Biology subjects

Shah, S. S.

Publications and source records attributed to Shah, S. S..

10 recordsLinked to original sources

bioRxiv2026

T6SS mutants exploit itaconate to support infection of phagocytes

Pseudomonas aeruginosa is a major cause of persistent pneumonias that are not readily cleared by seemingly appropriate antimicrobial therapy. We identified a reservoir of P. aeruginosa variants lacking expression of the H3-T6SS in patients with chronic but not acute pneumonia. A PAO1 {Delta}H3-T6 mutant caused increased infection in the murine lung as compared to the wild-type strain. The {Delta}H3 mutants exhibited increased transcription of genes involved in phagocytic uptake and respiration under conditions found in the phagolysosome, namely low O2, low pH and abundant itaconate. We confirmed increased intraphagocytic residence of the {Delta}H3 mutants and colocalization with LAMP1 within the phagolysosome of both bone marrow derived macrophages in vitro and in alveolar macrophages harvested directly from infected lungs. Persistence within macrophages required itaconate which preserved the viability of infected macrophages and boosted bacterial bioenergetics to optimize consumption of available carbon sources. Our findings demonstrate that selection for loss of H3-T6SS loss of function mutations promotes the metabolic versatility that enables P. aeruginosa to cause intractable pulmonary infection.

immunology↗

bioRxiv2025

Insulin-Like Growth Factor 1 Receptor Regulates Breast Cancer Cell Adhesion through Beta-1 Integrin

IntroductionThe insulin-like growth factor (IGF-1/IGF1R) pathway has been implicated in breast cancer aggressiveness; however, inhibition of this pathway has not been successful in clinical trials, indicating a lack of understanding about its role in TNBC metastasis. Recent studies have explored IGF1R involvement in integrin function and cancer cell adhesion dynamics. The goal of this study was to test the hypothesis that IGF1R itself regulates cancer cell adhesion. MethodsWe use MDA-MB-231 and Hs578T TNBC cell lines, siRNA-mediated knockdown, and adhesion assays to assess how IGF1R and integrin knockdowns impact cancer cell adhesion. Using xCELLigence E-plates, we quantify the effect of IGF-1 ligand stimulation versus IGF1R knockdown on functional cell adhesion. We also use HUVEC human endothelial cells to determine how IGF1R regulates adhesion to the endothelium. ResultsWe found that IGF-1 stimulation increased MDA-MB-231 TNBC adhesion, which was reversed by the IGF1R tyrosine kinase inhibitor BMS-754807 and the ligand-dependent receptor internalization inhibitor dansylcadaverine. Unexpectedly, IGF1R knockdown also potently stimulated cell adhesion. Concomitant {beta}1 integrin knockdown reversed the increased cell adhesion after both IGF-1 stimulation or IGF1R knockdown, indicating that the increased adhesion is {beta}1 integrin dependent. This was also seen via immunocytochemistry when cells were seeded on fibronectin. Finally, inhibiting IGF1R signaling also reduced MDA-MB-231 cell adhesion to HUVEC endothelial cells. DiscussionBoth IGF-1 stimulation and IGF1R knockdown in TNBC cells promote cell adhesion, which seems paradoxical. However, the commonality of both interventions is removal of IGF1R from the cell surface, since IGF-1 stimulation causes IGF1R internalization and intracellular trafficking. Blocking IGF1R signaling using a tyrosine kinase IGF1R inhibitor preserves IGF1R on the cell surface. Thus, we propose a model whereby surface-bound IGF1R inhibits {beta}1 integrin function and blocks cell adhesion. This model is supported further by our finding that treatment of MDA-MB-231 cells with dansylcadaverine, which inhibits ligand-mediated receptor internalization, blocked the effect of IGF-1 on adhesion. These findings may explain why selective IGF1R receptor antagonists, which downregulate IGF1R protein upon chronic administration, were unsuccessful in the clinical setting.

