Biology subjects

Mason, J.

Publications and source records attributed to Mason, J..

31 records · Page 2Linked to original sources

bioRxiv2020

Piperacillin/tazobactam resistant, cephalosporin susceptible Escherichia coli bloodstream infections driven by multiple resistance mechanisms across diverse sequence types

Resistance to piperacillin/tazobactam (TZP) in Escherichia coli has predominantly been associated with mechanisms that confer resistance to third generation cephalosporins. Recent reports have identified E. coli strains with phenotypic resistance to piperacillin/tazobactam but susceptibility to third generation cephalosporins (TZP-R/3GC-S). In this study we sought to determine the genetic diversity of this phenotype in E. coli (n = 58) isolated between 2014-2017 at a single tertiary hospital in Liverpool, UK, as well as the associated resistance mechanisms. We compare our findings to a UK-wide collection of invasive E. coli isolates (n = 1509) with publicly available phenotypic and genotypic data. These data sets included the TZP-R/3GC-S phenotype (n = 68), a piperacillin/tazobactam and third generation cephalosporin-susceptible (TZP-S/3GC-S, n = 1271) phenotypes. The TZP-R/3GC-S phenotype was displayed in a broad range of sequence types which was mirrored in the same phenotype from the UK-wide collection, and the overall diversity of invasive E. coli isolates. The TZP-R/3GC-S isolates contained a diverse range of plasmids, indicating multiple acquisition events of TZP resistance mechanisms rather than clonal expansion of a particular plasmid or sequence type. The putative resistance mechanisms were equally diverse, including hyperproduction of TEM-1, either via strong promoters or gene amplification, carriage of inhibitor resistant {beta}-lactamases, and an S133G b/aCTX-M-15 mutation detected for the first time in clinical isolates. Several of these mechanisms were present at a lower abundance in the TZP-S/3GC-S isolates from the UK-wide collection, but without the associated phenotypic resistance to TZP. Our findings highlight the complexity of this cryptic phenotype and the need for continued phenotypic monitoring, as well as further investigation to improve detection and prediction of the TZP-R/3GC-S phenotype from genomic data.

microbiology↗

bioRxiv2020

Sex and Power: sexual dimorphism in trait variability and its eco-evolutionary and statistical implications

Biomedical and clinical sciences are experiencing a renewed interest in the fact that males and females differ in many anatomic, physiological, and behavioral traits. Sex differences in trait variability, however, are yet to receive similar recognition. In medical science, mammalian females are assumed to have higher trait variability due to estrous cycles (the estrus-mediated variability hypothesis); historically in biomedical research, females have been excluded for this reason. Contrastingly, evolutionary theory and associated data support the greater male variability hypothesis. Here, we test these competing hypotheses in 218 traits measured in >26,900 mice, using meta-analysis methods. Neither hypothesis could universally explain patterns in trait variability. Sex-bias in variability was trait-dependent. While greater male variability was found in morphological traits, females were much more variable in immunological traits. Sex-specific variability has eco-evolutionary ramifications including sex-dependent responses to climate change, as well as statistical implications including power analysis considering sex difference in variance.

evolutionary biology↗

bioRxiv2020

Within-patient evolution to piperacillin/tazobactam resistance in a clinical isolate of Escherichia coli due to IS26-mediated amplification of blaTEM-1B

A novel phenotype of Escherichia coli and Klebsiella pneumoniae resistant to piperacillin/tazobactam (TZP), but susceptible to carbapenems and 3rd generation cephalosporins has recently emerged. The resistance mechanism of this phenotype has been identified as hyperproduction of the {beta}-lactamase blaTEM, however the mechanism of hyperproduction in isolates lacking promoter region mutations is not well understood. We sought to understand this mechanism by focussing on a pair of isolates obtained from an individual patient across two infection episodes and displaying within-patient evolution to TZP resistance. Following confirmation that the two isolates were clonal, we found that the TZP-resistant isolate hyperproduced a {beta}-lactamase but lacked mutations within {beta}-lactamase promoter regions. Hybrid assembly of long and short sequencing reads of the two isolates revealed both harboured a novel IS26-flanked composite transposon containing several antibiotic resistance genes, including blaTEM-1B, which was designated Tn6762. These resistance genes are also found to be present on a translocatable unit which had excised from Tn6762 in the TZP-resistant isolate. By replicating the evolutionary event leading to TZP resistance we were able to observe excision of the translocatable unit from Tn6762 following exposure to TZP and capture the TU in a plasmid containing a copy of IS26. Subsequent amplification of the TU, and by extension blaTEM-1B, leads to {beta}-lactamase hyperproduction and TZP resistance. Despite a significant increase in gene copy number (P value = <0.0001), we found that the TZP-resistant isolate was as fit as the susceptible ancestor. This mechanism of gene amplification, and the subsequent hyperproduction, of blaTEM-1B is an important consideration when using genomic data to predict resistance/susceptibility to TZP.

