Determination of qPCR Reference Genes Suitable for Normalizing Gene Expression in a Canine Model of Duchenne Muscular Dystrophy

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 established mdx mouse in many aspects, including gene expression. Analysis of expression in muscle plays a key role in the study of DMD, allowing monitoring and assessment of disease progression, evaluation of novel biomarkers and gauging of therapeutic intervention efficacy. Appropriate normalization of expression data via carefully selected reference genes is consequently essential for accurate quantitative assessment. Unlike the expression profile of healthy skeletal muscle, the dystrophic muscle environment is highly dynamic: transcriptional profiles of dystrophic muscle might alter with age, disease progression, disease severity, genetic background and between muscle groups. Objectives:The aim of this work was to identify reference genes suitable for normalizing gene expression in healthy and dystrophic dogs under various comparative scenarios. Methods:Using the delta-E50 MD canine model of DMD, we assessed a panel of candidate reference genes for stability of expression across healthy and dystrophic animals, at different ages and in different muscle groups. Results:We show that the genes HPRT1, SDHA and RPL13a appear universally suitable for normalizing gene expression in healthy and dystrophic canine muscle, while other putative reference genes are exceptionally poor, and in the case of B2M, actively disease-correlated. Conclusions:Our findings suggest consistent cross-sample normalization is possible even throughout the dynamic progression of dystrophic pathology, and furthermore highlight the importance of empirical determination of suitable reference genes for neuromuscular diseases

Designing for interaction immediacy to enhance social skills of children with autism

Children with Autism Spectrum Disorder often require therapeutic interventions to support engagement in effective social interactions. In this paper, we present the results of a study conducted in three public schools that use an educational and behavioral intervention for the instruction of social skills in changing situational contexts. The results of this study led to the concept of interaction immediacy to help children maintain appropriate spatial boundaries, reply to conversation initiators, disengage appropriately at the end of an interaction, and identify potential communication partners. We describe design principles for Ubicomp technologies to support interaction immediacy and present an example design. The contribution of this work is twofold. First, we present an understanding of social skills in mobile and dynamic contexts. Second, we introduce the concept of interaction immediacy and show its effectiveness as a guiding principle for the design of Ubicomp applications

GRAF1 deficiency blunts sarcolemmal injury repair and exacerbates cardiac and skeletal muscle pathology in dystrophin-deficient mice

Background The plasma membranes of striated muscle cells are particularly susceptible to rupture as they endure significant mechanical stress and strain during muscle contraction, and studies have shown that defects in membrane repair can contribute to the progression of muscular dystrophy. The synaptotagmin-related protein, dysferlin, has been implicated in mediating rapid membrane repair through its ability to direct intracellular vesicles to sites of membrane injury. However, further work is required to identify the precise molecular mechanisms that govern dysferlin targeting and membrane repair. We previously showed that the bin–amphiphysin–Rvs (BAR)–pleckstrin homology (PH) domain containing Rho-GAP GTPase regulator associated with focal adhesion kinase-1 (GRAF1) was dynamically recruited to the tips of fusing myoblasts wherein it promoted membrane merging by facilitating ferlin-dependent capturing of intracellular vesicles. Because acute membrane repair responses involve similar vesicle trafficking complexes/events and because our prior studies in GRAF1-deficient tadpoles revealed a putative role for GRAF1 in maintaining muscle membrane integrity, we postulated that GRAF1 might also play an important role in facilitating dysferlin-dependent plasma membrane repair. Methods We used an in vitro laser-injury model to test whether GRAF1 was necessary for efficient muscle membrane repair. We also generated dystrophin/GRAF1 doubledeficient mice by breeding mdx mice with GRAF1 hypomorphic mice. Evans blue dye uptake and extensive morphometric analyses were used to assess sarcolemmal integrity and related pathologies in cardiac and skeletal muscles isolated from these mice. Results Herein, we show that GRAF1 is dynamically recruited to damaged skeletal and cardiac muscle plasma membranes and that GRAF1-depleted muscle cells have reduced membrane healing abilities. Moreover, we show that dystrophin depletion exacerbated muscle damage in GRAF1-deficient mice and that mice with dystrophin/GRAF1 double deficiency phenocopied the severe muscle pathologies observed in dystrophin/dysferlin-double null mice. Consistent with a model that GRAF1 facilitates dysferlin-dependent membrane patching, we found that GRAF1 associates with and regulates plasma membrane deposition of dysferlin. Conclusions Overall, our work indicates that GRAF1 facilitates dysferlin-dependent membrane repair following acute muscle injury. These findings indicate that GRAF1 might play a role in the phenotypic variation and pathological progression of cardiac and skeletal muscle degeneration in muscular dystrophy patients

