Biology subjects

Hunter, D.

Publications and source records attributed to Hunter, D..

4 recordsLinked to original sources

bioRxiv2018

Effects of Pathological Mutations on the Prion-Like Polymerisation of MyD88

A novel concept has emerged whereby the higher-order self-assembly of proteins provides a simple and robust mechanism for signal amplification. This appears to be a universal signalling mechanism within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns need to trigger a strong, binary response within cells. Previously, multiple structural studies have been limited to single domains, expressed and assembled at high protein concentrations. We therefore set out to develop new in vitro strategies to characterise the behaviour of full-length proteins at physiological levels. In this study we focus on the adaptor protein MyD88, which contains two domains with different self-assembly properties: a TIR domain that can polymerise similarly to the TIR domain of Mal, and a Death Domain that has been shown to oligomerise with helical symmetry in the Myddosome complex. To visualize the behaviour of full-length MyD88 without purification steps, we use single-molecule fluorescence coupled to eukaryotic cell-free protein expression. These experiments demonstrate that at low protein concentration, only full-length MyD88 forms prion-like polymers. We also demonstrate that the metastability of MyD88 polymerisation creates the perfect binary response required in innate signalling: the system is silenced at normal concentrations but upstream signalling creates a \"seed\" that triggers polymerisation and amplification of the response. These findings pushed us to re-interpret the role of polymerisation in MyD88-related diseases and we studied the impact of disease-associated point mutations L93P, R196C and L252P/L265P at the molecular level. We discovered that all mutations completely block the ability of MyD88 to polymerise. We also confirm that L252P, a gain-of-function mutation, allows the MyD88 mutant to form extremely stable oligomers, even when expressed at low nanomolar concentrations. Thus, our results are consistent with and greatly add to the findings on the Myddosomes digital all-or-none responses and the behaviour of the oncogenic mutation of MyD88.

molecular biology↗

bioRxiv2017

Effects of mechanical loading on cortical defect repair using a novel mechanobiological model of bone healing

Mechanical loading is an important aspect of post-surgical care. The timing of load application relative to the injury event is thought to differentially regulate repair depending on the stage of healing. Here, we show using a novel mechanobiological model of cortical defect repair that daily loading (5 N peak load, 2 Hz, 60 cycles, 4 consecutive days) during hematoma consolidation and inflammation disrupts the injury site and activates cartilage formation on the periosteal surface adjacent to the defect. We also show that daily loading during the matrix deposition phase enhances both bone and cartilage formation at the defect site, while loading during the remodeling phase results in an enlarged woven bone regenerate. All loading regimens resulted in abundant cellular proliferation within the regenerate and at the periosteal surface and fibrous tissue formation directly above the defect. Stress was concentrated at the edges of the defect during exogenous loading, and finite element (FE)-modeled longitudinal strain ({varepsilon}zz) values along the anterior and posterior borders of the defect (~2200 {varepsilon}) were an order of magnitude larger than strain values on the proximal and distal borders (~50-100 {varepsilon}). These findings demonstrate that all phases of cortical defect healing are sensitive to physical stimulation. In addition, the proposed novel mechanobiological model offers several advantages including its technical simplicity and its well-characterized and spatially confined repair program, making effects of physical and biological interventions more easily assessed.

bioengineering↗

bioRxiv2017

Biocuration as an undergraduate training experience: Improving the annotation of the insect vector of Citrus greening disease

The Asian citrus psyllid (Diaphorina citri Kuwayama) is the insect vector of the bacterium Candidatus Liberibacter asiaticus (CLas), the pathogen associated with citrus Huanglongbing (HLB, citrus greening). HLB threatens citrus production worldwide. Suppression or reduction of the insect vector using chemical insecticides has been the primary method to inhibit the spread of citrus greening disease. Accurate structural and functional annotation of the Asian citrus psyllid genome, as well as a clear understanding of the interactions between the insect and CLas, are required for development of new molecular-based HLB control methods. A draft assembly of the D. citri genome has been generated and annotated with automated pipelines. However, knowledge transfer from well-curated reference genomes such as that of Drosophila melanogaster to newly sequenced ones is challenging due to the complexity and diversity of insect genomes. To identify and improve gene models as potential targets for pest control, we manually curated several gene families with a focus on genes that have key functional roles in D. citri biology and CLas interactions. This community effort produced 530 manually curated gene models across developmental, physiological, RNAi regulatory, and immunity-related pathways. As previously shown in the pea aphid, RNAi machinery genes putatively involved in the microRNA pathway have been specifically duplicated. A comprehensive transcriptome enabled us to identify a number of gene families that are either missing or misassembled in the draft genome. In order to develop biocuration as a training experience, we included undergraduate and graduate students from multiple institutions, as well as experienced annotators from the insect genomics research community. The resulting gene set (OGS v1.0) combines both automatically predicted and manually curated gene models. All data are available on https://citrusgreening.org/.

genomics↗

bioRxiv2016

A comprehensive survey of genetic variation in 20,691 subjects from four large cohorts

The Nurses Health Study (NHS), Nurses Health Study II (NHSII), Health Professionals Follow Up Study (HPFS) and the Physicians Health Study (PHS) have collected detailed longitudinal data on multiple exposures and traits for approximately 310,000 study participants over the last 35 years. Over 160,000 study participants across the cohorts have donated a DNA sample and to date, 20,691 subjects have been genotyped as part of genome-wide association studies (GWAS) of twelve primary outcomes. However, these studies utilized six different GWAS arrays making it difficult to conduct analyses of secondary phenotypes or share controls across studies. To allow for secondary analyses of these data, we have created three new datasets merged by platform family and performed imputation using a common reference panel, the 1,000 Genomes Phase I release. Here, we describe the methodology behind the data merging and imputation and present imputation quality statistics and association results from two GWAS of secondary phenotypes (body mass index (BMI) and venous thromboembolism (VTE)).\n\nWe observed the strongest BMI association for the FTO SNP rs55872725 ({beta}=0.45, p=3.48x10-22), and using a significance level of p=0.05, we replicated 19 out of 32 known BMI SNPs. For VTE, we observed the strongest association for the rs2040445 SNP (OR=2.17, 95% CI: 1.79-2.63, p=2.70x10-15), located downstream of F5 and also observed significant associations for the known ABO and F11 regions. This pooled resource can be used to maximize power in GWAS of phenotypes collected across the cohorts and for studying gene-environment interactions as well as rare phenotypes and genotypes.

epidemiology↗