Biology subjects

McBride, R.

Publications and source records attributed to McBride, R..

9 recordsLinked to original sources

bioRxiv2025

Comprehensive LC-MS/MS Data Acquisition in Metabolomics via Maximum Bipartite Matching

BackgroundIn untargeted metabolomics studies, liquid chromatography tandem mass spectrometry (LC-MS/MS) is a powerful analytical platform. The fragmentation spectra produced can be used as "molecular fingerprints" to identify unknown metabolites. However, the high number of analytes that may be co-eluting limits the number of fragmentation spectra that can be collected and potentially identified, presenting a serious bottleneck for many studies. There is a need for new fragmentation strategies which are comprehensive, interpretable and robust, meaning they produce high-quality fragmentation spectra for as many analytes as possible while operating within the constraints of notoriously noisy mass spectrometry data. ResultsWe present a data acquisition workflow which uses a bipartite graph to represent the relationship between opportunities for fragmentation and desired fragmentation targets. This method allows a schedule for data acquisition to be optimally allocated by a standard algorithm. We augment this existing technique by allowing it to solve for multiple samples collectively, allowing it to optimise target intensity (and hence spectral quality) via the use of a weighted matching and by assigning leftover scans redundantly to improve robustness. We also show how this workflow can be used flexibly to generate inclusion windows for Data-Dependent Acquisition (DDA) methods. Our experiments show that several thousand peaks identified in a realistic biological sample can be targeted using only two LC-MS/MS runs. We also further investigate the trade-off between offline workflows and DDA methods by exposing our target list of peaks to realistic variation across samples. We find in those circumstances that our new method has performance (measured by number of peaks targeted comparable to state-of-the-art DDA methods). However, this competitive performance is only possible with our additions to the base maximum matching technique, which provide extra resistance against inter-sample variations. ConclusionsWe have proposed a workflow for LC-MS/MS data acquisition which can be used flexibly for entirely pre-scheduled acquisition or which may generate inclusion windows for online DDA methods. Our results show that the maximum matching workflow with our improvements is state-of-the-art where pre-scheduling is concerned, and in future this foundation may be developed to build more powerful DDA methods which can action the promise of truly comprehensive data acquisition.

bioinformatics↗

bioRxiv2024

Bovine H5N1 influenza virus binds poorly to human-type sialic acid receptors

Clade 2.3.4.4b highly pathogenic H5N1 avian influenza (HPAI) viruses started circulating widely in lactating dairy cattle in the United States at the end of 2023. Avian influenza viruses enter cells after binding to glycan receptors with terminally linked 2-3 sialic acid, whereas human influenza viruses typically bind to glycan receptors terminally linked 2-6 sialic acid in the upper respiratory tract. Here, we evaluated the receptor binding properties of hemagglutinin (HA) trimers from a clade 2.3.4.4b avian isolate (A/American Wigeon/South Carolina/22-000345-001/2021) and a cattle isolate (A/dairy cattle/Texas/24-008749-002-v/2024). Using two different methods, we found that both of the 2.3.4.4b H5s bound efficiently to glycan receptors with terminally linked 2-3 sialic acid with no detectable binding to glycan receptors with terminally linked 2-6 sialic acid. Our data suggest that clade 2.3.4.4b H5N1 viruses bind poorly to human receptors. It will be important to continue evaluating receptor binding properties of these viruses as they evolve in cattle.

microbiology↗

bioRxiv2024

Receptors Determine Lytic Phage Host Range in Pseudomonas aeruginosa

Limited phage host range remains one of the obstacles to the widespread use of phage therapy against bacterial infections. Here, we perform a genome-wide association study (GWAS) using Pseudomonas aeruginosa clinical isolates collected from people with cystic fibrosis (pwCF) to identify bacterial genes associated with resistance or susceptibility to two lytic phages, OMKO1 and LPS-5, recently used in a clinical trial in pwCF. Results were validated with transposon mutagenesis experiments and functional assays. Genes associated with flagellum assembly and lipopolysaccharide biosynthesis are essential for infection by OMKO1 and LPS-5, respectively, consistent with functional studies implicating these molecules as receptors for these phages. Notably, the presence of bacterial genes encoding phage defense mechanisms is not predictive of phage susceptibility. Instead, the relative abundance of defense elements is associated with the number of temperate phages within bacterial genomes. Together, our findings highlight the central role of receptors in determining phage host range.

microbiology↗

bioRxiv2024

The contribution of neutrophils to bacteriophage clearance and pharmacokinetics in vivo