cancer biology↗

bioRxiv2025

Investigating diurnal effects and joint nest defense behavior in Herring Gulls

Nest defense in birds is vital for the protection of their young, but can prove energetically costly. Birds often show plasticity in nest defense depending on factors such as threat level, mate presence, nest stage, and body condition. Such factors can vary over temporal extents ranging from a diel cycle to a breeding season or an individuals lifetime. Understanding diurnal variation in nest defense intensity can be particularly useful when studying a breeding population to help investigators minimize nest disturbance, yet few studies have explored diurnal variation in nest defense intensity. Here we investigated how nest defense by Herring Gulls (Larus smithsonianus) varies with time of day. We simulated predatory threat at 26 Herring Gull nests during four different times of day: "Early Morning" (0530-0600), "Late Morning" (1000-1030), "Afternoon" (1400-1430), and "Evening" (1900-1930). Based on previous findings of diurnal activity patterns in a Herring Gull, we predicted that nest defense intensity--measured as aggressiveness of response and latency to calm--would be greater during the early morning and late evening than at other times. Contrary to our prediction, we found that the time of day did not affect nest defense intensity in Herring Gulls. However, independent of time of day, we found that when both mates were present at the nest, aggression scores were elevated. Our results suggest that joint nest defense in Herring Gulls permits greater aggressiveness towards predators, perhaps due to division of labor or lowered risk of complete nest failure if one parent is injured or killed. Further, our results indicate that researchers can minimize nest disturbance and accompanying stress by limiting research activities at Herring Gull nests when both parents are present.

animal behavior and cognition↗

bioRxiv2025

Regulation of airway fumarate by host and pathogen promotes S. aureus pneumonia

Staphylococcus aureus is a leading cause of healthcare-associated pneumonia, contributing significantly to morbidity and mortality worldwide. As a ubiquitous colonizer of the upper respiratory tract, S. aureus must undergo substantial metabolic adaptation to achieve persistent infection in the distinctive microenvironment of the lung. We observed that fumC, which encodes the enzyme that converts fumarate to malate, is highly conserved with low mutation rates in S. aureus isolates from chronic lung infections. Fumarate, a pro-inflammatory metabolite produced by macrophages during infection, is regulated by the host fumarate hydratase (FH) to limit inflammation. Here, we demonstrate that fumarate, which accumulates in the chronically infected lung, is detrimental to S. aureus, blocking primary metabolic pathways such as glycolysis and oxidative phosphorylation (OXPHOS). This creates a metabolic bottleneck that drives staphylococcal FH (FumC) activity for airway adaptation. FumC not only degrades fumarate but also directs its utilization into critical pathways including the tricarboxylic acid (TCA) cycle, gluconeogenesis and hexosamine synthesis to maintain metabolic fitness and form a protective biofilm. Itaconate, another abundant immunometabolite in the infected airway enhances FumC activity, in synergy with fumarate. In a mouse model of pneumonia, a {Delta}fumC mutant displays significant attenuation compared to its parent and complemented strains, particularly in fumarate- and itaconate-replete conditions. Our findings underscore the pivotal role of immunometabolites in promoting S. aureus pulmonary adaptation.

microbiology↗

bioRxiv2024

Comprehensive analysis of CXXX sequence space reveals that S. cerevisiae GGTase-I mainly relies on a2X substrate determinants

Many proteins undergo a post-translational lipid attachment, which increases their hydrophobicity, thus strengthening their membrane association properties or aiding in protein interactions. Geranylgeranyltransferase-I (GGTase-I) is an enzyme involved in a three-step post-translational modification (PTM) pathway that attaches a 20-carbon lipid group called geranylgeranyl at the carboxy-terminal cysteine of proteins ending in a canonical CaaL motif (C - cysteine, a - aliphatic, L - often leucine, but can be phenylalanine, isoleucine, methionine, or valine). Genetic approaches involving two distinct reporters were employed in this study to assess S. cerevisiae GGTase-I specificity, for which limited data exists, towards all 8000 CXXX combinations. Orthogonal biochemical analyses and structure-based alignments were also performed to better understand the features required for optimal target interaction. These approaches indicate that yeast GGTase-I best modifies the Cxa[L/F/I/M/V] sequence that resembles but is not an exact match for the canonical CaaL motif. We also observed that minor modification of non-canonical sequences is possible. A consistent feature associated with well-modified sequences was the presence of a non-polar a2 residue and a hydrophobic terminal residue, which are features recognized by mammalian GGTase-I. These results thus support that mammalian and yeast GGTase-I exhibit considerable shared specificity. Article SummaryThis work investigates yeast GGTase-I specificity through genetics, high throughput sequencing, and two distinct reporter systems. This approach allows for comprehensive evaluation of all CXXX sequence space, which has not been possible with earlier approaches. We identified CXXX sequences supporting geranylgeranylation that differ from the historically defined CaaL sequence often cited in the literature as the GGTase-I target motif, and our results indicate that the last two amino acids of the target motif largely dictate GGTase-I specificity.

genetics↗

bioRxiv2022

Co-occurring mutations in the POLE exonuclease and non-exonuclease domains define a unique subset of highly mutagenic tumors.

