Biology subjects

McGowan, J.

Publications and source records attributed to McGowan, J..

15 recordsLinked to original sources

bioRxiv2026

Long-read single-cell genomics: resolving chimeras in multiple displacement amplification

Multiple displacement amplification (MDA) enables whole-genome amplification from single cells, but introduces chimeric artifacts that severely compromise downstream analyses, particularly with long-read sequencing. Here, we systematically evaluate long-read PacBio HiFi sequencing of MDA amplified DNA from single cells using the model green alga Chlamydomonas reinhardtii. We show that MDA-derived libraries exhibit highly uneven coverage and extreme chimera rates impacting up to 70% of reads, leading to thousands of artefactual structural variants and misassemblies when assembled using algorithms designed for bulk sequencing. To overcome these challenges, we developed lrSAGA (long-read Single Amplified Genome Assembly), a novel tool to assemble long-read MDA sequencing datasets. Assemblies generated using lrSAGA are more complete, more contiguous, and have 75-95% fewer misassemblies compared to conventional assembly algorithms. Although overall contiguity is limited by MDA coverage dropouts, we demonstrate that up to 68% of the C. reinhardtii genome can be accurately assembled from just a single haploid cell. We further validated lrSAGA using published Oxford Nanopore and PacBio HiFi data from single or half Caenorhabditis elegans worms, generating accurate and highly complete assemblies. Applying our approach to single protist cells isolated from environmental water samples, we performed PacBio HiFi single-cell genome sequencing of four uncultivated microbial eukaryotes: an amoeboflagellate from the Naegleria genus, a flagellate from the Bodo genus, and two deep-branching flagellates from the enigmatic CRuMs supergroup, Collodictyon triciliatum and Diphylleia rotans. From single cells, we generated high-quality draft genome assemblies estimated to be 70-84% complete, demonstrating the potential of long-read single-cell genomics to unlock genome diversity from uncultivated microbial eukaryotes.

genomics↗

bioRxiv2026

Innate immune sensing of cell traversal by Plasmodium sporozoites drives protective T cell responses

Attenuated Plasmodium sporozoites elicit robust adaptive immune responses that protect against parasite challenge, yet how they engage the innate immune system remains poorly understood. To obtain sterile preparation of sporozoites we used flow cytometry to isolate pure GFP+ P. berghei and P. falciparum sporozoites, which were sorted onto murine bone marrow-derived macrophages (BMDMs) or human PBMCs to assess innate immune activation. Sporozoites induced a distinctive activation pattern resembling cell wounding responses, consistent with their ability to traverse host cells en route to the liver. Traversal-deficient sporozoites failed to activate BMDMs and showed impaired priming of protective CD8+ T cell immunity. This wounding response was independent of inflammasome or Toll-like receptor signaling but required {gamma}{delta} T cells, which are known to support CD8+ T cell responses against Plasmodium. These findings reveal a previously unappreciated innate sensing mechanism triggered by cell traversal that underpins the potent immunogenicity of Plasmodium sporozoites.

immunology↗

bioRxiv2025

De novo design of protein nanoparticles with integrated functional motifs

Computational design of self-assembling proteins has long relied on pre-existing structures and sequences, fundamentally limiting control over their structural and functional properties. Recent machine learning-based methods have transformed our ability to design functional small de novo proteins and oligomers, yet methods to design large de novo protein assemblies with structures tailored to specific applications are still underexplored. Here, we develop a generalizable method for designing de novo symmetric protein complexes that incorporate target functional motifs into their structures. We report 34 new protein nanoparticles that form on-target assemblies with cubic point group symmetries. The nanoparticles exhibit a wide variety of backbones that were designed with atom-level accuracy, as evidenced by several cryo-EM and crystal structures that reveal minimal deviations from the design models. We use the method to generate a de novo antigen-tailored nanoparticle vaccine that elicits robust immune responses in mice. These results establish a generalizable approach that can be used to design functional self-assembling protein complexes with structures tailored to specific applications.

biochemistry↗

bioRxiv2025

Single-cell sequencing reveals unexpected genetic diversity among Bodo spp. flagellates and their bacterial endosymbionts

