Biology subjects

Masud, S.

Publications and source records attributed to Masud, S..

8 recordsLinked to original sources

bioRxiv2025

A Novel dual Influenza virus and SARS-CoV-2 neutralisation assay

Influenza and SARS-CoV-2 are co-circulating during the traditional influenza season giving rise to potential co-infections; currently estimated at 2.4%. Additionally, this means that the uptake of both vaccines (including SARS-CoV-2 boosters) will be encouraged within the same timeframe. Currently, patients receive separate vaccines in different arms on the same or different days. Induction of neutralising antibodies (NAbs) is used as the measure of efficacy for SARS-CoV-2 vaccinations while, the hemagglutination inhibition (HI) assay is the gold standard for seasonal influenza vaccine assessment. However, with the advent of novel universal influenza vaccine and dual SARS-CoV-2/Influenza vaccine approaches it is possible that functional NAb assays will become an essential component of a vaccine assessment toolbox. This assay is capable of distinguishing NAb responses during both dual vaccination development, and as part of co-infection and imprinting studies. Taking advantage of existing pseudotyped viruses developed by us, we present herein a dual neutralisation assay, which utilises 2 different luciferase (Renilla and Firefly) reporter lentiviral vectors. We configured assays with various combinations of SARS-CoV-2 and influenza subtype/variant combinations and compared sensitivity of this dual approach to neutralisation levels seen in single virus pseudotype Micro-neutralisation (pMN) in a double-blinded test of monoclonal antibody cocktails, and subsequently with a pre-screened serum panel. Mono and dual neutralisation pMN identified correctly positive and negative samples and IC50 values were all within 2-fold of each other, suggesting that specificity and sensitivity are retained post-multiplex. As we have taken a pseudovirus based approach there is the potential to tailor to specific vaccination candidates or currently circulating strains of each virus. The use of this versatile assay could form part of the toolbox of analysis of dual vaccination candidates and investigating responses during co-infection including studies assessing the impact of immune imprinting in the future.

immunology↗

bioRxiv2025

Excessive Postnatal Smooth Muscle Differentiation in a Lung Specific Model of TBX4-related Pulmonary Hypertension

Heterozygous TBX4 variants are the second most common genetic cause of pediatric pulmonary hypertension (PH), yet the mechanisms underlying the pathophysiology of TBX4-related lung disease remain poorly understood. We developed a lung mesenchyme-specific Tbx4 loss of function (Tbx4cKO) mouse model that bypasses embryonic lethality to investigate TBX4-related lung disease. Echocardiography of adult Tbx4cKO mice demonstrated significant hemodynamic changes consistent with PH. Three-dimensional whole-mount analysis of embryonic day 18.5 lungs revealed reduced lobe volumes and decreased distance between pleural edges and muscularized vessels. In adult Tbx4cKO lungs, high-resolution spatial quantitation identified extensive vascular remodeling characterized by significant medial thickening, distal muscularization of small diameter arteries, and extension of muscularized vessels into normally non-muscularized subpleural zones. Contrary to previous reports suggesting vascular simplification with Tbx4 loss, our comprehensive three-dimensional approach demonstrated an elaborated arterial tree with pathologic muscularization. Additional heterozygous loss of Tbx5 (Tbx4cKO;Tbx5het) exacerbated vascular phenotypes without worsening hemodynamic parameters. We also documented dysregulated airway smooth muscle patterning and prominent subpleural smooth muscle bands that share molecular features with myofibroblasts and airway smooth muscle cells, echoing pathologic findings in human TBX4 syndrome lung tissue. Collectively, our findings identify TBX4 as a critical suppressor of smooth muscle differentiation across multiple pulmonary compartments. This model recapitulates key features of human TBX4 syndrome and reveals mild developmental underpinnings with subsequent progressive postnatal smooth muscle dysregulation, highlighting a postnatal window during which therapeutic regulation of mesenchymal differentiation may be beneficial.

genetics↗

bioRxiv2024

Peripheral opioid receptor antagonism alleviates fentanyl-induced cardiorespiratory depression and is devoid of aversive effects