microbiology↗

bioRxiv2019

Glutamine deprivation regulates the origin and function of cancer cell exosomes

Exosomes are secreted extracellular vesicles (EVs) carrying diverse cargos, which can modulate recipient cell behaviour. They are thought to derive from intraluminal vesicles formed in late endosomal multivesicular bodies (MVBs). An alternate exosome formation mechanism, which is conserved from fly to human, is described here, with exosomes carrying unique cargos, including the GTPase Rab11, generated in Rab11-positive recycling endosomal MVBs. Release of these exosomes from cancer cells is increased by reducing Akt/mechanistic Target of Rapamycin (mTORC1) signalling or depleting the key metabolic substrate glutamine, which diverts membrane flux through recycling endosomes. The resulting vesicles promote tumour cell proliferation and turnover, and modulate blood vessel networks in xenograft mouse models in vivo. Their growth-promoting activity, which is also observed in vitro, is Rab11a-dependent, involves ERK-MAPK-signalling and is inhibited by antibodies against Amphiregulin, an EGFR ligand concentrated on these vesicles. Therefore, glutamine depletion or mTORC1 inhibition stimulates release of Rab11a-exosomes with pro-tumorigenic functions, which we propose promote stress-induced tumour adaptation.

cancer biology↗

bioRxiv2019

The transcription factor E2A drives neural differentiation in pluripotent cells

The intrinsic mechanisms that link extracellular signalling to the onset of neural differentiation are not well understood. In pluripotent mouse cells, BMP blocks entry into the neural lineage via transcriptional upregulation of Inhibitor of Differentiation (Id) factors. We have previously identified that the major binding partner of Id proteins in pluripotent cells is the basic helix-loop-helix (bHLH) transcription factor (TF), E2A. Id1 can prevent E2A from forming heterodimers with bHLH TFs or from forming homodimers. Here, we show that overexpression of a forced E2A homodimer is sufficient to drive robust neural commitment in pluripotent cells, even under non-permissive conditions. Conversely, we find that E2A null cells display a defect in their neural differentiation capacity. E2A acts as an upstream activator of neural lineage genes, including Sox1 and Foxd4, and as a repressor of Nodal signalling. Our results suggest a crucial role for E2A in establishing neural lineage commitment in pluripotent cells.

developmental biology↗

bioRxiv2019

A direct role for SNX9 in the biogenesis of filopodia

Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and in invadopodia. In cells, we discover SNX9 at specialised filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia.

cell biology↗

bioRxiv2019

Constrained actin dynamics emerges from variable compositions of actin regulatory protein complexes

Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To begin to understand how diverse regulatory proteins work together to generate actin-rich structures we tracked the assembly of actin regulators and their relative proportions in a cell-free system that generates filopodia-like structures (FLS). We found that heterogeneous mixtures of regulators could give rise to morphologically similar structures and that the FLS actin bundles exhibited simple dynamic behaviour of growth and shrinkage. To explain these observations, we combined experiment with theory, and found that stochastic fluctuations between redundant actin regulatory subcomplexes can account for the actin dynamics. Comparing the localizations of a variety of endogenous actin regulators in Drosophila embryos and distributions of filopodia lengths yielded similar conclusions of heterogenous actin regulatory complexes and filopodia lengths governed by a stochastic growth process. Our results explain how weakly-associating assemblies of regulatory proteins can produce robust functional outcomes.

cell biology↗

bioRxiv2019

Non-invasive characterization of human bone marrow by cell free messenger-RNA reveals response to growth factor stimulation and hematopoietic reconstitution after transplantation

Circulating cell free mRNA (cf-mRNA) holds great promise as a non-invasive diagnostic biomarker. However, the biological origin of cf-mRNA is still not well understood, limiting the clinical applications of this technology. Here, we use the bone marrow (BM) and pharmacologic manipulation of its resident cells as a window to study the origin of cf-mRNA. Using NGS-based profiling, we show that cf-mRNA is enriched in transcripts derived from the BM compared to circulating cells. Further, BM ablation experiments followed by hematopoietic stem cell transplants in cancer patients show that cf-mRNA levels reflect the transcriptional activity of BM resident hematopoietic lineages during marrow reconstitution. Finally, by stimulating specific BM cell populations in vivo using growth factor therapeutics (i.e. EPO, G-CSF), we show that cf-mRNA reveals dynamic functional changes in growing cell types, suggesting that, unlike other cell-free nucleic acids, cf-mRNA is secreted from living cells, rather than exclusively from apoptotic cells. Our results shed new light on the biology of cf-mRNA and demonstrate its potential applications in clinical practice.

molecular biology↗

bioRxiv2018

Non-enzymatic roles of human RAD51 at stalled replication forks

The central recombination enzyme RAD51 has been implicated in replication fork processing and restart in response to replication stress. Here, we use a separation-of-function allele of RAD51 that retains DNA binding, but not strand exchange activity, to reveal mechanistic aspects of RAD51s roles in the response to replication stress. We find that cells lacking RAD51 strand exchange activity protect replication forks from MRE11-dependent degradation, as expected from previous studies. Unexpectedly we find that RAD51s strand exchange activity is not required to convert stalled forks to a form that can be degraded by DNA2. Such conversion was shown previously to require replication fork reversal, supporting a model in which fork reversal depends on a non-enzymatic function of RAD51. We also show RAD51 promotes replication restart by both strand exchange-dependent and strand exchange-independent mechanisms.

