Biology subjects

Hawkins, N.

Publications and source records attributed to Hawkins, N..

6 recordsLinked to original sources

bioRxiv2026

A Direct DNA Extraction Workflow for Metabarcoding Fungal Bioaerosols from Adhesive Samplers

Monitoring fungal bioaerosols is essential for understanding their impacts on human health and crop productivity. However, current monitoring approaches rely on culture-dependent techniques, which underestimate the diversity of fungal exposures. Adhesive-coated films can effectively capture airborne particles but have been regarded as unsuitable for DNA downstream analysis, owing to the lack of standardised DNA extraction methods for the adhesive capture surface. Here, we present an optimised DNA extraction protocol for adhesive film, which we developed and validated to enable direct and culture-independent ITS2-based metabarcoding of fungal bioaerosols collected on a 96 well plate sealing film ( Sticky sampler). This validated approach was applied to field samples collected from four agricultural farms and from a domestic garden to evaluate seasonal and spatial changes in fungal bioaerosols. Our analysis uncovered pronounced site-specific contrasts linked to geography and land management, as well as clear seasonal shifts in garden fungal communities. This study demonstrates that adhesive samplers, combined with optimised DNA extraction, provide a practical and inexpensive tool for fungal bioaerosol research by resolving ecologically meaningful spatiotemporal dynamics of airborne fungi. This protocol opens new opportunities for highly scalable surveillance of environmental bioaerosols.

microbiology↗

bioRxiv2024

Novel Mouse Model of Alternating Hemiplegia of Childhood Exhibits Prominent Motor and Seizure Phenotypes

Pathogenic variants in ATP1A3 encoding the neuronal Na/K-ATPase cause a spectrum of neurodevelopmental disorders including alternating hemiplegia of childhood (AHC). Three recurrent ATP1A3 variants are associated with approximately half of known AHC cases and mouse models of two of these variants (p.D801N, p.E815K) replicated key features of the human disorder, which include paroxysmal hemiplegia, dystonia and seizures. Epilepsy occurs in 40-50% of individuals affected with AHC, but detailed investigations of seizure phenotypes were limited in the previously reported mouse models. Using gene editing, we generated a novel AHC mouse expressing the third most recurrent ATP1A3 variant (p.G947R) to model neurological phenotypes of the disorder. Heterozygous Atp1a3-G947R (Atp1a3G947R) mice on a pure C57BL/6J background were born at a significantly lower frequency than wildtype (WT) littermates, but in vitro fertilization or outcrossing to a different strain (C3HeB/FeJ) generated offspring at near-Mendelian genotype ratios, suggesting a defect in reproductive fitness rather than embryonic lethality. Heterozygous mutant mice were noticeably smaller and exhibited premature lethality, hyperactivity, anxiety-like behaviors, severe motor dysfunction including low grip strength, impaired coordination with abnormal gait and balance, and cooling-induced hemiplegia and dystonia. We also observed a prominent seizure phenotype with lower thresholds to chemically (flurothyl, kainic acid) and electrically induced seizures, post-handling seizures, sudden death following seizures, and abnormal EEG activity. Together, our findings support face validity of a novel AHC mouse model with quantifiable traits including co-morbid epilepsy that will be useful as an in vivo platform for investigating pathophysiology and testing new therapeutic strategies for this rare neurodevelopmental disorder.

neuroscience↗

bioRxiv2021

Metabolic flux partitioning between the TCA cycle and glyoxylate shunt combined with a reversible methyl citrate cycle provide nutritional flexibility for Mycobacterium tuberculosis

