Biology subjects

Fletcher, N.

Publications and source records attributed to Fletcher, N..

8 recordsLinked to original sources

bioRxiv2026

Physics-aware measurement-supervised deep learning enables point spread function inversion in soft X-ray tomography

Soft X-ray tomography (SXT) is an emerging modality for whole-cell 3D imaging in near-native states. However, the effective spatial resolution is limited by optical artifacts characterized by the point spread function (PSF). Standard reconstruction methods force a compromise between structural sharpness and noise, failing to fully resolve these depth-dependent artifacts. By embedding experimentally measured, depth-variant PSFs into a differentiable forward model, we demonstrate a physics-aware computational optimization that bypasses these limitations to recover high-frequency cellular ultrastructure. The structural fidelity was validated using split-tilt Fourier ring correlation (FRC), alongside an experimental bead phantom tomogram, providing supporting evidence that the recovered high-frequency features reflect genuine specimen structure rather than fabricated artifacts. Our method effectively increases FRC spatial resolution and recovers cellular ultrastructure. Furthermore, under sparse-angular subsampling, the framework maintained spatial resolution using half the projection angles, a computational proxy pointing toward the potential for reduced radiation exposure in future acquisitions. This hardware-free, computational approach offers a route toward mitigating the optical and dosimetric constraints that currently limit nanoscale soft X-ray tomography.

Cell Biology↗

bioRxiv2024

The molecular arsenal of the key coastal bioturbator Hediste diversicolor faced with changing oceans

The importance of infaunal bioturbators for the functioning of marine ecosystems cannot be overstated. Inhabitants of estuarine and coastal habitats are expected to show resilience to fluctuations in seawater temperature and pH, which adds complexity to our understanding of the effects of global change drivers. Further, stress responses may be propagated through chemical cues within and across species, which may amplify the costs of life and alter species interactions. Research into the molecular mechanisms underlying this resilience has been limited by a lack of annotated genomes and associated molecular tools. In this study, we present the first chromosome-level, annotated draft genome of the marine ragworm Hediste diversicolor, specifically mapping genes important for chemical communication, sensing and pH homeostasis. Using these resources, we then evaluate the transcriptomic and behavioural responses of two distinct populations -- one field-sampled from Portugal (Ria Formosa) and one laboratory-acclimated and -bred from the United Kingdom (Humber) -- to changes in seawater pH, temperature, and odour cues from a low pH-stressed predator. Both populations displayed adaptive responses to future oceanic conditions, with targeted acid-base regulation in the Ria Formosa population experiment, and broader changes in metabolism and growth genes in the Humber population experiment. Chemical cues from stressed fish predators induced genes related to Schreckstoff biosynthesis in ragworms. Additionally, under future ocean conditions including increased temperature, the Humber population exhibited signs of cellular stress and damage. Our findings using the new annotated genome offer novel insights into the molecular arsenal of acid-base regulation which aids in predicting the impacts of an increasingly acidified and unstable ocean, and to transfer this knowledge to investigate these mechanisms in species with less tolerance.

genomics↗

bioRxiv2024

Deciphering transcriptional programming during lupin (Lupinus angustifolius) seed development using RNA-seq

Lupin (Lupinus spp.) seeds are valued for their high protein content (35-40%) for both human and animal consumption. Seed development in crop plants is a critical factor influencing both seed fate and yield, hence, understanding the molecular mechanisms of seed development is essential. This study conducted a transcriptome analysis of Narrow Leaf Lupin (NLL) during seed development stages (3, 6, 9, 12, 15, 18, and 21 days after flowering) to investigate transcriptional dynamics and identify key candidate genes that control seed development. Approximately 357 million sequencing reads were generated from nine samples from leave, flower and seed tissues, enabling the identification of 34,769 expressed genes. The analysis revealed dynamic gene expression, with early stages marked by high metabolic activity and later stages focusing on storage protein synthesis and nutrient reservoir formation. The differential expression patterns of seed storage protein genes, including cupin groups (, {beta}, {gamma}, and {delta} conglutins), were notable. The expression of and {beta} conglutins increased at later stages (15-21 days after flowering), supporting their role in grain filling and nutrient storage. Genes related to quinolizidine alkaloid biosynthesis, such as lysine/ornithine decarboxylase and purine permease transporter 1, showed late expression patterns suggesting alkaloid synthesis and transport during later stages. Many of the well-established transcription factors (TFs) known for their roles in seed development (bHLH, AP2, MYB, ERF, C2H2, NAC, WRKY, and C3H zinc finger families) showed differential expression, thus reinforcing the validity of our findings. These findings lay the groundwork for understanding the genetic and molecular mechanisms of seed development in lupin, contributing to enhanced crop management and breeding programs.