With the increasing prevalence of antimicrobial-resistant bacterial infections, there is great interest in using lytic bacteriophages (phages) to treat such infections. However, the factors that govern bacteriophage pharmacokinetics in vivo remain poorly understood. Here, we have examined the contribution of neutrophils, the most abundant phagocytes in the body, to the pharmacokinetics of intravenously administered bacteriophage in uninfected mice. A single dose of LPS-5, an antipseudomonal bacteriophage recently used in human clinical trials, was administered intravenously to both wild-type BALB/c and neutropenic ICR mice. Phage concentrations were assessed in peripheral blood and spleen at 0.5, 1, 2, 4, 8, 12, and 24 hours after administration by plaque assay and qPCR. We observed that the phage clearance is only minimally affected by neutropenia. Indeed, the half-life of phages in blood in BALB/c and ICR mice is 3.45 and 3.66 hours, respectively. These data suggest that neutrophil-mediated phagocytosis is not a major determinant of phage clearance. Conversely, we observed a substantial discrepancy in circulating phage levels over time when measured by qPCR versus plaque assay, suggesting that substantial functional inactivation of circulating phages occurs over time. These data indicate that circulating factors, but not neutrophils, inactivate intravenously administered phages.

pharmacology and toxicology↗

bioRxiv2024

Potential pandemic risk of circulating swine H1N2 influenza viruses

Influenza A viruses in swine have considerable genetic diversity and continue to pose a pandemic threat to humans. They were the source of the most recent influenza pandemic, and since 2010, novel swine viruses have spilled over into humans more than 400 times in the United States. Although these zoonotic infections generally result in mild illness with limited onward human transmission, the potential for sustained transmission of an emerging influenza virus between individuals due to lack of population level immunity is of great concern. Compiling the literature on pandemic threat assessment, we established a pipeline to characterize and triage influenza viruses for their pandemic risk and examined the pandemic potential of two widespread swine origin viruses. Our analysis revealed that a panel of human sera collected from healthy adults in 2020 has no cross-reactive neutralizing antibodies against an -H1 clade strain but do against a {gamma}-H1 clade strain. Swine H1N2 virus from the -H1 clade (-swH1N2) replicated efficiently in human airway cultures and exhibited phenotypic signatures similar to the human H1N1 pandemic strain from 2009 (H1N1pdm09). Furthermore, -swH1N2 was capable of efficient airborne transmission to both naive ferrets and ferrets with prior seasonal influenza immunity. Ferrets with H1N1pdm09 pre-existing immunity had reduced -swH1N2 viral shedding from the upper respiratory tract and cleared the infection faster. Despite this, H1N1pdm09-immune ferrets that became infected via the air could still onward transmit -swH1N2 with an efficiency of 50%. Taken together, these results indicate that this -swH1N2 strain has a higher pandemic potential, but a moderate level of impact since there is reduced replication fitness in animals with prior immunity.

microbiology↗

bioRxiv2023

Rapid assessment of changes in phage bioactivity using dynamic light scattering

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. Here, we use Dynamic Light Scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-year-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web-application (Phage-ELF) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and non-destructive tool for quality control of phage preparations in academic and commercial settings. Significance StatementPhages are promising for use in treating antibiotic-resistant infections, but their decay over time in refrigerated storage and higher temperatures has been a difficult barrier to overcome. This is in part because there are no suitable methods to monitor phage activity over time, especially in clinical settings. Here, we show that Dynamic Light Scattering (DLS) can be used to measure the physical state of phage preparations, which provides accurate and precise information on their lytic function - the key parameter underlying clinical efficacy. This study reveals a "structure-function" relationship for lytic phages and establishes DLS as a method to optimize the storage, handling, and clinical use of phages.

biophysics↗

bioRxiv2023

Engineered display of ganglioside-sugars on protein elicits a clonally and structurally constrained B cell response

