Biology subjects

O'Neill, P.

Publications and source records attributed to O'Neill, P..

13 recordsLinked to original sources

bioRxiv2026

Chromosome-level assembly of the Arabian killifish as a novel biomedical model species.

The Arabian killifish (Aphaniops dispar) is an excellent model for studying human diseases such as fungal infections and cancer progression. The embryos possess a transparent chorion for live imaging and display an extended period of independent feeding (13 days post-fertilisation). They also exhibit broad thermal tolerance which enables live imaging at physiologically relevant human temperatures. However, omics resources for this species remain limited and restricts its use in genomics studies. We generated a 1.45 GB high-quality reference assembly with 24 chromosome-level scaffolds and N50 score of 60.64 Mb by combining long-read, long-range, and short-read sequencing. The A. dispar genome exhibits a high level of homozygosity (95.6%) with BUSCO gene completeness scores of 99.2% and 97.5% for the Actinopterygii and Cyprinodontiformes lineages, respectively. The genome is highly repetitive (58.7%) and the majority of these are DNA transposons. The mitochondrial genome is 81.55% similar to that of Aphanius iberus (the Spanish toothcarp), with minor structural modifications. A. dispar exhibits highly conserved synteny with Fundulus heteroclitus within the Cyprinodontoidei clade. Together, these findings provide a valuable genomic resource for comparative genomics within the teleost community.

genomics↗

bioRxiv2026

Title: A de novo transcriptomic atlas of early embryo development in the Arabian killifish

The Arabian killifish (Aphaniops dispar) is new tractable vertebrate model system for developmental, ecological and biomedical research, including drug screening, pharmacological and infection biology studies. It is a relatively small euryhaline teleost with broad thermal tolerance and adaptability across a wide range of salinities from freshwater to hypersaline habitats. The embryos and early larvae are tolerant to environmental stressors and exhibit a delayed period of nutritional independence before hatching. This advantage offers an extended window for experimenting on the early developmental processes. Here, we describe time-course gene expression profiling of Arabian killifish embryos across nine developmental time points, from the 1-cell stage to the larval pre-hatching stage. Clustering of dynamic expression profiles for 27,564 Trinity genes revealed coordinated transcriptional modules corresponding to the maternal, blastula, maternal-to-zygotic transition (MZT)-related, gastrulation, organogenesis and larval maturation stages. The maternal stage displayed a highly distinct expression profile, dominated by maternal-specific transcripts that are rapidly degraded during the MZT. The later stages, from 48 hpf onward, revealed a shift from early regulatory mechanisms to the expression of organogenesis-related genes. The ZGA stage showed the conserved up-regulation of many zinc finger-associated genes, consistent with zebrafish and other teleost genomes. Overall, embryo development in A. dispar is slower than in zebrafish, with equivalent stages occurring several hours later. We propose a delayed onset of zygotic genome activation (ZGA) in the blastula stage, corresponding to 6 hpf in the Arabian killifish. Taken together, this study provides a transcriptomic resource for mining embryo development-related genes in the Arabian killifish.

genomics↗

bioRxiv2026

Prophage and metabolic determinants of Staphylococcus aureus survival to vancomycin identified via TraDIS screening

Reduced vancomycin susceptibility phenotypes in Staphylococcus aureus contribute to treatment failure, yet the genetic determinants of survival under inhibitory vancomycin exposure remain incompletely defined. We performed transposon directed insertion-site sequencing (TraDIS) on a methicillin resistant S. aureus (MRSA) ST398 mutant library following exposure to vancomycin at its minimum inhibitory concentration, identifying 52 genes whose disruption was associated with loss of population survival at inhibitory drug concentrations. Prophage associated loci were the largest functional group, spanning predicted structural and regulatory genes as well as multiple conserved hypothetical proteins. Targeted testing of defined transposon mutants in a USA300 background confirmed that disruption of selected loci impaired growth under vancomycin exposure. Our results highlight the contribution of diverse physiological processes, including metabolism, stress responses, and a prominent role for prophage-associated functions, rather than discrete resistance pathways. Together, these findings indicate that vancomycin tolerance is shaped by the general physiological state of the bacterial cell, including metabolic capacity and stress adaptation. ImportanceTreatment failure in Staphylococcus aureus infections often occurs in the absence of known antibiotic resistance determinants, suggesting that additional survival mechanisms influence therapeutic outcomes. In this study, we identify genetic determinants required for survival during inhibitory vancomycin exposure, revealing a broad role for metabolic functions, stress adaptation, and prophage-associated loci. The prominence of these diverse processes highlights that survival reflects global physiological adaptation rather than discrete resistance pathways. This insight underscores the need to consider cellular physiology and stress responses when developing strategies to prevent antibiotic tolerance and improve treatment efficacy.

microbiology↗

bioRxiv2024

Anatomy of a pest control failure: Introgression of cytochrome P450 337B3 alleles from invasive old-world bollworm into native corn earworm.