The utilisation of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection thereby identifying the Mtb specific metabolic pathways and enzymes required for carbon co-metabolism as potential drug targets. Metabolic flux represents the final integrative outcome of many different levels of cellular regulation that contribute to the flow of metabolites through the metabolic network. It is therefore critical that we have an in-depth understanding of the rewiring of metabolic fluxes in different conditions. Here, we employed 13C-metabolic flux analysis using stable isotope tracers (13C and 2H) and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly on physiologically relevant carbon sources in a steady state chemostat. We demonstrate that Mtb is able to efficiently co-metabolise combinations of either cholesterol or glycerol along with C2 generating carbon substrates. The uniform assimilation of the carbon sources by Mtb throughout the network indicated no compartmentalization of metabolism in these conditions however there were substrate specific differences in metabolic fluxes. This work identified that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle as the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide new insights into the metabolic architecture that affords adaptability of Mtb to divergent carbon substrates. ImportanceEach year more than 1 million people die of tuberculosis (TB). Many more are infected but successfully diagnosed and treated with antibiotics, however antibiotic-resistant TB isolates are becoming ever more prevalent and so novel therapies are urgently needed that can effectively kill the causative agent. Mtb specific metabolic pathways have been identified as an important drug target in TB. However the apparent metabolic plasticity of this pathogen presents a major obstacle to efficient targeting of Mtb specific vulnerabilities and therefore it is critical to define the metabolic fluxes that Mtb utilises in different conditions. Here, we used 13C-metabolic flux analysis to measure the metabolic fluxes that Mtb uses whilst growing on potential in vivo nutrients. Our analysis identified selective use of the metabolic network that included the TCA cycle, glyoxylate shunt and methyl citrate cycle. The metabolic flux phenotypes determined in this study improves our understanding about the co-metabolism of multiple carbon substrates by Mtb identifying a reversible methyl citrate cycle and the glyoxylate shunt as the critical metabolic nodes which underlie the nutritional flexibility of Mtb.

systems biology↗

bioRxiv2020

Enhanced synaptic transmission in the extended amygdala and altered excitability in an extended amygdala to brainstem circuit in a Dravet syndrome mouse model

ObjectiveDravet syndrome (DS) is a severe, early-onset epilepsy with an increased incidence of sudden death. Evidence of interictal breathing deficits in DS suggest that alterations in subcortical projections to brainstem nuclei may exist, which might be driving comorbidities in DS. The aim of this study was to determine if a subcortical structure, the bed nucleus of the stria terminalis (BNST) in the extended amygdala, is activated by seizures, exhibits changes in excitability, and expresses any alterations in neurons projecting to a brainstem nucleus associated with respiration, stress response and homeostasis. MethodsExperiments were conducted using F1 mice generated by breeding 129.Scn1a+/- mice with wildtype C57BL/6J mice. Immunohistochemistry was performed to quantify neuronal c-fos activation in DS mice after observed spontaneous seizures. Whole cell patch clamp and current clamp electrophysiology recordings were conducted to evaluate changes in intrinsic and synaptic excitability in the BNST. ResultsSpontaneous seizures in DS mice significantly enhanced neuronal c-fos expression in the BNST. Further, the BNST had altered AMPA/NMDA postsynaptic receptor composition and showed changes in spontaneous neurotransmission, with greater excitation and decreased inhibition. BNST to parabrachial nucleus (PBN) projection neurons exhibited intrinsic excitability in wildtype mice, while these projection neurons were hypoexcitable in DS mice. SignificanceThe findings suggest that there is altered excitability in neurons of the BNST, including BNST to PBN projection neurons, in DS mice. These alterations could potentially be driving comorbid aspects of DS outside of seizures, including respiratory dysfunction and sudden death. SIGNIFICANCE STATEMENTDravet syndrome (DS) is an early-onset epilepsy with an increased risk of sudden death. We determined that there are alterations in a subcortical nucleus, the bed nucleus of the stria terminalis (BNST) of the extended amygdala, in a murine DS model. The BNST is involved in stress, anxiety, feeding, and respiratory function. We found enhanced activation in the BNST after seizures and alterations in basal synaptic neurotransmission-with enhanced spontaneous excitatory and decreased spontaneous inhibitory postsynaptic events. Evaluating those neurons that project to the parabrachial nucleus (PBN), a nucleus with multiple homeostatic roles, we found them to be hypoexcitable in DS. Alterations in BNST to brainstem projections could be implicated in comorbid aspects of DS, including respiratory dysfunction and sudden death.

