John Hildyard

John Hildyard
Royal Veterinary College | RVC · Department of Comparative Biomedical Sciences

About

55
Publications
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Introduction
John Hildyard currently works in the Comparative Neuromuscular Diseases Laboratory at the Royal Veterinary College. The primary focus of his research is the fatal muscle-wasting disease Duchenne muscular dystrophy, though he also retains interests in other neuromuscular diseases, other disease models, and almost anything that captures his attention. Which is almost anything.

Publications

Publications (55)
Article
Full-text available
Background: The dystrophin gene has multiple isoforms: full-length dystrophin (dp427) is principally known for its expression in skeletal and cardiac muscle, but is also expressed in the brain, and several internal promoters give rise to shorter, N-terminally truncated isoforms with wider tissue expression patterns (dp260 in the retina, dp140 in th...
Preprint
Full-text available
Dystrophin plays a vital role in maintaining muscle health, yet low mRNA expression, lengthy transcription time and the limitations of traditional in-situ hybridization (ISH) methodologies mean that the dynamics of dystrophin transcription remain poorly understood. RNAscope is highly sensitive ISH method that can be multiplexed, allowing detection...
Article
Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational “hot spot” in the human DMD gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and ex...
Article
Background : Animal models of Duchenne muscular dystrophy (DMD) are essential to study disease progression and assess efficacy of therapeutic intervention, however dystrophic mice fail to display a clinically relevant phenotype, limiting translational utility. Dystrophin-deficient dogs exhibit disease similar to humans, making them increasingly imp...
Article
Background : Progression through mammalian embryogenesis involves many interacting cell types and multiple differentiating cell lineages. Quantitative polymerase chain reaction (qPCR) analysis of gene expression in the developing embryo is a valuable tool for deciphering these processes, but normalisation to stably-expressed reference genes is esse...
Article
Background: Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by mutations in the dystrophin gene. Due to their phenotypic similarity to human patients, large animal models are invaluable tools for pre-clinical trials. The DE50-MD dog is a relatively new model of DMD, and carries a therapeutically-tractable mutation lying w...
Preprint
Introduction Fibrosis is a key feature of many chronic myopathic disorders, such as in the muscle-wasting condition, Duchenne muscular dystrophy. Fibrosis disrupts skeletal muscle architecture, limits muscle function, impairs regeneration and might reduce efficacy of therapeutic interventions: quantifying muscle fibrosis is thus of key value in mon...
Article
Full-text available
Duchenne muscular dystrophy (DMD), a fatal musculoskeletal disorder, is associated with neurodevelopmental disorders and cognitive impairment caused by brain dystrophin deficiency. Dog models of DMD represent key translational tools to study dystrophin biology and to develop novel therapeutics. However, characterization of dystrophin expression and...
Article
Full-text available
Background: Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by mutations in the dystrophin gene. Due to their phenotypic similarity to human patients, large animal models are invaluable tools for pre-clinical trials. The DE50-MD dog is a relatively new model of DMD, and carries a therapeutically-tractable mutation lying w...
Article
Background : Progression through mammalian embryogenesis involves many interacting cell types and multiple differentiating cell lineages. Quantitative polymerase chain reaction (qPCR) analysis of gene expression in the developing embryo is a valuable tool for deciphering these processes, but normalisation to stably-expressed reference genes is esse...
Preprint
Full-text available
Mammalian embryogenesis is an intricate, tightly orchestrated process. Progression from zygote through somitogenesis and on to organogenesis and maturity involves many interacting cell types and multiple differentiating cell lineages. Quantitative PCR analysis of gene expression in the developing embryo is a valuable tool for deciphering these inte...
Article
Full-text available
Duchenne muscular dystrophy is an X-linked, recessive muscular dystrophy in which the absence of the dystrophin protein leads to fibrosis, inflammation and oxidative stress, resulting in loss of muscle tissue. Drug repurposing, i.e. using drugs already approved for other disorders, is attractive as it decreases development time. Recent studies sugg...
Article
Full-text available
Dystrophin plays a vital role in maintaining muscle health, yet low mRNA expression, lengthy transcription time and the limitations of traditional in-situ hybridization (ISH) methodologies mean that the dynamics of dystrophin transcription remain poorly understood. RNAscope is highly sensitive ISH method that can be multiplexed, allowing detection...
Article
Full-text available