Ganglioside sugars, as Tumour-Associated Carbohydrate Antigens (TACAs), are long-proposed targets for vaccination and therapeutic antibody production, but their self-like character imparts immunorecessive characteristics that classical vaccination approaches have to date failed to overcome. One prominent TACA, the glycan component of ganglioside GM3 (GM3g), is over-expressed on diverse tumours. To probe the limits of glycan tolerance, we used protein editing methods to display GM3g in systematically varied non-native presentation modes by attachment to carrier protein lysine sidechains using diverse chemical linkers. We report here that such presentation creates glycoconjugates that are strongly immunogenic in mice and elicit robust antigen-specific IgG responses specific to GM3g. Characterisation of this response by antigen-specific B cell cloning and phylogenetic and functional analyses suggests that such display enables the engagement of a highly restricted naive B cell class with a defined germline configuration dominated by members of the IGHV2 subgroup. Strikingly, structural analysis reveals that glycan features appear to be recognised primarily by antibody CDRH1/2, and despite the presence of an antigen-specific Th response and B cell somatic hypermutation, we found no evidence of affinity maturation towards the antigen. Together these findings suggest a reach-through model in which glycans, when displayed in non-self formats of sufficient distance from a conjugate backbone, may engage glycan ready V-region motifs encoded in the germline. Structural constraints define why, despite engaging the trisaccharide, antibodies do not bind natively-presented glycans, such as when linked to lipid GM3. Our findings provide an explanation for the long-standing difficulties in raising antibodies reactive with native TACAs, and provide a possible template for rational vaccine design against this and other TACA antigens. HighlightsO_LIGM3g synthetically coupled via a longer, orthogonal (from backbone) glycoconjugate (LOG) presentation format (thioethyl-lysyl-amidine) display elicits high-titre IgG responses in mice. C_LIO_LIThe germinal centre experience of LOG glycoconjugate-specific B cell responses is directly influenced by the protein backbone. C_LIO_LIStructural characterisation of the antibody response to LOGs reveals highly restricted germline-encoded glycan-engaging motifs that mediate GM3g recognition. C_LIO_LIFailure of antibodies to bind the native trisaccharide highlights barriers to be overcome for the rational design of anti-TACA antibodies. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/543556v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@b2111org.highwire.dtl.DTLVardef@a81b88org.highwire.dtl.DTLVardef@a78c6aorg.highwire.dtl.DTLVardef@1f3b4b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

TopNEXt: Automatic DDA Exclusion Framework for Multi-Sample Mass Spectrometry Experiments

MotivationLiquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) experiments aim to produce high quality fragmentation spectra which can be used to identify metabolites. However, current Data-Dependent Acquisition (DDA) approaches may fail to collect spectra of sufficient quality and quantity for experimental outcomes, and extend poorly across multiple samples by failing to share information across samples or by requiring manual expert input. ResultsWe present TopNEXt, a real-time scan prioritisation framework that improves data acquisition in multi-sample LC-MS/MS metabolomics experiments. TopNEXt extends traditional DDA exclusion methods across multiple samples by using a Region of Interest (RoI) and intensity-based scoring system. Through both simulated and lab experiments we show that methods incorporating these novel concepts acquire fragmentation spectra for an additional 10% of our set of target peaks and with an additional 20% of acquisition intensity. By increasing the quality and quantity of fragmentation spectra, TopNEXt can help improve metabolite identification with a potential impact across a variety of experimental contexts. AvailabilityTopNEXt is implemented as part of the ViMMS framework and the latest version can be found at https://github.com/glasgowcompbio/vimms. A stable version used to produce our results can be found at 10.5281/zenodo.7468914. Data can be found at 10.5525/gla.researchdata.1382. Contactr.mcbride.1@research.gla.ac.uk or vinny.davies@glasgow.ac.uk Supplementary informationSupplementary data are available at Bioarxiv online.

molecular biology↗

bioRxiv2023

Simulated-to-real Benchmarking of Acquisition Methods in Metabolomics

Data-Dependent and Data-Independent Acquisition modes (DDA and DIA, respectively) are both widely used to acquire MS2 spectra in untargeted liquid chromatography tandem mass spectrometry (LC-MS/MS) metabolomics analyses. Despite their wide use, little work has been attempted to systematically compare their performance due to the difficulty and cost of performing comparisons with real experimental data due to the lack of ground truth and the costs involved in running large number of acquisitions. Here, we present a systematic in-silico comparison of these two acquisition methods. To do so, we extended our Virtual Metabolomics Mass Spectrometer (ViMMS) framework with a DIA module. Our results show that the performance of these methods varies with the average number of co-eluting ions as the most important factor. At low numbers, DIA outperforms DDA, but at higher numbers, DDA has an advantage as DIA can no longer deal with the large amount of overlapping ion chromatograms. Results from simulation were further validated on an actual mass spectrometer, demonstrating that using ViMMS we can draw conclusions from simulation that translate well into the real world. Embedding this work within ViMMS also allows for researchers to easily simulate DDA and DIA LC-MS/MS runs, validate them on actual instrument, and potentially prototype novel methods that best combine the characteristics of both approaches. We believe that this work provides a useful guide to the choice of DIA or DDA for scientists involved in metabolomics data acquisition.

systems biology↗