Millions of Americans suffering from Opioid Use Disorders (OUD) face a high risk of fatal overdose due to opioid-induced respiratory depression (OIRD). Fentanyl, a powerful synthetic opioid, is a major contributor to the rising rates of overdose deaths. Reversing fentanyl overdoses has proved challenging due to its high potency and the rapid onset of OIRD. We assessed the contributions of central and peripheral mu opioid receptors (MORs) in mediating fentanyl-induced physiological responses. The peripherally restricted MOR antagonist naloxone methiodide (NLXM) both prevented and reversed OIRD to a degree comparable to that of naloxone (NLX), indicating substantial involvement of peripheral MORs to OIRD. Interestingly, NLXM-mediated OIRD reversal did not produce aversive behaviors observed after NLX. We show that neurons in the nucleus of the solitary tract (nTS), the first central synapse of peripheral afferents, exhibit a biphasic activity profile following fentanyl exposure. NLXM pretreatment attenuates this activity, suggesting that these responses are mediated by peripheral MORs. Together, these findings establish a critical role for peripheral MORs, including ascending inputs to the nTS, as sites of dysfunction during OIRD. Furthermore, selective peripheral MOR antagonism could be a promising therapeutic strategy for managing OIRD by sparing CNS-driven acute opioid-associated withdrawal and aversion observed after NLX. Significance StatementIn this study, we compare the central versus peripheral components underlying fentanyl-induced cardiorespiratory depression to prevent overdose deaths. Our data indicate that these effects are, at least partially, due to the activation of mu opioid receptors present in peripheral sites. These findings provide insight into peripheral contributions to fentanyl-induced overdoses and could potentially lead to the development of treatments selectively targeting the peripheral system, sparing individuals from the CNS-driven acute opioid withdrawal generally observed with the use of naloxone.

physiology↗

bioRxiv2024

Purine and pyrimidine synthesis differently affect the strength of the inoculum effect for aminoglycoside and B-lactam antibiotics.

The inoculum effect has been observed for nearly all antibiotics and bacterial species. However, explanations accounting for its occurrence and strength are lacking. We previously found that growth productivity, which captures the relationship between [ATP] and growth, can account for the strength of the inoculum effect for bactericidal antibiotics. However, the molecular pathway(s) underlying this relationship, and therefore determining the inoculum effect, remain undiscovered. We show that nucleotide synthesis can determine the relationship between [ATP] and growth, and thus the strength of inoculum effect in an antibiotic class-dependent manner. Specifically, and separate from activity through the tricarboxylic acid cycle, we find that transcriptional activity of genes involved in purine and pyrimidine synthesis can predict the strength of the inoculum effect for {beta}-lactam and aminoglycosides antibiotics, respectively. Our work highlights the antibiotic class-specific effect of purine and pyrimidine synthesis on the severity of the inoculum effect and paves the way for intervention strategies to reduce the inoculum effect in the clinic.

microbiology↗

bioRxiv2023

The direction of local adaptation on pathogen exploitation rates reverses with environmental context

Host heterogeneity and spatial population structure each influence parasite evolution but may also interact because space structures contacts between host types. Here, we experimentally evolve granulosis virus in microcosms of its Plodia interpunctella (Indian meal moth) host that differ in both spatial structure and host genetic diversity. We control spatial structure by manipulating the viscosity of the food that the larvae live within and host genetic diversity by adding larvae from either a single or two inbred lines to opposite ends of the microcosm. We preserve spatial structure across passages and assay virus from different positions within the microcosm on both host genotypes. We find that the lower contact rates between host genotypes resulting from spatial structure can lead to the evolution of locally specialized virus, even when the host population is genetically diverse overall. We also find that spatial structure changes how viruses specialize: viruses evolved in well-mixed environments had lower exploitation of familiar hosts, while those in spatially structured environments exhibited higher exploitation of familiar hosts. These results demonstrate that spatial structure and host heterogeneity interact to shape pathogen specialization and that the evolutionary consequences of host diversity depends on the population structure.