The establishment of invasive species populations can threaten the ecological balance in naive habitats and impact agricultural production practices. Helicoverpa armigera (old-world bollworm, OWBW) and H. zea (corn earworm, CEW) were geographically separated prior to the 2013 report of OWBW invasion into South America. Introgression of OWBW-specific cytochrome P450 337B3 (CYP337B3) gene into CEW was repeatedly detected across South America and the Caribbean. Two hybrids were documented from Texas in 2019. In this study, screening insects collected in Olathe, Colorado, USA, where a failure of pyrethroids to control CEW damage to conventional sweetcorn in 2023 detected 28.6% of insects with the OWBW-specific CYP337B3 marker. Nucleotide sequencing of the CYP337B3 gene identified 73.1 and 26.9% of insects carried CYP337B3v2 and CYP337B3v6 alleles, respectively and 0.15 overall frequency of CYP337B3 alleles. Based on prior data for distinct phylogeographic origins of CYP337B3v2 and v6 alleles, our results indicate Olathe samples were derived from two different introductions; An uncertain source of the v6 allele that was initially reported in West Africa and possibly South American or Caribbean origin of the globally distributed v2 allele. One of the 1618 individuals screened also carried a ribosomal RNA internal transcribed spacer 1 (ITS1) derived from OWBW. Local selection pressures at the Olathe location imposed by repeated pyrethroid exposures are likely attributed to the prevalence of CYP337B3, where control practices hasten the accumulation of phenotypic resistance by adaptive introgression. Pyrethroid and other resistance factors carried by invasive OWBW may continue to impact CEW management tactics across the Americas.

genetics↗

bioRxiv2024

Long-term survival of asexual Zymoseptoria tritici spores in the environment

The fungal phytopathogen Zymoseptoria tritici, causal agent of the economically damaging Septoria tritici blotch of wheat, is different from most foliar fungal pathogens in that its germination occurs slowly and apparently randomly after arrival on the leaf surface and is followed by a potentially prolonged period of epiphytic growth and even reproduction, during which no feeding structures are formed by the fungus. Thus, understanding the cues for germination and the mechanisms that underpin survival in low-nutrient environments could provide key new avenues for disease control. In this work, we examine survival, culturability, and virulence of spores following transfer from a high nutrient environment to water. We find that a sub-population of Z. tritici spores can survive and remain virulent for at least 7 weeks in water alone, during which time multicellular structures split to single cells. The fungus relies heavily on stored lipids; however, if cell suspensions in water are dried, the cells survive without lipid utilisation. Changes in gene expression in the first hours after suspension in water reflect adaptation to stress, while longer term starvation (7 days) induces changes particularly in primary metabolism and cytochrome P450 (CYP) gene expression. Importantly, we also found that Z. tritici spores are equally or better able to survive in soil as in water, and that rain-splash occurring 49 days after soil inoculation can transfer cells to wheat seedlings growing in inoculated soil and cause Septoria leaf blotch disease.

microbiology↗

bioRxiv2023

Long-read transcript sequencing identifies differential isoform expression in the entorhinal cortex in a transgenic model of tau pathology

Increasing evidence suggests that alternative splicing plays an important role in Alzheimers disease (AD), a devastating neurodegenerative disorder involving the intracellular aggregation of hyperphosphorylated tau. We used long-read cDNA sequencing to profile transcript diversity in the entorhinal cortex of wild-type (WT) and transgenic (TG) mice harboring a mutant form of human tau. Whole transcriptome profiling showed that previously reported gene-level expression differences between WT and TG mice reflect changes in the abundance of specific transcripts. Ultradeep targeted long-read cDNA sequencing of genes implicated in AD revealed hundreds of novel isoforms and identified specific transcripts associated with the development of tau pathology. Our results highlight the importance of differential transcript usage, even in the absence of gene-level expression alterations, as a mechanism underpinning gene regulation in the development of neuropathology. Our transcript annotations and a novel informatics pipeline for the analysis of long-read transcript sequencing data are provided as a resource to the community.

