ORCID Profile
0000-0002-5709-6790
Current Organisation
University of Southampton
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Publisher: Springer Science and Business Media LLC
Date: 30-10-2020
DOI: 10.1038/S41522-020-00154-8
Abstract: Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and biofilm-associated antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and genes missed within existing assemblies by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq data sets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth and a screen with the Tn-mutant library in microtiter plates. The screen identified hitherto unknown genes involved in biofilm growth and antibiotic resistance. Experiments conducted with the flow chamber across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated the function of both known genes and genes identified in the Tn-mutant screens. Differential protein abundance data from planktonic cells versus biofilm confirmed the upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type VI secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance, and resistance evolution in biofilms.
Publisher: Elsevier BV
Date: 09-1998
DOI: 10.1016/S0163-4453(98)80170-9
Abstract: Arbitrarily primed PCR (AP-PCR) was utilized to genetically fingerprint 252 Pseudomonas aeruginosa strains isolated from the sputa of 50 cystic fibrosis (CF) patients attending the Cork CF clinic over a period of 3 years. Ten distinct P. aeruginosa strains were identified and the distribution, temporal trends and clinical impact of colonization with these in idual P. aeruginosa clones was studied. A number of random isolates from each AP-PCR group were analysed using pulsed field gel electrophoresis (PFGE) in order to confirm the discriminatory power of the AP-PCR technique. The majority of patients were colonized with a single strain over the time period of the study, but it was also possible to harbour two or more strains transiently or simultaneously. Four main strains were relatively evenly distributed throughout the CF population, and it was noted that patients from the same family or attending the same school tended to harbour the same P. aeruginosa clone. Disease severity was significantly associated with the age of the patient (P < 0.001), clearly indicating an increase in severity with increase in age. The general clinical status of the CF patients was not significantly associated with the P. aeruginosa variant isolated from their sputa. Lung status was defined by FEV1 measurement and chest X-ray score (CXR). The non parametric Kruskal-Wallis significance test of FEV1, CXR and age by colonizing P. aeruginosa clone indicated that FEV1 (P = 0.017), but not CXR (P = 0.19) or age (P = 0.842), differed significantly across the clones of P. aeruginosa isolated. Patients harbouring P. aeruginosa strains B, F or G clearly had lower FEV1 scores while those harbouring clones A, C, D or H generally had higher FEV1 scores. Thus, the sub-species variant of P. aeruginosa colonizing CF patients may be associated with the severity of progressive lung disease.
Publisher: Royal Society of Chemistry (RSC)
Date: 2021
DOI: 10.1039/D1LC00292A
Abstract: Pirouette coupling involves Dean flow for cell and reporter bead inertial ordering for efficient co-encapsulation, achieving a throughput of 1 million cells per hour, a 2.5% multiplet rate and a 70% cell capture efficiency.
Publisher: Elsevier BV
Date: 03-2021
Publisher: Cold Spring Harbor Laboratory
Date: 09-04-2021
DOI: 10.1101/2021.04.08.439026
Abstract: The future of single cell ersity screens involves ever-larger s le sizes, dictating the need for higher throughput methods with low analytical noise to accurately describe the nature of the cellular system. Current approaches are limited by the Poisson statistic, requiring dilute cell suspensions and associated losses in throughput. In this contribution, we apply Dean entrainment to both cell and bead inputs, defining different volume packets to effect efficient co-encapsulation. Volume ratio scaling was explored to identify optimal conditions. This enabled the co-encapsulation of single cells with reporter beads at rates of ~1 million cells/hour, while increasing assay signal-to-noise with cell multiplet rates of ~2.5% and capturing ~70% of cells. The method, called Pirouette-seq, extends our capacity to investigate biological systems. Pirouette-seq involves cell and reporter bead inertial ordering for efficient co-encapsulation, achieving a throughput of 1 million cells/hour, a 2.5% multiplet rate and a 70% cell capture efficiency.
Publisher: Elsevier BV
Date: 10-2020
Publisher: Frontiers Media SA
Date: 15-06-2021
DOI: 10.3389/FIMMU.2021.665312
Abstract: Langerhans cells (LCs) reside in the epidermis as a dense network of immune system sentinels, coordinating both immunogenic and tolerogenic immune responses. To determine molecular switches directing induction of LC immune activation, we performed mathematical modelling of gene regulatory networks identified by single cell RNA sequencing of LCs exposed to TNF-alpha, a key pro-inflammatory signal produced by the skin. Our approach delineated three programmes of LC phenotypic activation (immunogenic, tolerogenic or ambivalent), and confirmed that TNF-alpha enhanced LC immunogenic programming. Through regulon analysis followed by mutual information modelling, we identified IRF1 as the key transcription factor for the regulation of immunogenicity in LCs. Application of a mathematical toggle switch model, coupling IRF1 with tolerance-inducing transcription factors, determined the key set of transcription factors regulating the switch between tolerance and immunogenicity, and correctly predicted LC behaviour in LCs derived from different body sites. Our findings provide a mechanistic explanation of how combinatorial interactions between different transcription factors can coordinate specific transcriptional programmes in human LCs, interpreting the microenvironmental context of the local tissue microenvironments.