Background: The dystrophin gene has multiple isoforms: full-length dystrophin (dp427) is principally known for its expression in skeletal and cardiac muscle, but is also expressed in the brain, and several internal promoters give rise to shorter, N-terminally truncated isoforms with wider tissue expression patterns (dp260 in the retina, dp140 in th...
Article
Full-text available
The mdx mouse is the most widely-used animal model of the human disease Duchenne muscular dystrophy, and quantitative PCR analysis of gene expression in the muscles of this animal plays a key role in the study of pathogenesis and disease progression and in evaluation of potential therapeutic interventions. Normalization to appropriate stably-expres...
Data
Sample summary, Primer and qPCR validation. (DOCX)
Data
Raw Cq data. (A) Individual Cq values (•) for all 126 samples for each candidate reference gene (as indicated). Genes for which some samples provided no amplicons are indicated, along with number of missing datapoints. (B) Relative expression levels for each candidate reference gene in human tissues as reported by the Illumina bodymap project (expr...
Data
geNorm outputs for all dataset combinations. geNorm ranking by average expression stability M (left to right: least stable to most stable) for the entire dataset, or specific subsets (as indicated). Dashed line: M = 0.5 (threshold of stability for strong candidates). (TIF)
Data
Dataset averages. Arithmetic mean and standard deviations of the Cq values for each candidate gene: of the genes used, FBXW2 shows the greatest sample-to-sample variation, while AP3D1 and CSNK2A2 show the least. Shaded boxes: genes omitted from analysis. (DOCX)
Data
geNorm pairwise variation. Output of the geNorm algorithm for the entire dataset (or subcategory as indicated) showing reduction in pairwise variation with additional reference genes (e.g. V2/3: increasing from 2 -the best pair- to three). Values of 0.2 or lower are considered acceptable, thus three or four reference genes reduce variability but tw...
Data
Correlation matrix of RQ values. Spearman’s Rho values for RQ correlations (all genes). Bold: correlations between high scoring candidates (ACTB, RPL13a, CSNK2A2, AP3D1). Italics: correlations with P values greater than 0.0001 (all other correlations P<0.0001); CDC40 vs GAPDH = 0.0046; 18S vs SDHA = 0.0002; GAPDH vs B2M = 0.0003. (DOCX)
Data
Muscle and disease-specific expression patterns of high-scoring candidates. (A) Raw RQ values (●) for ACTB, RPL13a, CSNK2A2 and AP3D1 for the entire dataset, separated by healthy/dystrophic. (B) Normalized expression data (means + SEM) for ACTB, RPL13a, CSNK2A2 and AP3D1 separated by muscle type and healthy (green) vs dystrophic (red). *:P<0.05, **...
Data
FBXW2 vs BestKeeper. Raw Cq values for FBXW2 plotted against a BestKeeper derived from the entire dataset (upper panel), or against a BestKeeper derived from the dataset after removal of FBXW2 (lower panel). Boxes: Pearson correlation (r) and significance of correlation. (TIF)
Data
Reference gene selection algorithms: Detailed summary. (DOCX)
Data
BestKeeper analysis for all dataset combinations. Coefficient of correlation values for the reference gene candidates are shown for the entire dataset, or specific subsets (as indicated), ranked (left to right) from least stable to most stable. (TIF)
Data
Muscle-specific Normfinder rankings (grouped). Normfinder results for muscle-specific subsets grouped by different criteria (as indicated: top row; datasets, second row; criterion), ranked from highest scoring (lowest stability value) to lowest scoring. Grouped analysis also suggests the best pair of genes for normalization (third row), (not necess...
Data
Normfinder analysis (ungrouped) for all dataset combinations. Stability values (left to right: least stable to most stable) for the reference gene candidates are shown for the entire dataset or specific subsets (as indicated). (TIF)
Data
Age-specific Normfinder rankings (grouped). Normfinder results for age-specific subsets grouped by different criteria (as indicated: top row; datasets, second row; criterion), ranked from highest scoring (lowest stability value) to lowest scoring. Grouped analysis also suggests the best pair of genes for normalization (third row), (not necessarily...
Data
Age and disease-specific Normfinder rankings (grouped). Normfinder results for age-specific subsets separated by healthy/dystrophic and then grouped by different criteria (as indicated: top row; datasets, second row; criterion), ranked from highest scoring (lowest stability value) to lowest scoring. Grouped analysis also suggests the best pair of g...
Data
Normfinder ‘best pair’ combinations for all dataset/grouping combinations. (DOCX)
Data
Correlation matrix of Cq values. Pearson correlations (r) for raw Cq values (all genes). Bold: correlations between high scoring candidates (ACTB, RPL13a, CSNK2A2, AP3D1). Italics: correlations with P values greater than 0.0001 (all other correlations P<0.0001); CDC40 vs GAPDH = 0.0026; 18S vs SDHA = 0.0001 (DOCX)
Article
Full-text available
Background: Dogs with dystrophin-deficient muscular dystrophy are valuable models of the equivalent human disease, Duchenne Muscular Dystrophy (DMD): unlike the mdx mouse, these animals present a disease severity and progression that closely matches that found in human patients. Canine models are however less thoroughly characterised than the esta...
