Biology subjects

Stafford, G.

Publications and source records attributed to Stafford, G..

5 recordsLinked to original sources

bioRxiv2026

Repurposing clofarabine against enteric pathogens via metabolic starvation

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC) cause severe global diarrhoeal disease, exacerbated by rising antimicrobial resistance. Pathogenesis relies on "attaching and effacing" lesions, where bacteria generate actin pedestals to adhere, replicate, and destroy intestinal microvilli. To identify novel anti-virulence candidates targeting this process, we conducted a high-throughput phenotypic screen quantifying pedestal formation via high-content fluorescence microscopy. We identified the anticancer nucleotide analogue clofarabine as a potent inhibitor of EPEC growth and pathogenesis. Distinct from its oncology mechanism - inducing DNA chain termination and apoptosis - clofarabine competes with bacterial ATP-dependent processes to impede gene transcription, protein synthesis, and Type III Secretion System (T3SS) function. Remarkably, EPEC counteracts this inhibition by cleaving clofarabine, enabling restored protein synthesis and resumed growth. Furthermore, we report for the first time that clofarabine suppresses protein translation in human monocytes, suggesting an uncharacterised translational impact on mammalian cells. These findings reveal host and bacterial translation as interconnected vulnerabilities to purine antimetabolites, offering a novel strategy to disarm enteric pathogens while uncovering a bacterial enzymatic mechanism for neutralizing host-targeted therapeutics.

microbiology↗

bioRxiv2026

Structural Basis for Dual Peptidoglycan Hydrolysis by an E. faecium Minhovirus Tail Spike Lysin

Bacteriophages rely on breaching the bacterial cell wall as the first step of infection. We characterise ORF11, a putative tail-spike lysin from the 19 kbp Minhovirus SHEF14, a podovirus infecting Enterococcus faecium. Bioinformatic analyses indicate that ORF11 comprises four domains: a predicted glycosyl hydrolase (D1) a cysteine, histidine-dependent amidohydrolases/peptidases (CHAP, D4), separated by a helical linker (D2) and a CHAP-like domain (D3). This modular organisation is conserved among Enterococcus minhoviruses but differs markedly from analogous proteins in Copernicusvirus phage and related staphylococcal phage. ORF11 2.1 [A] crystal structure reveals an unusual dimeric assembly. The predicted glycosyl hydrolase and CHAP peptidase domains occupy opposite ends of the protein, bridged by the two other domains positioned at the dimer interface. Biochemical assays using recombinant ORF11 and LC-MS/MS confirmed dual peptidoglycan-degrading activity. ORF11 functions as both an N-acetylglucosaminidase and a D, D-endopeptidase, cleaving the bond between the D-alanine in position-4 and the D-aspartate residue at the end of the side chain. Together, these results provide the first structural description of a podovirus tail-spike lysin and demonstrate its bifunctional enzymatic activity. This dual action likely facilitates initial surface recognition and localised peptidoglycan degradation during infection of E. faecium, offering new insights into how minimal-genome phage target this clinically significant antimicrobial-resistant pathogen.

biochemistry↗

bioRxiv2025

Rolling out plaque-2-sequence: a single plaque sequencing approach enabling rapid, low-cost sequencing of phages directly from plaques