Publisher: Springer Science and Business Media LLC
Date: 16-01-2020
DOI: 10.1038/S41467-019-14125-X
Abstract: Langerhans cells (LC) can prime tolerogenic as well as immunogenic responses in skin, but the genomic states and transcription factors (TF) regulating these context-specific responses are unclear. Bulk and single-cell transcriptional profiling demonstrates that human migratory LCs are robustly programmed for MHC-I and MHC-II antigen presentation. Chromatin analysis reveals enrichment of ETS-IRF and AP1-IRF composite regulatory elements in antigen-presentation genes, coinciding with expression of the TFs, PU.1, IRF4 and BATF3 but not IRF8. Migration of LCs from the epidermis is accompanied by upregulation of IRF4, antigen processing components and co-stimulatory molecules. TNF stimulation augments LC cross-presentation while attenuating IRF4 expression. CRISPR-mediated editing reveals IRF4 to positively regulate the LC activation programme, but repress NF2EL2 and NF-kB pathway genes that promote responsiveness to oxidative stress and inflammatory cytokines. Thus, IRF4-dependent genomic programming of human migratory LCs appears to enable LC maturation while attenuating excessive inflammatory and immunogenic responses in the epidermis.
Publisher: Springer Science and Business Media LLC
Date: 14-08-2022
DOI: 10.1186/S13578-022-00864-W
Abstract: Different pathologies, affecting the skeletal system, were reported to display altered bone and/or cartilage innervation profiles leading to the deregulation of the tissue homeostasis. The patterning of peripheral innervation is achieved through the tissue-specific expression of attractive or repulsive axonal guidance cues in specific space and time frames. During the last decade, emerging findings attributed to the extracellular vesicles (EV) trading a central role in peripheral tissue innervation. However, to date, the contribution of EV in controlling bone innervation is totally unknown. Here we show that sensory neurons outgrowth induced by the bone resorbing cells—osteoclasts—is promoted by osteoclast-derived EV. The EV induced axonal growth is achieved by targeting epidermal growth factor receptor (EGFR)/ErbB2 signaling rotein kinase C phosphorylation in sensory neurons. In addition, our data also indicate that osteoclasts promote sensory neurons electrophysiological activity reflecting a possible pathway in nerve sensitization in the bone microenvironment, however this effect is EV independent. Overall, these results identify a new mechanism of sensory bone innervation regulation and shed the light on the role of osteoclast-derived EV in shaping/guiding bone sensory innervation. These findings provide opportunities for exploitation of osteoclast-derived EV based strategies to prevent and/or mitigate pathological uncontrolled bone innervation.
Publisher: Frontiers Media SA
Date: 20-09-2022
DOI: 10.3389/FIMMU.2022.892254
Abstract: Human epidermal Langerhans cells (LCs) maintain immune homeostasis in the skin. To examine transcriptional programming of human primary LCs during homeostasis, we performed scRNA-seq analysis of LCs before and after migration from the epidermis, coupled with functional assessment of their regulatory T cell priming capabilities. The analysis revealed that steady-state LCs exist in a continuum of maturation states and upregulate antigen presentation genes along with an immunoregulatory module including the genes IDO1 , LGALS1 , LAMTOR1, IL4I , upon their migration. The migration-induced transition in genomic state is accompanied by the ability of LCs to more efficiently prime regulatory T cell responses in co-culture assays. Computational analyses of the scRNAseq datasets using SCENIC and Partial Information Decomposition in Context identified a set of migration-induced transcription factors including IRF4, KLF6 and RelB as key nodes within a immunoregulatory gene regulatory network. These findings support a model in which efficient priming of immunoregulatory responses by LCs is dependent on coordinated upregulation of a migration-coupled maturation program with a immunoregulation-promoting genomic module.
Publisher: Cold Spring Harbor Laboratory
Date: 10-10-2019
DOI: 10.1101/800631
Abstract: Single-cell transcriptomics has sensitivity limits that restrict low abundance transcript identification, affects clustering and introduce artefact. Here, we describe Constellation DropSeq (C-DropSeq), a molecular transcriptome filter that delivers two orders of magnitude sensitivity gains by maximising read utility while reducing sequencing depth and costs. The simple and powerful method is broadly compatible with library preparation routines and was demonstrated by identifying and characterizing the activation of rare dendritic cell sub-populations.
Publisher: Elsevier BV
Date: 10-2020
Publisher: Springer Science and Business Media LLC
Date: 06-2023
DOI: 10.1038/S41588-023-01409-8
Abstract: The kidneys operate at the interface of plasma and urine by clearing molecular waste products while retaining valuable solutes. Genetic studies of paired plasma and urine metabolomes may identify underlying processes. We conducted genome-wide studies of 1,916 plasma and urine metabolites and detected 1,299 significant associations. Associations with 40% of implicated metabolites would have been missed by studying plasma alone. We detected urine-specific findings that provide information about metabolite reabsorption in the kidney, such as aquaporin (AQP)-7-mediated glycerol transport, and different metabolomic footprints of kidney-expressed proteins in plasma and urine that are consistent with their localization and function, including the transporters NaDC3 ( SLC13A3 ) and ASBT ( SLC10A2 ). Shared genetic determinants of 7,073 metabolite–disease combinations represent a resource to better understand metabolic diseases and revealed connections of dipeptidase 1 with circulating digestive enzymes and with hypertension. Extending genetic studies of the metabolome beyond plasma yields unique insights into processes at the interface of body compartments.
Publisher: Cold Spring Harbor Laboratory
Date: 07-02-2020
DOI: 10.1101/2020.02.06.936690
Abstract: Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and missed genes by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq datasets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth. Experiments conducted across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated both known and novel genes involved in biofilm growth and antibiotic resistance identified in screens of the mutant collection. Differential protein expression data from planktonic cells versus biofilm confirmed upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type six secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance and resistance evolution.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: Germany
No related grants have been discovered for Jonathan West.