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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 (69)
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...
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...
Gene editing and muscular dystrophy
Duchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness and a shortened life span. The disease is caused by mutations that reduce or prevent expression of dystrophin, an essential structural protein in skeletal and heart muscle. The gene editing technology CRISPR-Cas9 can correct disease...
Dystrophin mRNA is produced from a very large genetic locus and transcription of a single mRNA requires approximately 16 hours. This prolonged interval between transcriptional initiation and completion results in unusual transcriptional behaviour: in skeletal muscle, myonuclei express dystrophin continuously and robustly, yet degrade mature transcr...
Background
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by mutations in the dystrophin gene. DE50-MD dogs are an animal model of DMD used as a final translational model for evaluation of promising treatments. MicroRNA (miR) expressions in the muscle of DE50-MD dogs represent potential biomarkers, but stable reference m...
Background Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by mutations in the dystrophin gene resulting in cycles of muscle degeneration, inflammation and regeneration. The 6-minute walk test (6MWT) is a key functional outcome measure for DMD patient clinical trials and has been adapted for use in animal models of the di...
Duchenne muscular dystrophy (DMD) is a X-linked neuromuscular disorder arising from mutations in the dystrophin gene, leading to a progressive muscle wasting and disability. Currently there is no universal therapy, and there is thus a strong interest in preclinical studies for finding novel treatments. The most widely used and characterized mouse m...
Tendons are one of the major load-bearing tissues in the body; subjected to enormous peak stresses, and thus vulnerable to injury. Cellular responses to tendon injury are complex, involving inflammatory and repair components, with the latter employing both resident and recruited exogenous cell populations. Gene expression analyses are valuable tool...
Background Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by mutations in the dystrophin gene. DE50-MD dogs are a canine model of DMD used as final translational models for evaluation of promising treatments. MicroRNA (miR) expressions in the muscle of DE50-MD dogs represent potential biomarkers, but stable reference miR...
Tendons are one of the major load-bearing tissues in the body; subjected to enormous peak stresses, and thus vulnerable to injury. Cellular responses to tendon injury are complex, involving inflammatory and repair components, with the latter employing both resident and recruited exogenous cell populations. Gene expression analyses are valuable tool...
Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is associated with fatal muscle degeneration and atrophy. Patients have progressive reductions in skeletal muscle strength and resistance to eccentric muscle stretch. We assessed tibiotarsal joint (TTJ) flexor and extensor force dynamics, and resistance of dystrophic mus...
At 2.3 megabases in length, the dystrophin gene is enormous: transcription of a single mRNA requires approximately 16 h. Principally expressed in skeletal muscle, the dystrophin protein product protects the muscle sarcolemma against contraction-induced injury, and dystrophin deficiency results in the fatal muscle-wasting disease, Duchenne muscular...
At 2.3 megabases in length, the dystrophin gene is enormous: transcription of a single mRNA requires approximately 16 hours. Principally expressed in skeletal muscle, the dystrophin protein product protects the muscle sarcolemma against contraction-induced injury, and dystrophin deficiency results in the fatal muscle-wasting disease, Duchenne muscu...
Background: In addition to progressive, debilitating muscle degeneration, ~50% of patients with Duchenne muscular dystrophy (DMD) have associated cognitive and behavioural disorders secondary to deficiency of dystrophin protein in the brain. The brain expresses a variety of dystrophin isoforms (Dp427, Dp140 and Dp71) whose functions remain to be fu...
Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged...
Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease, caused by mutations in the dystrophin gene, characterised by cycles of muscle degeneration, inflammation and regeneration. Recently, there has been renewed interest specifically in drugs that ameliorate muscle inflammation in DMD patients. The DE50-MD dog is a model of DMD that cl...
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...
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...
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...
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...
Picrosirius Red (PSR) staining of skeletal muscle tissue sections permits histological evaluation of muscle fibrosis: muscle fibres are stained a vivid picric acid yellow, while connective tissue is stained a vibrant Sirius red. Connective tissue can also be visualised via brightfield, fluorescence or polarized light microscopy, but no standard app...
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...
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...
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...
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...
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...
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...
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...
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...
Sample summary, Primer and qPCR validation.
(DOCX)
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...
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)
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)
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...
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)
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, **...
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)
Reference gene selection algorithms: Detailed summary.
(DOCX)
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)
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...
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)
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...
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...
Normfinder ‘best pair’ combinations for all dataset/grouping combinations.
(DOCX)
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)
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...
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...
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...
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...
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...
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...
qPCR primers used for LARGE2.
(DOC)
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...
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)
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...
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...
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...
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...
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...
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...
