Biology subjects

Vaughan, A.

Publications and source records attributed to Vaughan, A..

13 recordsLinked to original sources

bioRxiv2024

Dynamics of Reactive Oxygen Species and Nitrogen Cycling in Soils as a Mechanism for Volatile Reactive Nitrogen Oxide Production

Heterotrophic bacteria and fungi are responsible for the decomposition of organic matter in soil. During this process, reactive oxygen species (ROS) can be produced directly and indirectly as extracellular byproducts of respiration. It is well known that nitrogen (N) cycle processes lead to the formation of volatile reactive nitrogen oxides (NOy), a group of climate-active gases that contribute to atmospheric chemistry and negatively impact human health. Primary microbial sources of NOy include ammonia- oxidising bacteria and denitrifying bacteria and fungi. Despite soil being a significant source of global NOy emissions, studies to date have primarily focused on N emissions from agricultural soils and there remains a large scope for investigating mechanisms of soil NOy production in a wider context in order to better constrain terrestrial and climate process models. Here, we propose a potential microbial mechanism involving ROS that could influence the production of NOy from soil. We utilised metagenomics and metatranscriptomics alongside continuous gas flux measurements and analysis of soil properties to evaluate the connection between microbial ROS and soil-sourced NOy. Our findings suggest that more NOy, particularly nitric oxide (NO), is produced in the presence of increased abundance of ammonia-oxidising and denitrifying taxa. NO can be lost to the environment or reacts with superoxide, an ROS produced via the enzymatic activity of soil organic matter (SOM) decomposers. This reaction produces peroxynitrite (ONOO-), which we have demonstrated to enhance nitrogen dioxide (NO2) emissions from soil. We have shown that the extent of NOy production through this pathway may be dependent on SOM composition, and the associated variability in carbon and nitrogen content.

microbiology↗

bioRxiv2023

Fecal microbial transfer and complex carbohydrates mediate protection against COPD

ObjectiveChronic obstructive pulmonary disease (COPD) is a major cause of global illness and death, most commonly caused by cigarette smoke. The mechanisms of pathogenesis remain poorly understood, limiting the development of effective therapies. The gastrointestinal microbiome has been implicated in chronic lung diseases via the gut-lung axis, but its role is unclear. DesignUsing an in vivo mouse model of cigarette smoke-induced COPD and fecal microbial transfer (FMT), we characterized the fecal microbiota using metagenomics, proteomics and metabolomics. Findings were correlated with airway and systemic inflammation, lung and gut histopathology, and lung function. Complex carbohydrates were assessed in mice using a high resistant starch diet, and in sixteen COPD patients using a randomized, double-blind, placebo-controlled pilot study of inulin supplementation. ResultsFMT alleviated hallmark features of COPD (inflammation, alveolar destruction, impaired lung function), gastrointestinal pathology and systemic immune changes. Protective effects were additive to smoking cessation. Disease features correlated with the relative abundance of Muribaculaceae, Desulfovibrionaceae and Lachnospiraceae family members. Proteomics and metabolomics identified downregulation of glucose and starch metabolism in cigarette smoke-associated microbiota, and supplementation of mice or human patients with complex carbohydrates improved disease outcomes. ConclusionThe gut microbiome contributes to COPD pathogenesis and can be targeted therapeutically. What is already known on this topicO_LIChanges in gut microbiota are associated with COPD but the underlying host and microbial mechanisms are unclear, limiting the therapeutic applications. C_LI What this study addsO_LIMicrobiome composition and metabolism is reproducibly correlated with lung and gastrointestinal pathology in experimental COPD. C_LIO_LIMicrobiome modifying interventions effectively alleviate disease, including protective effects supplementing smoking cessation. C_LIO_LINutritional interventions targeting the microbiome in COPD patients demonstrate efficacy in a small pilot study. C_LI How this study might affect research, practice or policyO_LIMicrobiome-targeting therapeutics and nutritional interventions may be developed for COPD, including as supplements to smoking cessation. C_LI

physiology↗

bioRxiv2023

Airway secretory cell-derived p63+ progenitors contribute to alveolar regeneration after sterile lung injury