Bodo is a cosmopolitan genus of free living bacterivorous single-celled flagellates in the class Kinetoplastea. Members of genus Bodo are considered the closest free-living relatives to the parasitic lineages Trypanosoma and Leishmania, the causative agents of the human diseases sleeping sickness, Chagas disease, and leishmaniasis. Currently, a single genome exists for the one formally described species in the genus, Bodo saltans. Previous studies on B. saltans have shown that it is dependent on an endosymbiotic bacterium from the order Holosporales, "Candidatus Bodocaedibacter vickermanii". Using single cell-sequencing, we isolated, sequenced, and assembled genomes for seven uncultured Bodo spp. cells from a single freshwater sample from Royal Leamington Spa, UK. By using comparative genomics, we show that these seven cells represent three potentially novel Bodo species and exhibit unexpected levels of diversity at the genome level. Our results indicate that Small Subunit (SSU) rDNA sequencing, often used to classify Bodo flagellates, is insufficient for determining species delimitation in this genus. In addition, we recovered a Holosporales bacterium genome from all seven Bodo spp. cells. Surprisingly, these seven bacterial endosymbionts also represent three potentially novel species and one novel genus of Holosporales bacteria. This diversity would be indistinguishable in routinely-used SSU ribosomal DNA (rDNA) metabarcoding or bulk sequencing pipelines, thus demonstrating the utility of using single-cell sequencing to reveal the level of genomic diversity within lineages of microbial eukaryotes and their cobionts.

microbiology↗

bioRxiv2025

From sequence to scaffold: computational design of protein nanoparticle vaccines from AlphaFold2-predicted building blocks

Self-assembling protein nanoparticles are being increasingly utilized in the design of next-generation vaccines due to their ability to induce antibody responses of superior magnitude, breadth, and durability. Computational protein design offers a route to novel nanoparticle scaffolds with structural and biochemical features tailored to specific vaccine applications. Although strategies for designing new self-assembling proteins have been established, the recent development of powerful machine learning-based tools for protein structure prediction and design provides an opportunity to overcome several of their limitations. Here, we leveraged these tools to develop a generalizable method for designing novel self-assembling proteins starting from AlphaFold2 predictions of oligomeric protein building blocks. We used the method to generate six new 60-subunit protein nanoparticles with icosahedral symmetry, and single-particle cryo-electron microscopy reconstructions of three of them revealed that they were designed with atomic-level accuracy. To transform one of these nanoparticles into a functional immunogen, we reoriented its termini through circular permutation, added a genetically encoded oligomannose-type glycan, and displayed a stabilized trimeric variant of the influenza hemagglutinin receptor binding domain through a rigid de novo linker. The resultant immunogen elicited potent receptor-blocking and neutralizing antibody responses in mice. Our results demonstrate the practical utility of machine learning-based protein modeling tools in the design of nanoparticle vaccines. More broadly, by eliminating the requirement for experimentally determined structures of protein building blocks, our method dramatically expands the number of starting points available for designing new self-assembling proteins. Significance StatementSelf-assembling protein nanoparticle vaccines have steadily gained traction in both academic and industry vaccine development over the last decade. Recent work has shown that computationally designing new self-assembling proteins allows the structural and functional features of nanoparticle vaccines to be precisely tailored, and that this can significantly affect the vaccine-elicited immune response. To date, designing such nanoparticle vaccines has required the use of known crystal structures as starting points. Here we show how new machine learning tools can be leveraged to design new self-assembling protein nanoparticle immunogens in the absence of experimentally determined structures of the building blocks that elicit strong immune responses in mice.

biochemistry↗

bioRxiv2025

Stabilization of H5 highly pathogenic avian influenza hemagglutinin improves vaccine-elicited neutralizing antibody responses

Transmission of highly pathogenic avian influenza from H5 clade 2.3.4.4b has expanded in recent years to infect large populations of birds and mammals, heightening the risk of a human pandemic. Influenza viruses adapted to transmission in birds and some other animals tend to have a less stable hemagglutinin (HA) than seasonal influenza viruses, enabling membrane fusion at comparatively high pH levels. Here, we combine five mutations within H5 HA that dramatically increase its melting temperature and promote stable closure of the HA trimer. Structural analysis by cryo-electron microscopy revealed that the stabilizing mutations create several new hydrophobic interactions, while maintaining local HA structure. We found that vaccinating mice with stabilized H5 HA immunogens resulted in higher hemagglutination inhibition and neutralization titers than non-stabilized comparators. Epitope mapping of vaccine-elicited polyclonal antibody responses using negative stain electron microscopy and deep mutational scanning showed that site E on the side of the HA receptor binding domain was immunodominant across all groups; however, the stabilized immunogens shifted responses toward the receptor binding site (RBS), eliciting a higher proportion of neutralizing antibodies. These findings highlight that H5 HA-stabilizing mutations enhance the quality of antibody responses across different vaccine formats, underscoring their potential to improve pandemic preparedness vaccines targeting viruses from this widely circulating clade. One Sentence SummaryVaccinating with stabilized H5 HA increases the proportion of humoral responses against neutralizing RBS epitopes.