molecular biology↗

bioRxiv2018

Sclerostin Deficiency Alters Peripheral B Lymphocyte Responses in Mice

Understanding how changes in bone physiology and homeostasis affect immune responses will inform how to retain strong immunity in patients with bone disease and in aged individuals. We previously identified sclerostin (Sost) as a mediator of cell communication between the skeletal and the immune system. Elevated bone mineral density in Sost-knockout (Sost-/-) mice contributes to an altered bone marrow microenvironment and adversely affects B cell development. B cells originate from hematopoietic stem cells within the bone marrow and mature in peripheral lymphoid organs to produce antibodies in response to infection and/or vaccination. In this study, we investigated whether the aberrant B cell development observed in the bone marrow of Sost-/- mice extends to peripheral B cells in the spleen during immune challenge, and if these changes were age-dependent. Concomitant with more severe changes in bone architecture, B cell development in the bone marrow and in the spleen worsened with age in Sost-/- mice. B cell responses to T-independent antigens were enhanced in young Sost-/- mice, whereas responses to T-dependent antigens were impaired. Our results support the hypothesis that the adverse effects of B cell development in the Sost-deficient bone marrow microenvironment extends to the peripheral B cell immune response to protein antigens, and suggest that the B cell response to routine vaccinations should be monitored regularly in patients being treated with sclerostin antibody therapy. In addition, our results open the possibility that Sost regulates the T-independent B cell response, which might be applicable to the improvement of vaccines towards non-protein antigens.

immunology↗

bioRxiv2018

PDX Finder: A Portal for Patient-Derived tumor Xenograft Model Discovery

Patient-derived tumor xenograft (PDX) mouse models are a versatile oncology research platform for studying tumor biology and for testing chemotherapeutic approaches tailored to genomic characteristics of individual patients tumors. PDX models are generated and distributed by a diverse group of academic labs, research organizations, multi-institution consortia, and contract research organizations. The distributed nature of PDX repositories and the use of different standards in the associated metadata presents a significant challenge to finding PDX models relevant to specific cancer research questions. The Jackson Laboratory and EMBL-EBI are addressing these challenges by co-developing PDX Finder, a comprehensive open global catalog of PDX models and their associated datasets. Within PDX Finder, model attributes are harmonized and integrated using a previously developed community minimal information standard to support consistent searching across the originating resources. Links to repositories are provided from the PDX Finder search results to facilitate model acquisition and/or collaboration. The PDX Finder resource currently contains information for more than 1900 PDX models of diverse cancers including those from large resources such as the Patient-Derived Models Repository, PDXNet, and EurOPDX. Individuals or organizations that generate and distribute PDXs are invited to increase the \"findability\" of their models by participating in the PDX Finder initiative at www.pdxfinder.org.

cancer biology↗

bioRxiv2018

Markov chain models of cancer metastasis

Abstract.We describe the use of Markov chain models for the purpose of quantitative forecasting of metastatic cancer progression. Each site (node) in the Markov network (directed graph) is an organ site where a secondary tumor could develop with some probability. The Markov matrix is an N x N matrix where each entry represents a transition probability of the disease progressing from one site to another during the course of the disease. The initial state-vector has a 1 at the position corresponding to the primary tumor, and 0s elsewhere (no initial metastases). The spread of the disease to other sites (metastases) is modeled as a directed random walk on the Markov network, moving from site to site with the estimated transition probabilities obtained from longitudinal data. The stochastic model produces probabilistic predictions of the likelihood of each metastatic pathway and corresponding time sequences obtained from computer Monte Carlo simulations. The main challenge is to empirically estimate the N^2 transition probabilities in the Markov matrix using appropriate longitudinal data.

cancer biology↗

bioRxiv2017

Filopodyan: An Open-Source Pipeline For The Analysis Of Filopodia

Filopodia have important sensory and mechanical roles in motile cells. The recruitment of actin regulators, such as ENA/VASP proteins, to sites of protrusion underlies diverse molecular mechanisms of filopodia formation and extension. We developed Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia and at their tips and bases concurrently with their morphological and dynamic properties. Filopodyan supports high-throughput phenotype characterization as well as detailed interactive editing of filopodia reconstructions through an intuitive graphical user interface. Our highly customizable pipeline is widely applicable, capable of detecting filopodia in four different cell types in vitro and in vivo. We use Filopodyan to quantify the recruitment of ENA and VASP preceding filopodia formation in neuronal growth cones, and uncover a molecular heterogeneity whereby different filopodia display markedly different responses to changes in the accumulation of ENA and VASP fluorescence in their tips over time.\n\neTOC summary: Urban[c]i[c] et al. developed an open-source platform called Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia, at their tips, and bases, concurrently with their morphological and dynamic properties. This customizable tool therefore enables researchers to determine the relationship between protein localization and filopodium behavior.

cell biology↗