Lung injury activates epithelial stem or progenitor cells for alveolar repair and regeneration. However, the origin and fate of injury-induced progenitors are poorly defined. Here, we report that p63-expressing progenitors emerge upon bleomycin-induced lung injury. These p63+ progenitors proliferate rapidly and differentiate into alveolar type 1 (AT1) and type 2 (AT2) cells through distinct trajectories. Dual recombinase-mediated sequential genetic lineage tracing reveals that p63+ progenitors originate from airway secretory cells and subsequently generate alveolar cells. Functionally, p63 activation is required for efficient alveolar regeneration from secretory cells. Our study identifies a secretory cell-derived p63+ progenitor that contributes to alveolar repair, indicating a potential therapeutic avenue for lung regeneration after injury.

cell biology↗

bioRxiv2022

Expansion Sequencing of RNA Barcoded Neurons in the Mammalian Brain: Progress and Implications for Molecularly Annotated Connectomics

Mapping and molecularly annotating mammalian neural circuits is challenging due to the inability to uniquely label cells while also resolving subcellular features such as synaptic proteins or fine cellular processes. We argue that an ideal technology for connectomics would have the following characteristics: the capacity for robust distance-independent labeling, synaptic resolution, molecular interrogation, and scalable computational methods. The recent development of high-diversity cellular barcoding with RNA has provided a way to overcome the labeling limitations associated with spectral dyes, however performing all-optical circuit mapping has not been demonstrated because no method exists to image barcodes throughout cells at synaptic-resolution. Here we show ExBarSeq, an integrated method combining in situ sequencing of RNA barcodes, immunostaining, and Expansion Microscopy coupled with an end-to-end software pipeline that automatically extracts barcode identities from large imaging datasets without data processing bottlenecks. As a proof of concept, we applied ExBarSeq to thick tissue sections from mice virally infected with MAPseq viral vectors and demonstrated the extraction of 50 barcoded cells in the visual cortex as well as cell morphologies uncovered via immunostaining. The current work demonstrates high resolution multiplexing of exogenous barcodes and endogenous synaptic proteins and outlines a roadmap for molecularly annotated connectomics at a brain-wide scale.

neuroscience↗

bioRxiv2020

Bulk Segregant Approaches to Nutritional Genomics in Plasmodium falciparum

What genes determine growth and nutrient utilization in asexual blood-stage malaria parasites? Competition experiments between a lab-adapted African parasite (NF54), and a recently isolated Asian parasite (NHP4026) reveal contrasting outcomes in different media: NF54 outcompetes NHP4026 in media containing human serum, while NHP4026 outcompetes NF54 in media containing AlbuMAX, a lipid-rich bovine serum formulation. We conducted parasite genetic crosses and compared genome-wide allele frequency changes in progeny populations cultured in media containing serum or AlbuMAX: this bulk segregant analysis (BSA) reveals three quantitative trait loci (QTL) underlying differential growth. The strongest QTL (chromosome 13) contains EBA-140: competition experiments between EBA-140-knockout and isogenic wildtype parasites showed fitness reversals in the two media types, validating this locus as the causative gene. These results (i) demonstrate the effectiveness of BSA for dissecting fitness traits in Plasmodium falciparum, and (ii) reveal an intimate link between red blood cell invasion and nutrient composition of growth media.

