Effects of compounds on C. elegans DMD model health

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the dystrophin gene. The dystrophin gene encodes a cytoskeletal protein with the same name that is responsible for ensuring the strength, stability, and functionality of myofibers. In DMD the dystrophin protein is either absent or there are insufficient levels of functional dystrophin resulting in progressive muscular damage and degeneration. This results in muscular weakness, motor delays, loss of ambulation, and a shortened life expectancy due to respiratory impairment and cardiomyopathy. Treatment options are limited and mainly focused on alleviating symptoms; they are not a cure. The main therapeutic treatment used are glucocorticoids, these can be used for a couple of years, but treatment is often ceased due to undesirable side effects. An emerging therapy is the use of exon-skipping but currently these can only be used for patients amenable to skipping of exons 45, 51, and 53 (approximately 30% of patients). There is therefore a great need for alternative therapies. This thesis uses C. elegans as a model for DMD. We demonstrate throughout that the C. elegans DMD model has clinical relevance as it shares some of the underlying pathophysiology that are also displayed in patients, including mitochondrial dysfunction and calcium dysregulation. It also has several clinically relevant phenotypes that can be exploited including movement and strength decline and changes in gait. Finally, the current standard treatment used to treat patients with DMD, prednisone, has also been identified as being beneficial in the DMD C. elegans model as well. Hydrogen sulfide (H2S) compounds were trialled as a potential treatment for DMD in this thesis. The rationale behind this was that H2S compounds had been demonstrated previously to improve lifespan in ageing animals. There are some similarities between ageing and DMD muscle, the former being associated with sarcopenia and the latter with progressive muscle degeneration. It therefore seemed reasonable to expect an improvement in the DMD animals given there was one in ageing animals. In Chapter 3, we started by trialling a non-targeted H2S compound sodium GYY4137 (NaGYY) and showed that this compound does improve movement, strength, and gait in the DMD model. The basis of this improvement was likely mitochondrial, and the mechanism of action was like that of prednisone. In Chapter 4, to confirm the basis of this we then used a mitochondrially targeted H2S compound, AP39, and demonstrated that this compound was also able to improve movement and strength in DMD animals. This provided further evidence to suggest that at least part of the mechanism of NaGYY was through improvements in mitochondrial dysfunction. We then further probed the mechanism of action of AP39 in the mitochondria and established that AP39 is likely donating electrons to complex III of the electron transport chain (ETC) and thus causing an increase in ATP content. In Chapter 5, we show that there is a decline in the gene expression of enzymes responsible for sulfur metabolism that are resulting in a H2S deficit. We also demonstrate that supplementing sulfur in a different way (via sulfur containing amino acids) is also beneficial in the C. elegans DMD model. This highlights a potential novel underlying pathophysiology of DMD in a defective sulfur metabolism pathway and the potential of using H2S as a biomarker for disease progression. To conclude we have shown that supplementing H2S compounds and sulfur containing amino acids are potential treatments for DMD and potential alternatives to prednisone. We have also demonstrated that manipulation of the sulfur metabolism pathway warrants further study in DMD. Future work includes trialling these therapies in the DMD mouse model and beyond and identifying whether the defective sulfur metabolism pathway and H2S deficit corresponds with higher organisms