neuroscience↗

bioRxiv2023

A dirofilariasis mouse model for heartworm preclinical research

Use of experimental cats and dogs in veterinary heartworm preclinical drug research is increasing. As a potential alternative primary in vivo heartworm preventative drug screen, we assessed lymphopenic mice with ablation of the interleukin-2/7 common gamma chain ({gamma}c) as susceptible to the larval development phase of D. immitis. Non-obese diabetic (NOD) Severe Combined ImmunoDeficient (SCID){gamma}c-/-(NSG / NXG) mice consistently yielded viable D. immitis larvae at 2-4 weeks post-infection across multiple experiments, different batches of infectious larvae inoculates, different isolates of D. immitis and at independent laboratories. Mice did not display any overt clinical signs associated with infection up to 4 weeks. Developing larvae were found in subcutaneous and muscle fascia tissues, the natural site of this stage of heartworm in dogs. Larvae retrieved from NSG / NXG mice were mid-L4 stage of development. Compared with 14-day in vitro propagated larvae, in vivo derived L4 were significantly larger and contained expanded Wolbachia endobacteria titres, determined by QPCR and Fluorescent in situ Hybridisation (FISH). We established an ex vivo 6-day L4 paralytic screening system against nematodicidal agents (moxidectin, levamisole) which highlighted discrepancies in relative drug sensitivities in comparison with in vitro reared L4 D. immitis. We demonstrated effective depletion of Wolbachia by 70-90% in D. immitis L4 following 2-7 day oral in vivo exposures of NSG / NXG infected mice with doxycycline or the rapid-acting investigational anti-Wolbachia drug, AWZ1066S. We validated the NSG / NXG mouse model as a filaricide drug screen by in vivo treatments with single injections of moxidectin, which mediated 60-88% reduction in L4 larvae at 14-28 days. Future adoption of the mouse model as a first-line efficacy screen will benefit end-user laboratories conducting research and development of novel heartworm preventatives via increased access, rapid turnaround and reduced costs whilst simultaneously decreasing need for experimental cat or dog use.

microbiology↗

bioRxiv2023

Protein-specific signal peptides for mammalian vector engineering

Expression of recombinant proteins in mammalian cell factories relies on synthetic assemblies of genetic parts to optimally control flux through the product biosynthetic pathway. In comparison to other genetic part-types, there is a relative paucity of characterized signal peptide components, particularly for mammalian cell contexts. In this study, we describe a toolkit of signal peptide elements, created using bioinformatics-led and synthetic design approaches, that can be utilized to enhance production of biopharmaceutical proteins in Chinese Hamster Ovary cell factories. We demonstrate, for the first time in a mammalian cell context, that machine learning can be used to predict how discrete signal peptide elements will perform when utilized to drive ER translocation of specific single chain protein products. For more complex molecular formats, such as multichain monoclonal antibodies, we describe how a combination of in silico and targeted design rule-based in vitro testing can be employed to rapidly identify product-specific signal peptide solutions from minimal screening spaces. The utility of this technology is validated by deriving vector designs that increase product titers [≥] 1.8x, compared to standard industry systems, for a range of products, including a difficult-to-express monoclonal antibody. The availability of a vastly expanded toolbox of characterized signal peptide parts, combined with streamlined in silico/in vitro testing processes, will permit efficient expression vector re-design to maximize titers of both simple and complex protein products. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/532380v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@b0d655org.highwire.dtl.DTLVardef@1c76ae8org.highwire.dtl.DTLVardef@13bae76org.highwire.dtl.DTLVardef@13ae35b_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Nutrient and salt depletion synergistically boosts glucose metabolism in individual Escherichia coli cells

The interaction between a cell and its environment shapes fundamental intracellular processes such as cellular metabolism. In most cases growth rate is treated as a proximal metric for understanding the cellular metabolic status. However, changes in growth rate might not reflect metabolic variations in individuals responding to environmental fluctuations. Here we use single-cell microfluidics-microscopy combined with transcriptomics, proteomics and mathematical modelling to quantify the accumulation of glucose within Escherichia coli cells. In contrast to the current consensus, we reveal that environmental conditions which are comparatively unfavourable for growth, where both nutrients and salinity are depleted, increase glucose accumulation rates in individual bacteria and population subsets. We find that these changes in metabolic function are underpinned by variations at the translational and posttranslational level but not at the transcriptional level and are not dictated by changes in cell size. The metabolic response-characteristics identified greatly advance our fundamental understanding of the interactions between bacteria and their environment and have important ramifications when investigating cellular processes where salinity plays an important role.