genetics↗

bioRxiv2020

Optimised use of Oxford Nanopore Flowcells for Hybrid Assemblies

Hybrid assemblies are highly valuable for studies of Enterobacteriaceae due to their ability to fully resolve the structure of mobile genetic elements, such as plasmids, which are involved in the carriage of clinically important genes (e.g. those involved in AMR/virulence). The widespread application of this technique is currently primarily limited by cost. Recent data has suggested that non-inferior, and even superior, hybrid assemblies can be produced using a fraction of the total output from a multiplexed nanopore (Oxford Nanopore Technologies [ONT]) flowcell run. In this study we sought to determine the optimal minimal running time for flowcells when acquiring reads for hybrid assembly. We then evaluated whether the ONT wash kit might allow users to exploit shorter running times by sequencing multiple libraries per flowcell. After 24 hours of sequencing, most chromosomes and plasmids had circularised and there was no benefit associated with longer running times. Quality was similar at 12 hours suggesting shorter running times are likely to be acceptable for certain applications (e.g. plasmid genomics). The ONT wash kit was highly effective in removing DNA between libraries. Contamination between libraries did not appear to affect subsequent hybrid assemblies, even when the same barcodes were used successively on a single flowcell. Utilising shorter run-times in combination with between-library nuclease washes allows at least 36 Enterobacteriaceae isolates to be sequenced per flowcell, significantly reducing the per isolate sequencing cost. Ultimately this will facilitate large-scale studies utilising hybrid assembly advancing our understanding of the genomics of key human pathogens. Data SummaryO_LIRaw sequencing data is available via NCBI under project accession number PRJNA604975. Sample accession numbers are provided in table S1. C_LIO_LIAssemblies are available via Figshare https://doi.org/10.6084/m9.figshare.11816532.v1. C_LI Impact StatementMost existing sequencing data has been acquired from short-read platforms (eg. Illumina). For some species of bacteria, clinically important genes, such as those involved in antibiotic resistance and/or virulence, are carried on plasmids. Whilst Illumina sequencing is highly accurate, it is generally unable to resolve complete genomic structures due to repetitive regions. Hybrid assembly uses long reads to scaffold together short-read contigs, maximising the benefits of both technologies. A major limiting factor to using hybrid assemblies at scale is the cost of sequencing the same isolate with two different technologies. Here we show that high-quality hybrid assemblies can be created for most isolates using significantly shorter run-times than are currently standard. We demonstrate that a simple washing step allows several libraries to be run on the same flowcell, facilitating the ability to take advantage of shorter running times. Adding nuclease means that contamination between libraries is minimal and has no significant effect on the quality of subsequent hybrid assemblies. This approach reduces the cost of acquiring long reads by >30%, paving the way for large-scale studies utilising hybrid assemblies which will likely significantly enhance our understanding of the genomics of important human pathogens.

bioinformatics↗

bioRxiv2019

Genomic epidemiology of a complex, multi-species plasmid-borne blaKPC carbapenemase outbreak in Enterobacterales in the UK, 2009-2014

Carbapenem resistance in Enterobacterales is a public health threat. Klebsiella pneumoniae carbapenemase (encoded by alleles of the blaKPC family) is one of the commonest transmissible carbapenem resistance mechanisms worldwide. The dissemination of blaKPC has historically been associated with distinct K. pneumoniae lineages (clonal group 258 [CG258]), a particular plasmid family (pKpQIL), and a composite transposon (Tn4401). In the UK, blaKPC has caused a large-scale, persistent outbreak focused on hospitals in North-West England. This outbreak has evolved to be polyclonal and poly-species, but the genetic mechanisms underpinning this evolution have not been elucidated in detail; this study used short-read whole genome sequencing of 604 blaKPC-positive isolates (Illumina) and long-read assembly (PacBio)/polishing (Illumina) of 21 isolates for characterisation. We observed the dissemination of blaKPC (predominantly blaKPC-2; 573/604 [95%] isolates) across eight species and more than 100 known sequence types. Although there was some variation at the transposon level (mostly Tn4401a, 584/604 (97%) isolates; predominantly with ATTGA-ATTGA target site duplications, 465/604 [77%] isolates), blaKPC spread appears to have been supported by highly fluid, modular exchange of larger genetic segments amongst plasmid populations dominated by IncFIB (580/604 isolates), IncFII (545/604 isolates) and IncR replicons (252/604 isolates). The subset of reconstructed plasmid sequences also highlighted modular exchange amongst non-blaKPC and blaKPC plasmids, and the common presence of multiple replicons within blaKPC plasmid structures (>60%). The substantial genomic plasticity observed has important implications for our understanding of the epidemiology of transmissible carbapenem resistance in Enterobacterales, for the implementation of adequate surveillance approaches, and for control.\n\nIMPORTANCEAntimicrobial resistance is a major threat to the management of infections, and resistance to carbapenems, one of the \"last line\" antibiotics available for managing drug-resistant infections, is a significant problem. This study used large-scale whole genome sequencing over a five-year period in the UK to highlight the complexity of genetic structures facilitating the spread of an important carbapenem resistance gene (blaKPC) amongst a number of bacterial species that cause disease in humans. In contrast to a recent pan-European study from 2012-2013(1), which demonstrated the major role of spread of clonal blaKPC-Klebsiella pneumoniae lineages in continental Europe, our study highlights the substantial plasticity in genetic mechanisms underpinning the dissemination of blaKPC. This genetic flux has important implications for: the surveillance of drug resistance (i.e. making surveillance more difficult); detection of outbreaks and tracking hospital transmission; generalizability of surveillance findings over time and for different regions; and for the implementation and evaluation of control interventions.

