Biology subjects

O'Brien, P.

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

9 recordsLinked to original sources

bioRxiv2025

The earliest changes in the translatome upon human T cell activation

Stimulation of resting T cells triggers a rapid transition into an activated state, characterized by significant changes in phenotype, metabolism and secretory function. To explore the earliest responses, we examined transcriptional and translational changes in isolated human T cells within the first four hours post-stimulation. Polyclonal stimulation initially upregulates cytokine genes while downregulating certain transcription factors and regulators. Subsequently, selective mRNA translation occurs on 5-terminal oligopyrimidine (TOP) motif-containing mRNAs and ATF4, alongside global transcriptional reprogramming involving alternative transcription and splicing. Notably, both stimulated and unstimulated T cells undergo dynamic gene expression changes over time, reflecting adaptation to in vitro conditions and the loss of in vivo homeostatic signals. This includes heightened translation initiation stringency, particularly in non-stimulated cells. Thus, stress responses induced by standard T cell isolation protocols must be considered when investigating T cell signaling and activation.

immunology↗

bioRxiv2025

Evaluating transboundary connectivity to support cross-border conservation between Canada and the United States

Landscape connectivity is considered critical for maintaining biodiversity. Many jurisdictions have identified the importance of considering connectivity in land use plans, and connectivity has recently been included as a metric in international conservation agreements. Consequently, there is a need for measures of connectivity that can be applied at national and international scales; however, evaluating connectivity across international boundaries remains a challenge due to inconsistencies in mapping data, and differences in sociopolitical systems. Canada and the United States of America (USA) share a long international border, and thus, there is a need and opportunity to develop transboundary connectivity plans for this international region. We extended a previously published pan-Canadian multi-species connectivity model into the USA to produce a seamless and high-resolution omnidirectional connectivity map for the two countries. We identify several significant animal movement corridors across the transboundary region and show that about 20% of connectivity hotspots in the region are covered by protected areas. We also demonstrate the potential of the map for identifying important areas for wildlife movement and the spread of zoonotic diseases. Our map will be useful for supporting transboundary connectivity conservation between Canada and the USA and our modelling approach can easily be applied to other countries to support their own connectivity initiatives.

ecology↗

bioRxiv2023

Using sentinel nodes to evaluate changing connectivity in a protected areas network

It has been recognized that well-connected networks of protected areas are needed to halt the continued loss of global biodiversity. The recently signed Kunming-Montreal biodiversity agreement commits countries to protecting 30% of terrestrial lands in well-connected networks of protected areas by 2030. To meet these ambitious targets, land-use planners and conservation practitioners will require tools to identify areas important for connectivity and track future changes. In this study we present methods using circuit theoretic models with a subset of sentinel park nodes to evaluate connectivity for a protected areas network. We assigned a lower cost to natural areas within protected areas, under the assumption that animal movement within parks should be less costly given the regulation of activities. We found that by using mean pairwise effective resistance (MPER) as an indicator of overall network connectivity, we were able to detect changes in a parks network in response to simulated land-use changes. As expected, MPER increased with the addition of high-cost developments and decreased with the addition of new, low-cost protected areas. We tested our sentinel node method by evaluating connectivity for the protected areas network in the province of Ontario, Canada. Our method can help provide protected areas ecologists and planners with baseline estimates of connectivity for a given protected areas network and an indicator that can be used to track changes in connectivity in the future.

ecology↗

bioRxiv2022

Integrating carbon stocks and wildlife connectivity for nature-based climate solutions

Actions to protect against biodiversity loss and climate change will require a framework that addresses synergies between these interrelated issues. In this study we present methods for identifying areas important for the implementation of nature-based climate solutions and biodiversity conservation by intersecting high resolution spatial data for carbon storage and terrestrial connectivity. We explored the spatial congruence of carbon and connectivity in Ontario, Canada and examined effectiveness of current protected areas coverage. We found a weak positive relationship between carbon stocks and terrestrial connectivity; however, our maps revealed large hotspots, with high values of both indices, throughout the boreal forest and northern peatlands and smaller, isolated hotspots in the settled landscapes of the south. Location of hotspots varied depending on whether we considered forest or soil carbon. Further, our results show that current protected and conserved areas in Ontario only cover 13% of landscapes with the highest values for both carbon storage and connectivity. Protection or restoration of areas that maximize the co-benefits of carbon storage and connectivity would make significant contributions towards ambitious national targets to reduce greenhouse gas emissions and conserve biodiversity.

ecology↗

bioRxiv2021

Areas Important for Ecological Connectivity Throughout Canada

Governments around the world have acknowledged the importance of conserving ecological connectivity to help reverse the decline of biodiversity. In this study we employed recent methodological developments in circuit theory to conduct the first pan-Canadian analysis of multi-species connectivity for all terrestrial regions of the country, at a spatial grain sufficient to support local land-management decisions. We developed a movement cost surface with a limited number of thematic categories using the most recently updated land cover data available for the country. We divided the country into 17 tiles and used a wall-to-wall, omnidirectional mode of Circuitscape on each tile in order to assess ecological connectivity throughout entire landscapes as opposed to strictly among protected areas. The resulting raw current density map of Canada revealed heterogenous patterns of current density across the country, strongly influenced by geography, natural barriers, and human development. We included a validation analysis of the output current density map with independent wildlife data from across the country and found that mammal and herpetofauna locations were predicted by areas of high current density. We believe our current density map can be used to identify areas important for connectivity throughout Canada and thereby contribute to efforts to conserve biodiversity.