neuroscience↗

bioRxiv2020

Characterisation of factors contributing to the performance of nonwoven fibrous matrices as substrates for adenovirus vectored vaccine stabilisation

The global network of fridges and freezers known as the "cold chain" can account for a significant proportion of the total cost of vaccination and is susceptible to failure. Cost-efficient techniques to enhance stability of vaccines could prevent such losses and improve vaccination coverage, particularly in low income countries. We have previously reported a novel, potentially less expensive thermostabilisation approach using a combination of simple sugars and glass micro-fibrous matrix, achieving an excellent recovery of vaccines after storage at supraphysiological temperatures. This matrix is, however, prone to fragmentation and currently not suitable for clinical translation. Here, we report an investigation of alternative, potentially GMP compatible, fibrous matrices. A number of commercially-available matrices permitted good protein recovery, quality of sugar glass and moisture content of the dried product but did not achieve the thermostabilisation performance of the original glass fibre matrix. We therefore further investigated physical and chemical characteristics of the glass fibre matrix and its components. Our investigation shows that the polyvinyl alcohol present in the glass fibre matrix assists vaccine stability. This finding enabled us to develop a custom-produced matrix with encouraging performance, as an initial step towards a biocompatible matrix for clinical translation. We discuss the path to transfer of the technology into clinical use, including potential obstacles.

pharmacology and toxicology↗

bioRxiv2018

In search of non-coding driver mutations by deep sequencing of regulatory elements in colorectal cancer

Large-scale whole cancer-genome sequencing projects have led to the identification of a handful of cis-regulatory driver mutations in cancer genomes. However, recent studies have demonstrated that very large cancer cohorts will be required in order to identify low frequency non-coding drivers. To further this endeavour, in this study, we performed highdepth sequencing across 95 colorectal cancers and matched normal samples using a unique target capture sequencing (TCS) assay focusing on over 35 megabases of gene regulatory elements. We first assessed coverage and variant detection capability from our TCS data, and compared this with a sample that was additionally whole-genome sequenced (WGS). TCS enabled substantially deeper sequencing and thus we detected 51% more somatic single nucleotide variants (n = 2,457) and 144% more somatic insertions and deletions (n = 39) by TCS than WGS. Variants obtained from TCS data were suitable for somatic mutational signature detection, enabling us to define the signatures associated with germline deleterious variants in MSH6 and MUTYH in samples within our cohort. Finally, we surveyed regulatory mutations to find putative drivers by assessing variant recurrence and function, identifying some regulatory variants that may influence oncogenesis. Our study demonstrates TCS to be a sequencing-efficient alternative to traditional WGS, enabling improved coverage and variant detection when seeking to identify variants at specific loci among larger cohorts. Interestingly, we found no candidate variants that have a clear driver function, suggesting that regulatory drivers may be rare in a colorectal cancer cohort of this size.\n\nAuthor SummaryIn recent years, some cancer research focus has turned towards the role of somatic mutations in the 98% of the genome that is non-coding. To investigate such mutations, we performed deep sequencing of regulatory regions and a selection of coding genes across 95 colorectal cancer and matched-normal samples. To determine the ability of our targeted deep sequencing methodology to accurately detect variants, we compared our results with those from a sample that was additionally whole-genome sequenced. We found target capture sequencing to enable greater sequencing depth, allowing the detection of 51% and 144% more somatic single nucleotide and insertion/deletion mutations, respectively. Our study here demonstrates target capture sequencing to be a useful approach for researchers seeking to identify variants at specific loci among larger cohorts. Our results also enabled the generation of mutational signatures, implicating deleterious germline single nucleotide variants in coding exons of MSH6 and MUTYH in samples within our cohort. Finally, we surveyed regulatory elements in search of somatic cancer driver mutations. We identified some regulatory variants that may influence oncogenesis, but found no candidate variants with clear driver function. These findings suggest that regulatory driver mutations may be rare in a colorectal cancer cohort of this size.

biochemistry↗