evolutionary biology↗

bioRxiv2021

Dram1 confers resistance to Salmonella infection

Dram1 is a stress and infection inducible autophagy modulator that functions downstream of transcription factors p53 and NF{kappa}B. Using a zebrafish embryo infection model, we have previously shown that Dram1 provides protection against the intracellular pathogen Mycobacterium marinum by promoting the p62-dependent xenophagy of bacteria that have escaped into the cytosol. However, the possible interplay between Dram1 and other anti-bacterial autophagic mechanisms remains unknown. Recently, LC3-associated phagocytosis (LAP) has emerged as an important host defense mechanism that requires components of the autophagy machinery and targets bacteria directly in phagosomes. Our previous work established LAP as the main autophagic mechanism by which macrophages restrict growth of Salmonella Typhimurium in a systemically infected zebrafish host. We therefore employed this infection model to investigate the possible role of Dram1 in LAP. Morpholino knockdown or CRISPR/Cas9-mediated mutation of Dram1 led to reduced host survival and increased bacterial burden during S. Typhimurium infections. In contrast, overexpression of dram1 by mRNA injection curtailed Salmonella replication and reduced mortality of the infected host. During the early response to infection, GFP-Lc3 levels in transgenic zebrafish larvae correlated with the dram1 expression level, showing over two-fold reduction of GFP-Lc3-Salmonella association in dram1 knockdown or mutant embryos and an approximately 30% increase by dram1 overexpression. Since LAP is known to require the activity of the phagosomal NADPH oxidase, we used a Salmonella biosensor strain to detect bacterial exposure to reactive oxygen species (ROS) and found that the ROS response was largely abolished in the absence of dram1. Together, these results demonstrate the host protective role of Dram1 during S. Typhimurium infection and suggest a functional link between Dram1 and the induction of LAP.

immunology↗

bioRxiv2019

The autophagic response to Staphylococcus aureus provides an intracellular niche in neutrophils

Staphylococcus aureus is a major human pathogen causing multiple pathologies, from cutaneous lesions to life-threatening sepsis. Although neutrophils contribute to immunity against S. aureus, multiple lines of evidence suggest that these phagocytes can provide an intracellular niche for staphylococcal dissemination. However, the mechanism of neutrophil subversion by intracellular S. aureus remains unknown. Targeting of intracellular pathogens by autophagy is recognised as an important component of host innate immunity, but whether autophagy is beneficial or detrimental to S. aureus-infected hosts remains controversial. Here, using larval zebrafish we show that S. aureus is rapidly decorated by the autophagy marker Lc3 following engulfment by macrophages and neutrophils. Upon phagocytosis by neutrophils, Lc3-positive, non-acidified spacious phagosomes are formed. This response is dependent on phagocyte NADPH oxidase as both cyba knockdown and diphenyleneiodonium (DPI) treatment inhibited Lc3 decoration of phagosomes. Importantly, NADPH oxidase inhibition diverted neutrophil S. aureus processing into tight acidified vesicles, which resulted in increased host resistance to the infection. Some intracellular bacteria within neutrophils were also tagged by p62-GFP fusion protein and loss of p62 impaired host defence. Taken together, we have shown that intracellular handling of S. aureus by neutrophils is best explained by Lc3-associated phagocytosis (LAP), which appears to provide an intracellular niche for bacterial pathogenesis, while the selective autophagy receptor p62 is host-protective. The antagonistic roles of LAP and p62-mediated pathways in S. aureus-infected neutrophils may explain the conflicting reports relating to anti-staphylococcal autophagy and provide new insights for therapeutic strategies against antimicrobial resistant staphylococci.

immunology↗

bioRxiv2017

Evaluation and Design of Genome-wide CRISPR/Cas9 Knockout Screens

The adaptation of CRISPR/Cas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs), targeting human protein-coding genes and encoded in viral vectors, have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we re-analyze 17 genome-scale knockout screens in human cell lines from three research groups using three different genome-scale gRNA libraries, using the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm to identify essential genes, to refine and expand our previously defined set of human core essential genes, from 360 to 684 genes. We use this expanded set of reference Core Essential Genes (CEG2), plus empirical data from six CRISPR knockout screens, to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

systems biology↗