Social Stories™ to alleviate challenging behaviour and social difficulties exhibited by children with autism spectrum disorder in mainstream schools : design of a manualised training toolkit and feasibility study for a cluster randomised controlled trial with nested qualitative and cost-effectiveness components

BACKGROUND: A Social Story™ (Carol Gray) is a child-friendly intervention that is used to give children with autism spectrum disorders (ASDs) social information in situations where they have social difficulties. Limited evidence mainly using single-case designs suggests that they can reduce anxiety and challenging behaviour. OBJECTIVES: The objectives were to conduct a systematic review, use this to develop a manualised intervention and run a feasibility trial to inform a fully powered randomised controlled trial (RCT) on their clinical effectiveness and cost-effectiveness in schools. DESIGN: This is a three-stage study following the Medical Research Council framework for complex interventions. Specifically, it involved a theoretical phase, a qualitative stage and a feasibility trial stage. SETTING: Qualitative interviews and focus groups took place in Child and Adolescent Mental Health Service and primary care settings. The feasibility study took place in 37 local mainstream schools. PARTICIPANTS: Fifty children (aged 5-15 years) in mainstream school settings with a diagnosis of ASD were entered into the trial. For each child, an associated teacher and parent was also recruited. INTERVENTIONS: The intervention was a goal-setting session followed by a manualised toolkit (including a training session) for creating Social Stories™ for use with school-aged children. The comparator treatment was a goal-setting session followed by an attention control. Both arms received treatment as usual. MAIN OUTCOME MEASURES: Outcomes tested as part of the feasibility study included child- and proxy-completed questionnaires for mental health, quality of life and goal-based outcome measures. Adults additionally completed behaviour diaries and the parental stress index. RESULTS: The review found that the research into social stories is predominantly based in the USA, carried out in under-12-year-olds and using single-case designs. Most studies either did not follow established Social Story criteria or did not report if they did. The assessment of effectiveness presents a largely positive picture but is limited by methodological issues. There were no adequate RCTs and insufficient information to assess a number of important sources of potential bias in most studies. A manualised intervention was produced using an iterative process between user focus groups and a writing team, and assessed in the feasibility study. All 50 participant groups were recruited within the study time frame. Two outcome measures, the Social Responsiveness Scale-2 and the custom-made goal-based measure, showed high levels of completion rates and appeared to be capturing social and behaviour skills targeted by the use of Social Stories. Detailed recommendations for a full trial are provided. LIMITATIONS: Blinding of participants was not feasible. Treatment fidelity was not assessed because of low levels of story return rates. CONCLUSIONS: The study showed that a fully powered RCT is feasible with an extended geographical footprint. A large amount of data and information has helped to inform the design of this RCT, which will be the subject of a future research grant application. Future work could focus on developing an appropriate blinded outcome measure for this population. STUDY REGISTRATION: This study is registered as PROSPERO CRD42011001440. TRIAL REGISTRATION: Current Controlled Trials ISRCTN96286707. FUNDING: This project was funded by the NIHR Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 20, No. 6. See the NIHR Journals Library website for further project information

EMQN best practice guidelines for genetic testing in dystrophinopathies.