immunology↗

bioRxiv2024

An operational framework to map Essential Life Support Areas (ELSAs) for biodiversity, climate, and sustainable development

Almost all countries are making increasingly bold commitments to halt and reverse biodiversity loss, minimise the impacts of climate change, and transition to more sustainable development. The effective achievement of many of these commitments relies on integrated spatial planning frameworks that are adaptable to national circumstances, priorities and capabilities. This need is formally recognized by Target 1 of the Kunming-Montreal Global Biodiversity Framework (GBF), which specifies that all areas should be under such planning. Here, we describe the development and application of an operational framework for national-level integrated spatial planning: Essential Life Support Areas (ELSAs). This framework facilitates the identification of areas that - if protected, restored, or sustainably managed - can support the achievement of national commitments to biodiversity, climate, and sustainable development. The process of mapping ELSAs relies heavily on leadership by national experts and stakeholders and the integration of spatial data using systematic conservation planning tools. We showcase the ELSA process carried out for Ecuador, where the use of real-time scenario analyses enabled diverse stakeholder groups to collaborate to assess national priorities for nature, climate, and sustainable development, view trade-offs and synergies, and arrive at a spatial plan to guide national action. ELSA presented an actionable approach for Ecuador, and 12 other pilot countries, to create a spatial plan aimed at fulfilling their national and international commitments to nature, including to the GBF.

ecology↗

bioRxiv2024

Multiple Independent Genetic Code Reassignments of the UAG Stop Codon in Phyllopharyngean Ciliates

The translation of nucleotide sequences into amino acid sequences, governed by the genetic code, is one of the most conserved features of molecular biology. The standard genetic code, which uses 61 sense codons to encode one of the 20 standard amino acids and 3 stop codons (UAA, UAG, and UGA) to terminate translation, is used by most extant organisms. The protistan phyla Ciliophora (the ciliates) are an unusual exception to this norm, exhibiting the greatest diversity of non-canonical nuclear genetic code variants and evidence of repeated changes in code. In this study, we report the discovery of multiple independent genetic code changes within the Phyllopharyngea class of ciliates. By mining publicly available ciliate genome datasets, we discovered that three ciliate species from the TARA Oceans eukaryotic metagenome dataset use the UAG codon to putatively encode leucine. We identified novel suppressor tRNA genes in two of these genomes. Phylogenomics analysis revealed that these three uncultivated taxa form a monophyletic lineage within the Phyllopharyngea class. Expanding our analysis by reassembling published phyllopharyngean genome datasets led to the discovery that the UAG codon had also been reassigned to putatively code for glutamine in Hartmannula sinica and Trochilia petrani. Phylogenomics analysis suggests that this occurred via two independent genetic code change events. These data demonstrate that the reassigned UAG codons have widespread usage as sense codons within the phyllopharyngean ciliates. Furthermore, we show that the function of UAA is firmly fixed as the preferred stop codon. These findings shed light on the evolvability of the genetic code in understudied microbial eukaryotes.

evolutionary biology↗

bioRxiv2022

Identification of a Non-Canonical Ciliate Nuclear Genetic Code Where UAA and UAG Code for Different Amino Acids