microbiology↗

bioRxiv2021

Novel inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives

The global emergence of SARS-CoV-2 has triggered numerous efforts to develop therapeutic options for COVID-19 pandemic. The main protease of SARS-CoV-2 (Mpro), which is a critical enzyme for transcription and replication of SARS-CoV-2, is a key target for therapeutic development against COVID-19. An organoselenium drug called ebselen has recently been demonstrated to have strong inhibition against Mpro and antiviral activity but its molecular mode of action is unknown preventing further development. We have examined the binding modes of ebselen and its derivative in Mpro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of ebselen derivatives. A free selenium atom bound with cysteine 145 of Mpro catalytic dyad has been revealed by crystallographic studies of Mpro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct, formation of a phenolic by-product is confirmed by mass spectrometry. The target engagement of these compounds with an unprecedented mechanism of SARS-CoV-2 Mpro inhibition suggests wider therapeutic applications of organo-selenium compounds in SARS-CoV-2 and other zoonotic beta-corona viruses.

biophysics↗

bioRxiv2020

Full-length transcript sequencing of human and mouse identifies widespread isoform diversity and alternative splicing in the cerebral cortex

Alternative splicing is a post-transcriptional regulatory mechanism producing multiple distinct mRNA molecules from a single pre-mRNA. Alternative splicing has a prominent role in the central nervous system, impacting neurodevelopment and various neuronal functions as well as being increasingly implicated in brain disorders including autism, schizophrenia and Alzheimers disease. Standard short-read RNA-Seq approaches only sequence fragments of the mRNA molecule, making it difficult to accurately characterize the true nature of RNA isoform diversity. In this study, we used long-read isoform sequencing (Iso-Seq) to generate full-length cDNA sequences and map transcript diversity in the human and mouse cerebral cortex. We identify widespread RNA isoform diversity amongst expressed genes in the cortex, including many novel transcripts not present in existing genome annotations. Alternative splicing events were found to make a major contribution to RNA isoform diversity in the cortex, with intron retention being a relatively common event associated with nonsense-mediated decay and reduced transcript expression. Of note, we found evidence for transcription from novel (unannotated genes) and fusion events between neighbouring genes. Although global patterns of RNA isoform diversity were found to be generally similar between human and mouse cortex, we identified some notable exceptions. We also identified striking developmental changes in transcript diversity, with differential transcript usage between human adult and fetal cerebral cortex. Finally, we found evidence for extensive isoform diversity in genes associated with autism, schizophrenia and Alzheimers disease. Our data confirm the importance of alternative splicing in the cerebral cortex, dramatically increasing transcriptional diversity and representing an important mechanism underpinning gene regulation in the brain. We provide this transcript level data as a resource to the scientific community.

genomics↗

bioRxiv2020

Positively selected modifications in the pore of TbAQP2 allow pentamidine to enter Trypanosoma brucei

Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2s unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant but exclude most other diamidine drugs. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.

biochemistry↗

bioRxiv2019

Transcriptional signatures of progressive neuropathology in transgenic tau and amyloid mouse models.

The onset and progression of Alzheimers disease (AD) is characterized by increasing intracellular aggregation of hyperphosphorylated tau protein and accumulation of {beta}-amyloid (A{beta}) in the neocortex. Despite recent success in identifying genetic risk factors for AD the transcriptional mechanisms involved in disease progression are not fully understood. We used transgenic mice harbouring human tau (rTg4510) and amyloid precursor protein (J20) mutations to investigate transcriptional changes associated with the development of both tau and amyloid pathology. Using highly-parallel RNA sequencing we profiled transcriptional variation in the entorhinal cortex at four time points identifying robust genotype-associated differences in entorhinal cortex gene expression in both models. We quantified neuropathological burden across multiple brain regions in the same individual mice, identifying widespread changes in gene expression paralleling the development of tau pathology in rTg4510 mice. Differentially expressed transcripts included genes associated with familial AD from genetic studies of human patients, and genes annotated to both common and rare variants identified in GWAS and exome-sequencing studies of late-onset sporadic AD. Systems-level analyses identified discrete co-expression networks associated with the progressive accumulation of tau, with these enriched for genes and pathways previously implicated in the neuro-immunological and neurodegenerative processes driving AD pathology. Finally, we report considerable overlap between tau-associated networks and AD-associated co-expression modules identified in the human cortex. Our data provide further support for an immune-response component in the accumulation of tau, and reveal novel molecular pathways associated with the progression of AD neuropathology.

genomics↗