Somatic POLE mutations in the exonuclease domain (ExoD) are prevalent in colorectal cancer (CRC), endometrial cancer (EC), and others and typically lead to dramatically increased tumor mutation burden (TMB). To understand whether non-ExoD mutations also play a role in mutagenesis, we assessed TMB in 447/14541 POLE-mutated CRCs, ECs, and ovarian cancers (OC) based on classification TMB-High (TMB-H) or TMB-Low (TMB-L). TMB-H tumors were segregated as POLE ExoD driver, POLE ExoD driver plus POLE Variant, and POLE Variant TMB-H. Intriguingly, TMB was highest in tumors bearing POLE ExoD driver plus POLE Variant (p<0.001 in CRC and EC, p<0.05 in OC). Integrated analysis of AlphaFold2-modeled POLE models and quantitative estimate of stability indicated that multiple variants had significant impact on functionality. These data indicate that co-occurring POLE variants categorize a unique subset of POLE-driven tumors defined by ultra-high TMB, which has implications for abundance of tumor neoantigens, therapeutic response, and patient outcomes. SignificanceSomatic POLE ExoD driver mutations cause proofreading deficiency that induces high tumor mutation burden (TMB). This study defines a novel modifier role for non-ExoD mutations in POLE ExoD-driven tumors, associated with ultra-high TMB. These data may inform acquisition of tumor neoantigens, tumor classification, therapeutic response, and patient outcomes.

cancer biology↗

bioRxiv2021

Identification of Cancer-Associated Fibroblasts in Glioblastoma and Defining Their Pro-tumoral Effects

Despite their identification in some cancers, pro-tumoral cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of primary glioblastoma cultures yielded cells with CAF morphology, CAF transcriptomic profile, and mesenchymal lineage in single-cell RNA-seq. Glioblastoma CAFs were attracted to glioblastoma stem cells (GSCs) and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF-{beta} and TGF-{beta} as mediators of GSC effects on CAFs, and osteopontin and hepatocyte growth factor as mediators of CAF-induced GSC enrichment. Glioblastoma CAFs also induced M2 macrophage polarization by producing the EDA fibronectin variant. Glioblastoma CAFs were enriched in the subventricular zone which houses neural stem cells that produce GSCs. Including CAFs in GSC-derived xenografts induced in vivo growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.

cancer biology↗

bioRxiv2020

A super-resolution protocol to correlate structural underpinnings of fast second-messenger signalling in primary cell types

Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals.

biophysics↗

bioRxiv2020

Role of c-Met/β1 integrin complex in the metastatic cascade

Metastases cause 90% of human cancer deaths. The metastatic cascade involves local invasion, intravasation, extravasation, metastatic site colonization, and proliferation. While individual mediators of these processes have been investigated, interactions between these mediators remain less well defined. We previously identified a structural complex between receptor tyrosine kinase c-Met and {beta}1 integrin in metastases. Using novel cell culture and in vivo assays, we found that c-Met/{beta}1 complex induction promotes breast cancer intravasation and adhesion to the vessel wall, but does not increase extravasation. These effects may be driven by the ability of the c-Met/{beta}1 complex to increase mesenchymal and stem cell characteristics. Multiplex transcriptomic analysis revealed upregulated Wnt and hedgehog pathways after c-Met/{beta}1 complex induction. A {beta}1 integrin point mutation that prevented binding to c-Met reduced intravasation. OS2966, a therapeutic antibody disrupting c-Met/{beta}1 binding, decreased invasion and mesenchymal gene expression and morphology of breast cancer cells. Bone-seeking breast cancer cells exhibited higher c-Met/{beta}1 complex levels than parental controls and preferentially adhere to tissue-specific matrix. Patient bone metastases demonstrated higher c-Met/{beta}1 levels than brain metastases. Thus, the c-Met/{beta}1 complex drives breast cancer cell intravasation and preferential affinity for bone tissue-specific matrix. Pharmacological targeting of the complex may prevent metastases, particularly osseous metastases.

cancer biology↗

bioRxiv2018

Dynamic Processing of Displacement Loops During Recombinational DNA Repair

Displacement-loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo, which enabled studying the kinetics of their formation and defining the network of D-loop formation and reversal pathways. Nascent D-loops are detected within 2 hrs of DSB formation and extended over the next 2 hrs in a system allowing break-induced replication. The majority of D-loops are disrupted in wild type cells by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a novel layer of HR control in cells relying on nascent D-loop dynamics, revealing unsuspected complexities, and identifying a surprising role for a conserved Rad54 paralog.

molecular biology↗