plant biology↗

bioRxiv2024

A modular encapsulation system for precision delivery of proteins, nucleic acids and therapeutics

Targeted nanoparticles have the potential to revolutionize therapeutics for medical applications. Here, we demonstrate the utility of a flexible precision nanovesicle delivery system for functional delivery of DNA, RNA, proteins and drugs into target cells. Nanovesicles generated by the membrane sculpting protein caveolin, termed caveospheres, can be loaded with RNA, DNA, proteins or drugs post-synthesis or incorporate genetically-encoded cargo proteins during production without the need for protein purification. Functionalized fluorescently-labeled caveospheres form a modular system that shows high stability in biological fluids, specific uptake by target-positive cells, and can deliver proteins, drugs, DNA, and mRNA directly to the cytoplasm and nuclei of only the target cells. The negligible level of off-target transduction and uniform levels of targeted expression demonstrates advantages of the system over lipid-mediated gene delivery. Caveospheres can also be engineered to mimic viral particles by displaying the SARS-CoV-2-RBD protein, enabling targeted delivery to human bronchial epithelial cells. We demonstrate their application as a targeted transfection system for cells in culture, and critically, their efficacy in precision tumor killing in vivo.

bioengineering↗

bioRxiv2024

Limited genomic signatures of population collapse in the critically endangered black abalone (Haliotis cracherodii)

The black abalone, Haliotis cracherodii, is a large, long-lived marine mollusc that inhabits rocky intertidal habitats along the coast of California and Mexico. In 1985, populations were impacted by a bacterial disease known as withering syndrome (WS) that wiped out >90% of individuals, leading to the species designation as critically endangered. Current conservation strategies include restoring diminished populations by translocating healthy individuals. However, population collapse on this scale may have dramatically lowered genetic diversity and strengthened geographic differentiation, making translocation-based recovery contentious. Additionally, the current prevalence of WS is unknown. To address these uncertainties, we sequenced and analyzed the genomes of 133 black abalone individuals from across their present range. We observed no spatial genetic structure among black abalone, with the exception of a single chromosomal inversion that increases in frequency with latitude. Genetic divergence between sites is minimal, and does not scale with either geographic distance or environmental dissimilarity. Genetic diversity appears uniformly high across the range. Despite this, however, demographic inference confirms a severe population bottleneck beginning around the time of WS onset, highlighting the temporal offset that may occur between a population collapse and its potential impact on genetic diversity. Finally, we find the bacterial agent of WS is equally present across the sampled range, but only in 10% of individuals. The lack of genetic structure, uniform diversity, and prevalence of WS bacteria indicates that translocation could be a valid and low-risk means of population restoration for black abalone species recovery.

evolutionary biology↗

bioRxiv2023

SARS-CoV-2 infects neurons, astrocytes, choroid plexus epithelial cells and pericytes of the human central nervous system

SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, is associated with a range of neurological manifestations including haemorrhage, thrombosis and ischaemic necrosis and encephalitits. However, the mechanism by which this occurs is unclear. Neurological disease associated with SARS-CoV-2 infection has been proposed to occur following direct infection of the central nervous system and/or indirect sequelae as a result of peripheral inflammation. We profiled ACE2 and TMPRSS2 in brain tissue from five healthy human donors, and observed expression of these proteins in astrocytes, neurons and choroid plexus epithelium within frontal cortex and medulla. Primary human astrocytes, neurons and choroid plexus epithelial cells supported productive SARS-CoV-2 infection in an ACE2- dependent manner. Infected cells supported the full viral lifecycle, releasing infectious virus particles. In contrast, primary brain microvascular endothelial cells, pericytes and microglia were refractory to SARS-CoV-2 infection. These data support a model whereby SARS-CoV-2 is neurotropic, and this may in part explain the neurological sequelae of infection. ImportanceA subset of patients with COVID-19 develop neurological symptoms, but the underlying cause is poorly understood. We observed that cells within normal human brain express the SARS-CoV-2 entry factors ACE-2 and TMPRRS2, with expression mainly observed within astrocytes, neurons and choroid plexus epithelium. Primary human astrocytes, neurons and choroid plexus epithelial cells cultured in vitro supported the full SARS-CoV-2 life cycle with a range of SARS-CoV-2 variants. This study demonstrates that cells of the human central nervous system express SARS-CoV-2 entry factors in vivo and support viral infection in vitro, thus supporting a model where neurological symptoms seen in some COVID-19 patients may be as a result of direct viral infection of the central nervous system. Furthermore, these data highlight the importance of investigating the ability of therapeutics to clear virus from this potential reservoir of infection.

microbiology↗

bioRxiv2023

First Eurasian cases of SARS-CoV-2 seropositivity in a free-ranging urban population of wild fallow deer

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects wildlife. Recent studies highlighted that variants of concern (VOC) may expand into novel animal reservoirs with the potential for reverse zoonosis. North American white-tailed deer are the only deer species in which SARS-CoV-2 has been documented, raising the question whether further reservoir species exist as new VOC emerge. Here, we report the first cases of deer SARS-CoV-2 seropositivity in Eurasia, in a city population of fallow deer in Dublin, Ireland. Deer were seronegative in 2020 (circulating variant in humans: Alpha), one animal was seropositive in 2021 (Delta variant), and 57% of animals tested in 2022 were seropositive (Omicron variant). Ex vivo, a clinical isolate of Omicron BA.1 infected fallow deer precision cut lung slice type-2 pneumocytes, also a major target of infection in human lungs. Our findings suggest a change in host tropism as new variants emerged in the human reservoir, highlighting the importance of continued wildlife disease monitoring and limiting human-wildlife contacts. Teaser: This study is the first report of SARS-CoV-2 seropositivity in fallow deer, highlighting expansion of viral variants into new host reservoirs.

microbiology↗

bioRxiv2019

Saxitoxin and tetrodotoxin bioavailability increases in future oceans

Increasing atmospheric levels of carbon dioxide are largely absorbed by the worlds oceans, decreasing surface water pH1. In combination with increasing ocean temperatures, these changes have been identified as a major sustainability threat to future marine life2. Interactions between marine organisms are known to depend on biomolecules, but the influence of oceanic pH on their bioavailability and functionality remains unexplored. Here we show that global change significantly impacts two ecological keystone molecules3 in the ocean, the paralytic toxins saxitoxin (STX) and tetrodotoxin (TTX). Increasing temperatures and declining pH increase the abundance of the toxic forms of these two neurotoxins in the water. Our geospatial global model highlights where this increased toxicity could intensify the devastating impact of harmful algal blooms on ecosystems in the future, for example through an increased incidence of paralytic shellfish poisoning (PSP). We also use these results to calculate future saxitoxin toxicity levels in Alaskan clams, Saxidomus gigantea, showing critical exceedance of limits save for consumption. Our findings for TTX and STX exemplarily highlight potential consequences of changing pH and temperature on chemicals dissolved in the sea. This reveals major implications not only for ecotoxicology, but also for chemical signals mediating species interactions such as foraging, reproduction, or predation in the ocean with unexplored consequences for ecosystem stability and ecosystem services.

pharmacology and toxicology↗