ecology↗

bioRxiv2021

Genetic and pharmacological causes of germline hypermutation

Mutation in the germline is the source of all evolutionary genetic variation and a cause of genetic disease. Previous studies have shown parental age to be the primary determinant of the number of new germline mutations seen in an individuals genome. Here we analysed the genome-wide sequences of 21,879 families with rare genetic diseases and identified 12 hypermutated individuals with between two and seven times more de novo single nucleotide variants (dnSNVs) than expected. In most of these families (8/12) the excess mutations could be attributed to the father. We determined that two of these families had genetic drivers of germline hypermutation, with the fathers carrying damaging genetic variation in known DNA repair genes, causing distinctive mutational signatures. For five families, by analysing clinical records and mutational signatures, we determined that paternal exposure to chemotherapeutic agents prior to conception was a key driver of hypermutation. Our results suggest that the germline is well protected from mutagenic effects, hypermutation is rare and relatively modest in degree and that most hypermutated individuals will not have a genetic disease.

genomics↗

bioRxiv2021

Design and proof-of-concept for targeted phage-based COVID-19 vaccination strategies with a streamlined cold-free supply chain

Development of effective vaccines against Coronavirus Disease 2019 (COVID-19) is a global imperative. Rapid immunization of the world human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and many different vaccine approaches are being pursued to meet this task. Engineered filamentous bacteriophage (phage) have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the design, development, and initial evaluation of targeted phage-based vaccination approaches against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) by using dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. Towards a unique phage- and AAVP-based dual-display candidate approach, we first performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein for display on the recombinant major capsid coat protein pVIII. Targeted phage particles carrying one of these epitopes induced a strong and specific humoral response. In an initial experimental approach, when these targeted phage particles were further genetically engineered to simultaneously display a ligand peptide (CAKSMGDIVC) on the minor capsid protein pIII, which enables receptor-mediated transport of phage particles from the lung epithelium into the systemic circulation (termed "dual-display"), they enhanced a systemic and specific spike (S) protein-specific antibody response upon aerosolization into the lungs of mice. In a second line of investigation, we engineered targeted AAVP particles to deliver the entire S protein gene under the control of a constitutive cytomegalovirus (CMV) promoter, which induced tissue-specific transgene expression stimulating a systemic S protein-specific antibody response. As proof-of-concept preclinical experiments, we show that targeted phage- and AAVP-based particles serve as robust yet versatile enabling platforms for ligand-directed immunization and promptly yield COVID-19 vaccine prototypes for further translational development. SignificanceThe ongoing COVID-19 global pandemic has accounted for over 2.5 million deaths and an unprecedented impact on the health of mankind worldwide. Over the past several months, while a few COVID-19 vaccines have received Emergency Use Authorization and are currently being administered to the entire human population, the demand for prompt global immunization has created enormous logistical challenges--including but not limited to supply, access, and distribution--that justify and reinforce the research for additional strategic alternatives. Phage are viruses that only infect bacteria and have been safely administered to humans as antibiotics for decades. As experimental proof-of-concept, we demonstrated that aerosol pulmonary vaccination with lung-targeted phage particles that display short epitopes of the S protein on the capsid as well as preclinical vaccination with targeted AAVP particles carrying the S protein gene elicit a systemic and specific immune response against SARS-CoV-2 in immunocompetent mice. Given that targeted phage- and AAVP-based viral particles are sturdy yet simple to genetically engineer, cost-effective for rapid large-scale production in clinical grade, and relatively stable at room temperature, such unique attributes might perhaps become additional tools towards COVID-19 vaccine design and development for immediate and future unmet needs.

microbiology↗

bioRxiv2020

Multiplexed experimental strategies for fragment library screening using SPR biosensors

Surface plasmon resonance biosensor technology (SPR) is ideally suited for fragment-based lead discovery. However, generally suitable experimental procedures or detailed protocols are lacking, especially for structurally or physico-chemically challenging targets or when tool compounds are lacking. Success depends on accounting for the features of both the target and the chemical library, purposely designing screening experiments for identification and validation of hits with desired specificity and mode-of-action, and availability of orthogonal methods capable of confirming fragment hits. By adopting a multiplexed strategy, the range of targets and libraries amenable to an SPR biosensor-based approach for identifying hits is considerably expanded. We here illustrate innovative strategies using five challenging targets and variants thereof. Two libraries of 90 and 1056 fragments were screened using two different flow-based SPR biosensor systems, allowing different experimental approaches. Practical considerations and procedures accounting for the characteristics of the proteins and libraries, and that increase robustness, sensitivity, throughput and versatility are highlighted.

biophysics↗

bioRxiv2020

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments was progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

biophysics↗