The genetic code is one of the most highly conserved features across life. Only a few lineages have deviated from the "universal" genetic code. Amongst the few variants of the genetic code reported to date, the codons UAA and UAG virtually always have the same translation, suggesting that their evolution is coupled. Here, we report the genome and transcriptome sequencing of a novel ciliate, belonging to the Oligohymenophorea class, where the translation of the UAA and UAG stop codons have changed to specify different amino acids. Genomic and transcriptomic analyses revealed that UAA has been reassigned to encode lysine, while UAG has been reassigned to encode glutamic acid. We identified multiple suppressor tRNA genes with anticodons complementary to the reassigned codons. We show that the retained UGA stop codon is enriched in the 3UTR immediately downstream of the coding region of genes, suggesting that there is functional drive to maintain tandem stop codons. Using a phylogenomics approach, we reconstructed the ciliate phylogeny and mapped genetic code changes, highlighting the remarkable number of independent genetic code changes within the Ciliophora group of protists. According to our knowledge, this is the first report of a genetic code variant where UAA and UAG encode different amino acids.

genomics↗

bioRxiv2021

Targeted, collaborative biodiversity conservation in the global ocean can benefit fisheries economies

Marine protected areas (MPAs) are key to averting continued loss of species and ecosystem services in our oceans, but concerns around economic trade-offs hamper progress. Here we provide optimized planning scenarios for global MPA networks that secure species habitat while minimizing impacts on fisheries revenues. We found that MPA coverage requirements differ vastly among nations, and that two-thirds of nations benefit economically from a collaborative approach. Immediate global protection of marine biodiversity habitat comes with losses of ~19% of total fisheries revenues, but international cooperation in concert with high seas protection improves economic losses for most countries, safeguards all species, and could save ~5B USD annually worldwide. Nations and fishery economies both share benefits from a coordinated approach to conserving marine biodiversity, with direct relevance to current international policies.

ecology↗

bioRxiv2021

An objective-based prioritization approach to improve trophic complexity through ecological restoration

O_LIReassembling ecological communities and rebuilding habitats through active restoration treatments requires curating the selection of plant species to use in seeding and planting mixes. Ideally, these mixes should be assembled based on attributes that support ecosystem function and services, promote plant and animal species interactions and ecological networks in restoration while balancing project constraints. Despite these critical considerations, it is common for species mixes to be selected opportunistically. Reframing the selection of seed mixes for restoration around ecological objectives is essential for success but accessible methods and tools are needed to support this effort. C_LIO_LIWe developed a framework to optimize species seed mixes based on prioritizing plant species attributes to best support different objectives for ecosystem functions, services, and trophic relationships such as pollination, seed dispersal, and herbivory. We compared results to approaches where plant species are selected to represent plant taxonomic richness, dominant species, and at random. We tested our framework for 176 plant species found in European alpine grasslands and identified 163 associated attributes affiliated to trophic relationships, ecosystem functions, and services. C_LIO_LIIn all cases, trophic relationships, ecosystem functions, and services can be captured more efficiently through objective-based prioritization using the functional identity of plant species. Solutions (plant species lists) can be compared quantitatively, in terms of costs, species, or objectives. We confirm that a random draw of plant species from the regional plant species pool cannot be assumed to support other trophic groups and ecosystem functions and services. C_LIO_LISynthesis and Applications. Our framework is presented as a proof of concept to help restoration practitioners better apply quantitative decision-support to plant species selection in order to meet ecological restoration outcomes. Our approach may be tailored to any restoration initiative and habitat where seeding or planting mixes will be applied in active treatments. As global priority and resources are increasingly placed into restoration, this approach could be advanced to help make efficient decisions for many stages of the restoration process. C_LI

ecology↗

bioRxiv2021

Delivering on Biden's 2030 conservation commitment

On January 27, 2021, President Biden signed an executive order, Tackling the Climate Crisis at Home and Abroad, committing the United States to various goals within his campaigns major climate policy, the Biden Plan for a Clean Energy Revolution and Environmental Justice. Included in this executive order is a commitment to "conserving at least 30 percent of [the United States] lands and oceans by 2030." This ambitious conservation target signals a promising direction for biodiversity in the United States. However, while the executive order outlines several goals for climate mitigation, the 30x30 target remains vague in its objectives, actions, and implementation strategies for protecting biodiversity. Biodiversity urgently needs effective conservation action, but it remains unclear where and what this 30% target will be applied to. Achieving different climate and biodiversity objectives will require different strategies and, in combination with the associated costs of implementation, will lead to different priority areas for conservation actions. Here, we illustrate what the 30% target could look like across four objectives reflective of the ambitious goals outlined in the executive order. We compile several variations of terrestrial protected area networks guided by these different objectives and examine the trade-offs in costs, ecosystem representation, and climate mitigation potential between each. We find little congruence in priority areas across objectives, emphasizing just how crucial it will be for the Biden administration to develop clear objectives and establish appropriate performance metrics from the outset to maximize both conservation and climate outcomes in support of the 30x30 target. We discuss important considerations that must guide the administrations conservation strategies in order to ensure meaningful conservation outcomes can be achieved over the next decade.