Dystrophinopathies are X-linked diseases, including Duchenne muscular dystrophy and Becker muscular dystrophy, due to DMD gene variants. In recent years, the application of new genetic technologies and the availability of new personalised drugs have influenced diagnostic genetic testing for dystrophinopathies. Therefore, these European best practice guidelines for genetic testing in dystrophinopathies have been produced to update previous guidelines published in 2010.These guidelines summarise current recommended technologies and methodologies for analysis of the DMD gene, including testing for deletions and duplications of one or more exons, small variant detection and RNA analysis. Genetic testing strategies for diagnosis, carrier testing and prenatal diagnosis (including non-invasive prenatal diagnosis) are then outlined. Guidelines for sequence variant annotation and interpretation are provided, followed by recommendations for reporting results of all categories of testing. Finally, atypical findings (such as non-contiguous deletions and dual DMD variants), implications for personalised medicine and clinical trials and incidental findings (identification of DMD gene variants in patients where a clinical diagnosis of dystrophinopathy has not been considered or suspected) are discussed

A non-terminal surgical procedure for chronic collection of exocrine pancreatic secretions from unrestrained dogs (Canis familiaris)

The ability of dogs to adaptively modulate secretion by the exocrine pancreas to match changes in the amounts and sources of macronutrients is poorly understood. We evaluated the use of re-entrant pancreatic catheters as a non-terminal, temporary approach for the chronic collection of exocrine pancreatic secretion using unrestrained dogs fed diets differing in composition. Re-entrant catheters were surgically placed in the accessory pancreatic duct of two adult mongrel dogs. Secretions were collected for 40 days, during which the dogs were fed three diets with different amounts and sources of macronutrients. The volume of secretion was recorded, protein content was measured, and the activities of trypsin, chymotrypsin, amylase, and lipase were assayed. Inter-dog variation was detected for the volume of secretion (ml/h) but not for protein content (mg/ml) or activities (U/ml) of the enzymes. The volume and composition of the secretion differed among diets. The responses were delayed about 4 days, were transient, and did not coincide with the changes in diet composition. We found that the re-entrant catheters were suitable for studying the exocrine pancreatic secretion of dogs. Our findings were inconclusive about the influence of diet but suggested that adult dogs have a limited and nonspecific response of pancreatic secretion

The superhealing MRL background improves muscular dystrophy

<p>Abstract</p> <p>Background</p> <p>Mice from the MRL or “superhealing” strain have enhanced repair after acute injury to the skin, cornea, and heart. We now tested an admixture of the MRL genome and found that it altered the course of muscle pathology and cardiac function in a chronic disease model of skeletal and cardiac muscle. Mice lacking γ-sarcoglycan (<it>Sgcg</it>), a dystrophin-associated protein, develop muscular dystrophy and cardiomyopathy similar to their human counterparts with limb girdle muscular dystrophy. With disruption of the dystrophin complex, the muscle plasma membrane becomes leaky and muscles develop increased fibrosis.</p> <p>Methods</p> <p>MRL/MpJ mice were bred with <it>Sgcg</it> mice, and cardiac function was measured. Muscles were assessed for fibrosis and membrane leak using measurements of hydroxyproline and Evans blue dye. Quantitative trait locus mapping was conducted using single nucleotide polymorphisms distinct between the two parental strains.</p> <p>Results</p> <p>Introduction of the MRL genome reduced fibrosis but did not alter membrane leak in skeletal muscle of the <it>Sgcg</it> model. The MRL genome was also associated with improved cardiac function with reversal of depressed fractional shortening and the left ventricular ejection fraction. We conducted a genome-wide analysis of genetic modifiers and found that a region on chromosome 2 was associated with cardiac, diaphragm muscle and abdominal muscle fibrosis.</p> <p>Conclusions</p> <p>These data are consistent with a model where the MRL genome acts in a dominant manner to suppress fibrosis in this chronic disease setting of heart and muscle disease.</p

A Social StoriesTm Intervention Package for Students with Autism in Inclusive Classroom Settings

A Social StoriesTM intervention package was used to teach 2 students with autism to read Social Stories, answer comprehension questions, and engage in role plays. Appropriate social behaviors increased and inappropriate behaviors decreased for both participants, and the effects were maintained for up to 10 months. This intervention package appears to be useful in inclusive classroom environments and does not require intensive supervision of the child's behavior