Rapid, accurate, and scalable sequencing of bacteriophage genomes is critical to advance phage therapy, build phage biobanks and understand phage genomic diversity. Current methods are based on sequencing and assembling complete bacteriophage genomes using short- or long-read technologies. However, current protocols require large DNA input and are cost prohibitive which limits their application to phage collections that typically are large and have low-biomass. In order to address this we have developed plaque-2-sequence, a robust and cost-effective workflow for high-throughput phage genome sequencing that will transform the speed and cost of attaining phage genomes. Plaque-2-sequence combines low-input transposase-based library preparation, amplification, nanopore sequencing and optimised assembly steps tailored to phage genomes. We applied the method to phages isolated on seven genetically diverse bacterial hosts; Escherichia, Pseudomonas, Synechococcus, Enterococcus, Klebsiella , Serratia and Enterobacter. High quality genome assemblies were validated using CheckV and benchmarking against previously sequenced phage isolates. Compared to standard Illumina sequencing, plaque-2-sequence offers [~]10-fold savings in sequencing price for individual labs. Furthermore, it substantially decreases the time required to produce a phage genome, once a plaque is obtained. Offering the ability to routinely obtain hundreds of phage genome sequences a week, with minimal hands-on time. Plaque-2-sequence enables systematic genomic characterisation of phage isolates, facilitating taxonomic classification, for the development of large scale phage biobanks. Impact StatementHere we have optimised a method for high-throughput sequencing of bacteriophage genomes from single plaques (plaque-2-sequence). We present a robust, high-throughput and cost-effective workflow. Plaque-2-sequence combines low-input transposase-based library preparation, amplification, nanopore sequencing and optimised assembly steps tailored to phage genomes. We demonstrate the scalability of this approach by sequencing over 100 phages from multiple bacterial hosts. This marks a step-change for the field, allowing phage genome sequencing to keep pace with phage isolation rates, and transforming how rapidly we can explore and understand phage genomic diversity.

microbiology↗

bioRxiv2023

Skeletons in the closet? Using a bibliometric lens to visualise phytochemical and pharmacological activities linked to Sceletium, a mood enhancer

Plants from the Sceletium genus (Aizoaceae) have been traditionally used by the Khoe-S[a]n people in southern Africa, mainly for thirst and hunger relief, pain reduction and spiritual purposes, particularly Sceletium tortuosum. The research on this species has seen rapid growth with advancements in analytical and pharmacological tools. The Web of Science (WoS) database was searched for articles related to Sceletium and Mesembrine. These data were additionally analysed by bibliometric software (VOSviewer) to generate term maps and author associations. The thematic areas with the most citations were, South African Traditional Medicine for mental health (110) and anxiolytic agents (75). Pioneer studies in the genus focused on chemical structural isolation, purification and characterization and techniques such as thin layer chromatography, liquid chromatography (HPLC, UPLC and more recently, LC-MS), gas chromatography mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) to study mesembrine alkaloids. Different laboratories have used a diverse range of extraction and pre-analytical methods that become routinely favoured in the analysis of the main metabolites (mesembrine, mesembranol, mesembranone and Sceletium A4) in their respective experimental settings. In contrast with previous reviews, this paper identified gaps in the research field, being a lack of toxicology assays, a deficit of clinical assessments, too few bioavailability studies and little to no investigation into the minor alkaloid groups found in Sceletium. Future studies are likely to see innovations in analytical techniques like leaf spray mass spectrometry and direct analysis in real-time ionization coupled with high-resolution time-of-flight mass spectrometry (DART-HR-TOF-MS) for rapid alkaloid identification and quality control purposes. While S. tortuosum has been the primary focus, studying other Sceletium species may aid in establishing chemotaxonomic relationships and addressing challenges with species misidentification. This research can benefit the nutraceutical industry and conservation efforts for the entire genus. At present, little to no pharmacological information is available in terms of the molecular physiological effects of mesembrine alkaloids in medical clinical settings. Research in these fields is expected to increase due to the growing interest in S. tortuosum as a herbal supplement and the potential development of mesembrine alkaloids into pharmaceutical drugs.

plant biology↗

bioRxiv2018

Bioinformatics workflows for genomic analysis of tumors from Patient Derived Xenografts (PDX): challenges and guidelines

Bioinformatics workflows for analyzing genomic data obtained from xenografted tumor (e.g., human tumors engrafted in a mouse host) must address several challenges, including separating mouse and human sequence reads and accurate identification of somatic mutations and copy number aberrations when paired normal DNA from the patient is not available. We report here data analysis workflows that address these challenges and result in reliable identification of somatic mutations, copy number alterations, and transcriptomic profiles of tumors from patient derived xenograft models. We validated our analytical approaches using simulated data and by assessing concordance of the genomic properties of xenograft tumors with data from primary human tumors in The Cancer Genome Atlas (TCGA). The commands and parameters for the workflows are available at https://github.com/TheJacksonLaboratory/PDX-Analysis-Workflows.

bioinformatics↗