manifest differently, with transient order and depleted solvent access, compared to those in Short IDRs. Using MD simulations of sequences with single missense variants, we identify stronger evidence for pathogenic effects in Long IDRs compared to Short IDRs. MDmis, when combined with conservation information, achieves strong predictive accuracy of pathogenicity of variants in Long IDRs. Overall, extracting information from MD simulations can help understand the drivers of predictive performance and elucidate biophysical behaviors affected by pathogenic variants.

bioinformatics↗

bioRxiv2025

Understanding Protein Language Model Scaling on Mutation Effect Prediction

Protein language models, and models that incorporate structure or homologous sequences, estimate sequence likelihood p(sequence) that reflects the protein fitness landscape and is commonly used in mutation effect prediction and protein design. It is widely believed in deep learning field that larger model performs better across tasks. However, for fitness prediction, language model performance declines beyond a certain size, raising concerns about their scalability. Here, we showed that model size, training dataset, and stochastic elements can bias the predicted p(sequence) away from real fitness. Model performance on fitness prediction depends on how well p(sequence) matches evolutionary patterns in homologs, which is best achieved at a moderate p(sequence) level for most proteins. At extreme predicted wild-type sequence likelihoods, models predict uniformly low or high likelihoods for nearly all mutations, failing to reflect the real fitness landscape. Notably, larger models tend to predict proteins with higher p(sequence), which may exceed the moderate range and thus reduce performance. Our findings clarify the scaling behavior of protein models on fitness prediction and provide practical guidelines for their application and future development.

bioinformatics↗

bioRxiv2024

The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

Background and AimsIntestinal epithelial barrier-integrity is essential for human health, and its disruption induces and exacerbates intestinal inflammatory disorders. While the cytoskeleton is critical for maintaining gut barrier-integrity, the role of the septins- the newest family of cytoskeletal proteins- is unknown. To address this knowledge gap, we evaluate the role of SEPT9- a critical component of the septin-cytoskeleton- in intestinal epithelial cell (IEC) barrier permeability and inflammation. MethodsWe developed SEPT9-NeonGreen knockin mice, inducible intestinal epithelial cell (IEC)-specific SEPT9 knockout (KO) mice, and SEPT9-KO human IEC lines. SEPT9 localization was analyzed using super-resolution microscopy. Barrier-integrity was assessed via transepithelial electrical resistance, FITC-dextran flux, and visualization of tight junction (TJ) and adherens junction (AJ) proteins. Dextran sodium sulfate-induced experimental colitis was evaluated in control and KO mice through measuring cytokine expression, immune cell infiltration, and IEC death. SEPT9 expression was examined in intestinal tissue of IBD patients. ResultsSEPT9 overlapped with TJs and AJs at IEC apical junctions. SEPT9 loss resulted in a leaky epithelial barrier due to mislocalization of junctional proteins. SEPT9 interacted with non-muscle myosin IIC (NMIIC) at the IEC apical-junctional actomyosin belt, and its ablation displaced NMIIC from IEC junctions. Loss of NMIIC also caused barrier disruption. SEPT9 KO mice exhibited increased susceptibility to experimental-colitis. SEPT9 expression was significantly reduced in intestinal mucosa of IBD patients. ConclusionSEPT9 regulates intestinal barrier integrity, supporting TJ and AJ assembly through NMIIC recruitment to the actomyosin belt. SEPT9 safeguards the intestinal mucosa during acute inflammation, and its reduced expression in IBD suggests a loss of this protective function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/629767v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@5141dcorg.highwire.dtl.DTLVardef@bae524org.highwire.dtl.DTLVardef@19c8eaorg.highwire.dtl.DTLVardef@d5b218_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

pyVIPER: A fast and scalable Python package for rank-based enrichment analysis of single-cell RNASeq data

SummarySingle-cell sequencing has revolutionized biomedical research by offering insights into cellular heterogeneity at unprecedented resolution. Yet, the low signal-to-noise ratio, characteristic of single-cell RNA sequencing (scRNASeq), challenges quantitative analyses. We have shown that gene regulatory network (GRN) analysis can help overcome this obstacle and support mechanistic elucidation of cellular state determinants, for example by using the VIPER algorithm to identify Master Regulator (MR) proteins from gene expression data. A key challenge, as the size and complexity of scRNASeq datasets grow, is the need for highly scalable tools supporting the analysis of large-scale datasets with up to hundreds of thousands of cells. To address it, we introduce pyVIPER, a fast, memory-efficient, and highly scalable Python toolkit for assessing protein activity in large-scale scRNASeq datasets. pyVIPER supports multiple enrichment analysis algorithms, data transformation/postprocessing modules, a novel data structure for GRNs manipulation, and seamless integration with AnnData, Scanpy and several widely adopted machine learning libraries. Compared to VIPER, benchmarking reveals orders of magnitude runtime reduction for large datasets--i.e., from hours to minutes-- thus supporting VIPER-based analysis of virtually any large-scale single-cell dataset, as well as integration with other Python-based tools. Availability and ImplementationpyVIPER is available on GitHub (https://github.com/alevax/pyviper) and PyPI (https://pypi.org/project/viper-in-python/). Contactav2729@cumc.columbia.edu Supplementary informationSupplementary data are available at Bioinformatics online. Accompanying data for the tutorials are available on Zenodo (https://zenodo.org/records/10059791).

bioinformatics↗

bioRxiv2024

Subconcussive head impacts sustained during American football alter gut microbiome diversity and composition

Subconcussive head impacts (SHIs) are a public health concern amongst at-risk populations. SHIs are hits to the head that do not typically generate symptoms and are unlikely to meet diagnostic criteria for mild traumatic brain injury (mTBI). Changes in the gut microbiome have been associated with mTBIs and implicated in both acute recovery from and neurodegenerative pathologies associated with repeated mTBI. This study monitored the gut microbiomes and head impact exposure of collegiate American football players across a competition season to determine if SHIs lead to acute and longer-term changes to the gut microbiome. SHI exposure correlates with changes in microbial diversity and composition three days post-exposure, the athletes gut microbiomes change significantly across the season, and mixed effects modeling provides evidence for cumulative effects of SHIs. These data provide the first evidence for a link between SHIs and changes in the diversity and composition of the gut microbiome.

microbiology↗

bioRxiv2019

Reconstruction of 1,000 projection neurons reveals new cell types and organization of long-range connectivity in the mouse brain

Neuronal cell types are the nodes of neural circuits that determine the flow of information within the brain. Neuronal morphology, especially the shape of the axonal arbor, provides an essential descriptor of cell type and reveals how individual neurons route their output across the brain. Despite the importance of morphology, few projection neurons in the mouse brain have been reconstructed in their entirety. Here we present a robust and efficient platform for imaging and reconstructing complete neuronal morphologies, including axonal arbors that span substantial portions of the brain. We used this platform to reconstruct more than 1,000 projection neurons in the motor cortex, thalamus, subiculum, and hypothalamus. Together, the reconstructed neurons comprise more than 75 meters of axonal length and are available in a searchable online database. Axonal shapes revealed previously unknown subtypes of projection neurons and suggest organizational principles of long-range connectivity.

neuroscience↗