ecology↗

bioRxiv2020

Areas of global importance for terrestrial biodiversity, carbon, and water

Summary paragraphTo meet the ambitious objectives of biodiversity and climate conventions, countries and the international community require clarity on how these objectives can be operationalized spatially, and multiple targets be pursued concurrently1. To support governments and political conventions, spatial guidance is needed to identify which areas should be managed for conservation to generate the greatest synergies between biodiversity and natures contribution to people (NCP). Here we present results from a joint optimization that maximizes improvements in species conservation status, carbon retention and water provisioning and rank terrestrial conservation priorities globally. We found that, selecting the top-ranked 30% (respectively 50%) of areas would conserve 62.4% (86.8%) of the estimated total carbon stock and 67.8% (90.7%) of all clean water provisioning, in addition to improving the conservation status for 69.7% (83.8%) of all species considered. If priority was given to biodiversity only, managing 30% of optimally located land area for conservation may be sufficient to improve the conservation status of 86.3% of plant and vertebrate species on Earth. Our results provide a global baseline on where land could be managed for conservation. We discuss how such a spatial prioritisation framework can support the implementation of the biodiversity and climate conventions.

ecology↗

bioRxiv2020

Taking a landscape approach to conservation goals: designing multi-objective landscapes

Designing landscapes to accommodate both humans and nature poses huge challenges, but is increasingly recognised as an essential component of conservation and land management. The land-sparing land-sharing framework has been proposed as a tool to address this challenge. However, it has been largely criticised for its simplicity. We provide a new conceptual framework amenable to the application of structured decision-making that moves beyond the dichotomy of land-sparing or land-sharing. Using this new framework, we present a general system model that can be used to make land management decisions for the conservation of species, ecosystem services and production land at different spatial scales. The model can be parameterised for specific systems using information about: the current state of the landscape, the rates of change between landscape states, and the cost and effectiveness of taking actions. To demonstrate the utility of the model we apply it to three different landscape types. Across our three case studies, we show that investment into one of three management actions (varying degrees of management and restoration) can move the system towards more biodiversity or more managed land depending on the objectives of the land manager. We show that the dynamic and flexible nature of the landscape is important to take into account rather than a static snapshot in time. Rather than focusing on establishing the perfect landscape with a set proportion dedicated to production and to biodiversity conservation, we argue that a more useful approach is to establish incremental movements towards a landscape that meets the goals of multiple objectives. Our framework can be used to illustrate to decision makers the costs and trade-offs of different actions and help them determine land management policy.

ecology↗

bioRxiv2018

Operationalizing ecological connectivity in spatial conservation planning with Marxan Connect

O_LIGlobally, protected areas are being established to protect biodiversity and to promote ecosystem resilience. The typical spatial conservation planning process leading to the creation of these protected areas focuses on representation and replication of ecological features, often using decision support systems such as Marxan. Unfortunately, Marxan currently requires manual input or specialised scripts to explicitly consider ecological connectivity, a property critical to metapopulation persistence and resilience.\nC_LIO_LI\"Marxan Connect\" is a new open source, open access Graphical User Interface (GUI) designed to assist conservation planners in the systematic operationalization of ecological connectivity in protected area network planning.\nC_LIO_LIMarxan Connect is able to incorporate estimates of demographic connectivity (e.g. derived from tracking data, dispersal models, or genetics) or structural landscape connectivity (e.g. isolation by resistance). This is accomplished by calculating metapopulation-relevant connectivity metrics (e.g. eigenvector centrality) and treating those as conservation features, or using the connectivity data as a spatial dependency amongst sites to be included in the prioritization process.\nC_LIO_LIMarxan Connect allows a wide group of users to incorporate directional ecological connectivity into conservation plans. The least-cost conservation solutions provided by Marxan Connect, combined with ecologically relevant post-hoc testing, are more likely to support persistent and resilient metapopulations (e.g. fish stocks) and provide better protection for biodiversity than if connectivity is ignored.\nC_LI

ecology↗