genomics↗

bioRxiv2019

Metagenomic Nanopore sequencing of influenza virus direct from clinical respiratory samples

Influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential of a diagnostic test for influenza which also provides insights on transmission, evolution and drug resistance, and simultaneously detects other viruses. We therefore set out to apply Oxford Nanopore Technology to metagenomic sequencing of respiratory samples. We generated influenza reads down to a limit of detection of 102-103 genome copies/ml in pooled samples, observing a strong relationship between the viral titre and the proportion of influenza reads (p = 4.7x10-5). Applying our methods to clinical throat swabs, we generated influenza reads for 27/27 samples with high-to-mid viral titres (Cycle threshold (Ct) values <30) and 6/13 samples with low viral titres (Ct values 30-40). No false positive reads were generated from 10 influenza-negative samples. Thus Nanopore sequencing operated with 83% sensitivity (95% CI 67-93%) and 100% specificity (95% CI 69-100%) compared to the current diagnostic standard. Coverage of full length virus was dependent on sample composition, being negatively influenced by increased host and bacterial reads. However, at high influenza titres, we were able to reconstruct >99% complete sequence for all eight gene segments. We also detected Human Coronavirus and generated a near complete Human Metapneumovirus genome from clinical samples. While further optimisation is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza and other respiratory viruses.

microbiology↗

bioRxiv2019

Genetic mapping of fitness determinants across the malaria parasite Plasmodium falciparum life cycle

Malaria is transmitted through female Anopheline mosquitoes where gamete fusion and meiosis occurs, and humans where parasites proliferate asexually. We describe a powerful approach to identify the genetic determinants of parasite fitness across both invertebrate and vertebrate life-cycle stages in human malaria parasite Plasmodium falciparum using bulk segregant analysis (BSA). We combined experimental genetic crosses using humanized mice, with selective whole genome amplification and BSA at multiple developmental stages in both mosquito and vertebrate host to examine parasite competition and identify genomic regions under selection. We generated crosses between artemisinin resistant (ART-R, kelch13-C580Y) and ART-sensitive (ART-S, kelch13-WT) parasite clones recently isolated from Southeast Asian patients. We then quantified genome-wide changes in allele frequency in the parasite progeny population from infected midgut and salivary glands of Anopheles stephensi mosquitoes, infected livers, emerging merozoites and aliquots of in vitro cultured progeny parasites at intervals over 30 days. Three striking results emerge: we observed (i) a strong skew (>80%) towards alleles from the ART-R parent in the mosquito stage, that dropped to [~]50% in the blood stage as selfed ART-R parasites were selected against; (ii) highly repeatable skews in allele frequencies across the genome in blood stage parasites; (iii) particularly strong selection (selection coefficient (s) [≤] 0.18/asexual cycle) against alleles from the ART-R parent at loci on chromosome 12 containing MRP2 and chromosome 14 containing ARPS10. This approach robustly identifies selected loci and has strong potential for identifying parasite genes that interact with the mosquito vector or compensatory loci involved in drug resistance.

evolutionary biology↗

bioRxiv2019

Reconciling the potentially irreconcilable? Genotypic and phenotypic amoxicillin-clavulanate resistance in Escherichia coli