Article
Full-text available
We have investigated a pathogenic mutation in D-amino acid oxidase (DAO), DAOR199W, associated with familial Amyotrophic Lateral Sclerosis (ALS) that impairs D-serine metabolism and causes protein aggregation, autophagy and cell death in motor neuron cell lines. These features are consistent with the pathogenic processes occurring in ALS but most i...
Article
Full-text available
Amyotrophic lateral sclerosis (ALS) is the most common adult-onset neuromuscular disorder characterised by selective loss of motor neurons leading to fatal paralysis. Current therapeutic approaches are limited in their effectiveness. Substantial advances in understanding ALS disease mechanisms has come from the identification of pathogenic mutation...
Data
Text A. Methodology: Genotyping and Gait Analysis. Figure A. DAO enzyme activity in spinal cord and brain from wild-type (WT) and DAOR199W transgenic mice (R199W). Activity measured as mUnits/mg tissue/lysate in brain and spinal cord from wild-type (WT) and DAOR199W transgenic mice (R199W). Figure B. D-serine immunoreactivity in lumbar spinal cord...
Article
Full-text available
LARGE is a glycosyltransferase involved in glycosylation of α-dystroglycan (α-DG). Absence of this protein in the LARGEmyd mouse results in α-DG hypoglycosylation, and is associated with central nervous system abnormalities and progressive muscular dystrophy. Up-regulation of LARGE has previously been proposed as a therapy for the secondary dystrog...
Data
LARGE-LV5 transgene corrects LARGEmyd cortical defects. Cortical coronal sections of brains from wild type, WT-LV5, LARGEmyd and LARGEmyd-LV5 mice (as indicated). a-d: Haematoxylin/Eosin staining. WT and WT-LV5 mice display normal laminar cortical arrangement (arrowheads), which is lost in LARGEmyd but restored by the LARGE-LV5 transgene. e-h: IIH6...
Data
qPCR primers used for LARGE2. (DOC)
Data
LARGE-LV5 transgene corrects LARGEmyd cerebellar defects. Cerebellar sections of brains from wild type, WT-LV5, LARGEmyd and LARGEmyd-LV5 mice (as indicated). a-d: Haematoxylin/Eosin staining. In WT and WT-LV5 mice (a, b), the molecular layer (Black arrowhead) and granular cell layer (White arrowhead) are readily apparent, with a single layer of la...
Data
LARGE-LV5 transgene confers IIH6 reactivity upon testis. IIH6 western blot of tissue lysates from testis of WT, LARGEmyd and LARGEmyd-LV5 mice (as indicated). (TIF)
Article
Full-text available
Exon-skipping via synthetic antisense oligonucleotides represents one of the most promising potential therapies for Duchenne muscular dystrophy (DMD), yet this approach is highly sequence-specific and thus each oligonucleotide is of benefit to only a subset of patients. The discovery that dystrophin mRNA is subject to translational suppression by t...
Article
Full-text available
Splice modulation therapy has shown great clinical promise in Duchenne muscular dystrophy, resulting in the production of dystrophin protein. Despite this, the relationship between restoring dystrophin to established dystrophic muscle and its ability to induce clinically relevant changes in muscle function is poorly understood. In order to robustly...
Article
Full-text available
The coordinated differentiation of myoblasts to mature muscle is essential for muscle development and repair, and study of the myogenic program in health and disease is critical to the understanding and treatment of muscle pathologies. Use of quantitative RT-PCR to analyse gene expression in cell culture models of muscle differentiation can be high...
Article
Full-text available
Dhh1 and Pat1 in yeast are mRNA decapping activators/translational repressors thought to play key roles in the transition of mRNAs from translation to degradation. However, little is known about the physical and functional relationships between these proteins and the translation machinery. We describe a previously unknown type of diauxic shift-depe...
Article
Two novel thiazolidine compounds, GW604714X and GW450863X, were found to be potent inhibitors of mitochondrial respiration supported by pyruvate but not other substrates. Direct measurement of pyruvate transport into rat liver and yeast mitochondria confirmed that these agents inhibited the mitochondrial pyruvate carrier (MPC) with K(i) values <0.1...
Article
Full-text available
Mitochondrial pyruvate transport is fundamental for metabolism and mediated by a specific inhibitable carrier. We have identified the yeast mitochondrial pyruvate carrier by measuring inhibitor-sensitive pyruvate uptake into mitochondria from 18 different Saccharomyces cerevisiae mutants, each lacking an unattributed member of the mitochondrial car...

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Projects

Projects (5)
Project
The Comparative Neuromuscular Diseases Laboratory directed by Professor Richard Piercy at the Royal Veterinary College houses the DE50-MD dog model of DMD. A natural history study has been completed to comprehensively characterise the DE50-MD dog, in advance of conducting pre-clinical trials using this model.
Project
To analye and compare the histopathology in various skeletal muscles in the canine model