Resistance to amoxicillin-clavulanate, a widely used beta-lactam/beta-lactamase inhibitor combination antibiotic, is rising globally, yet susceptibility testing remains challenging. To test whether whole-genome sequencing (WGS) could provide a more reliable assessment of susceptibility than traditional methods, we predicted resistance from WGS for 976 E. coli bloodstream infection isolates from Oxfordshire, UK, comparing against phenotypes from the BD Phoenix (calibrated against EUCAST guidelines). 339/976 (35%) isolates were amoxicillin-clavulanate resistant. Predictions based solely on beta-lactamase presence/absence performed poorly (sensitivity 23% (78/339)) but improved when genetic features associated with penicillinase hyper-production (e.g. promoter mutations, copy number estimates) were considered (sensitivity 82% (277/339); p<0.0001). Most discrepancies occurred in isolates with peri-breakpoint MICs. We investigated two potential causes; the phenotypic reference and the binary resistant/susceptible classification. We performed reference standard, replicated phenotyping in a random stratified subsample of 261/976 (27%) isolates using agar dilution, following both EUCAST and CLSI guidelines, which use different clavulanate concentrations. As well as disagreeing with each other, neither agar dilution phenotype aligned perfectly with genetic features. A random-effects model investigating associations between genetic features and MICs showed that some genetic features had small, variable and additive effects, resulting in variable resistance classification. Using model fixed-effects to predict MICs for the non-agar dilution isolates, predicted MICs were in essential agreement ({+/-}1 doubling dilution) with observed (BD Phoenix) MICs for 691/715 (97%) isolates. This suggests amoxicillin-clavulanate resistance in E. coli is quantitative, rather than qualitative, explaining the poorly reproducible binary (resistant/susceptible) phenotypes and suboptimal concordance between different phenotypic methods and with WGS-based predictions.

microbiology↗

bioRxiv2018

Nanopore metagenomic sequencing of full length human metapneumovirus (HMPV) within a unique sub-lineage

Human metapneumovirus (HMPV) has been recognized as an important pathogen which can cause a spectrum of respiratory tract disease. Here, we report Nanopore metagenomic sequencing of the first full length HMPV genome directly from a throat swab from a UK patient with complex lung disease and immunocompromise. We found a predominance (26.4%) of HMPV reads in the metagenomic sequencing data and consequently assembled the full genome at a high depth of coverage (mean 4,786). Through phylogenetic analyses, we identified this HMPV strain to originate from a unique genetic group in A2b, showing the presence of this group in the UK. Our study demonstrated the effectiveness of Nanopore metagenomic sequencing for diagnosing infectious diseases and recovering complete sequences for genomic characterization, highlighting the applicability of Nanopore sequencing in clinical settings.\n\nImportanceNanopore metagenomic sequencing has the potential to evolve as a point-of-care test for a range of infectious diseases. Here, we report the first full length human metapneumovirus (HMPV) genome in the UK sequenced by Nanopore from a non-invasive sample from an immunocompromised patient. We demonstrate the presence of HMPV from a unique genetic group not previously reported from the UK. Our study demonstrates the effectiveness of Nanopore sequencing for diagnosing an infection that was not detected by routine first-line tests in the clinical microbiology laboratory. We report sufficient genomic data to provide insight into the epidemiology of infection and with the potential to inform treatment decisions.

microbiology↗

bioRxiv2018

Klebsiella quasipneumonaiae provides a window into carbapenemase gene transfer, plasmid rearrangements and nosocomial acquisition from the hospital environment

Several emerging pathogens have arisen as a result of selection pressures exerted by modern healthcare. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intra-species transmission of the Klebsiella pneumoniae carbapenemase (KPC) gene, blaKPC, between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10 and five-year periods respectively. There were 32 blaKPC-positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from eight patients and 11 sink drains, with evidence for seven separate blaKPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subspecies quasipneumoniae (n=23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then probable transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique blaKPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases.

microbiology↗

bioRxiv2017

Categorical Representations Of Decision-Variables In Orbitofrontal Cortex

The brain creates internal representations of the external world in the form of neural activity, which is structured to support adaptive behavior. In many cortical regions, individual neurons respond to specific features that are matched to the function of each region and statistics of the world. In frontal cortex, however, neurons display baffling complexity, responding to a mixture of sensory, motor and other variables. Here we use an integrated new approach to understanding the architecture of higher-order cortical representations, and use this approach to show that discrete groups of orbitofrontal cortex (OFC) neurons encode distinct decision variables. Using rats engaged in a complex task combining perceptual and value guided decisions, we found that OFC neurons can be grouped into distinct, categorical response types. These categorical representations map directly onto decision-variables of a choice model explaining our behavioral data, such as reward size, decision confidence and integrated value. We propose that, like sensory neurons, frontal neurons form a sparse and over complete population representation aligned to the natural statistics of the world - in this case spanning the space of decision-variables required for optimal behavior.

neuroscience↗