Effects of compounds on C. elegans DMD model health

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the dystrophin gene. The dystrophin gene encodes a cytoskeletal protein with the same name that is responsible for ensuring the strength, stability, and functionality of myofibers. In DMD the dystrophin protein is either absent or there are insufficient levels of functional dystrophin resulting in progressive muscular damage and degeneration. This results in muscular weakness, motor delays, loss of ambulation, and a shortened life expectancy due to respiratory impairment and cardiomyopathy. Treatment options are limited and mainly focused on alleviating symptoms; they are not a cure. The main therapeutic treatment used are glucocorticoids, these can be used for a couple of years, but treatment is often ceased due to undesirable side effects. An emerging therapy is the use of exon-skipping but currently these can only be used for patients amenable to skipping of exons 45, 51, and 53 (approximately 30% of patients). There is therefore a great need for alternative therapies. This thesis uses C. elegans as a model for DMD. We demonstrate throughout that the C. elegans DMD model has clinical relevance as it shares some of the underlying pathophysiology that are also displayed in patients, including mitochondrial dysfunction and calcium dysregulation. It also has several clinically relevant phenotypes that can be exploited including movement and strength decline and changes in gait. Finally, the current standard treatment used to treat patients with DMD, prednisone, has also been identified as being beneficial in the DMD C. elegans model as well. Hydrogen sulfide (H2S) compounds were trialled as a potential treatment for DMD in this thesis. The rationale behind this was that H2S compounds had been demonstrated previously to improve lifespan in ageing animals. There are some similarities between ageing and DMD muscle, the former being associated with sarcopenia and the latter with progressive muscle degeneration. It therefore seemed reasonable to expect an improvement in the DMD animals given there was one in ageing animals. In Chapter 3, we started by trialling a non-targeted H2S compound sodium GYY4137 (NaGYY) and showed that this compound does improve movement, strength, and gait in the DMD model. The basis of this improvement was likely mitochondrial, and the mechanism of action was like that of prednisone. In Chapter 4, to confirm the basis of this we then used a mitochondrially targeted H2S compound, AP39, and demonstrated that this compound was also able to improve movement and strength in DMD animals. This provided further evidence to suggest that at least part of the mechanism of NaGYY was through improvements in mitochondrial dysfunction. We then further probed the mechanism of action of AP39 in the mitochondria and established that AP39 is likely donating electrons to complex III of the electron transport chain (ETC) and thus causing an increase in ATP content. In Chapter 5, we show that there is a decline in the gene expression of enzymes responsible for sulfur metabolism that are resulting in a H2S deficit. We also demonstrate that supplementing sulfur in a different way (via sulfur containing amino acids) is also beneficial in the C. elegans DMD model. This highlights a potential novel underlying pathophysiology of DMD in a defective sulfur metabolism pathway and the potential of using H2S as a biomarker for disease progression. To conclude we have shown that supplementing H2S compounds and sulfur containing amino acids are potential treatments for DMD and potential alternatives to prednisone. We have also demonstrated that manipulation of the sulfur metabolism pathway warrants further study in DMD. Future work includes trialling these therapies in the DMD mouse model and beyond and identifying whether the defective sulfur metabolism pathway and H2S deficit corresponds with higher organisms

Caenorhabditis elegans as a Model System for Duchenne Muscular Dystrophy

The nematode worm Caenorhabditis elegans has been used extensively to enhance our understanding of the human neuromuscular disorder Duchenne Muscular Dystrophy (DMD). With new arising clinically relevant models, technologies and treatments, there is a need to reconcile the literature and collate the key findings associated with this model

Mitochondrial dysfunction causes Ca2+ overload and ECM degradation–mediated muscle damage in C. elegans

Mitochondrial dysfunction impairs muscle health and causes subsequent muscle wasting. This study explores the role of mitochondrial dysfunction as an intramuscular signal for the extracellular matrix (ECM)–based proteolysis and, consequentially, muscle cell dystrophy. We found that inhibition of the mitochondrial electron transport chain causes paralysis as well as muscle structural damage in the nematode Caenorhabditis elegans. This was associated with a significant decline in collagen content. Both paralysis and muscle damage could be rescued with collagen IV overexpression, matrix metalloproteinase (MMP), and Furin inhibitors in Antimycin A–treated animal as well as in the C. elegans Duchenne muscular dystrophy model. Additionally, muscle cytosolic calcium increased in the Antimycin A–treated worms, and its down-regulation rescued the muscle damage, suggesting that calcium overload acts as one of the early triggers and activates Furin and MMPs for collagen degradation. In conclusion, we have established ECM degradation as an important pathway of muscle damage

Sulfur amino acid supplementation displays therapeutic potential in a C. elegans model of Duchenne muscular dystrophy

Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), a common muscle disease that manifests with muscle weakness, wasting, and degeneration. An emerging theme in DMD pathophysiology is an intramuscular deficit in the gasotransmitter hydrogen sulfide (H2S). Here we show that the C. elegans DMD model displays reduced levels of H2S and expression of genes required for sulfur metabolism. These reductions can be offset by increasing bioavailability of sulfur containing amino acids (L-methionine, L-homocysteine, L-cysteine, L-glutathione, and L-taurine), augmenting healthspan primarily via improved calcium regulation, mitochondrial structure and delayed muscle cell death. Additionally, we show distinct differences in preservation mechanisms between sulfur amino acid vs H2S administration, despite similarities in required health-preserving pathways. Our results suggest that the H2S deficit in DMD is likely caused by altered sulfur metabolism and that modulation of this pathway may improve DMD muscle health via multiple evolutionarily conserved mechanisms

Research Exploring Physical Activity in Care Homes (REACH): study protocol for a randomised controlled trial

Background: As life expectancy increases and the number of older people, particularly those aged 85 years and over, expands there is an increase in demand for long-term care. A large proportion of people in a care home setting spend most of their time sedentary, and this is one of the leading preventable causes of death. Encouraging residents to engage in more physical activity could deliver benefits in terms of physical and psychological health, and quality of life. This study is the final stage of a programme of research to develop and preliminarily test an evidence-based intervention designed to enhance opportunities for movement amongst care home residents, thereby increasing levels of physical activity. Methods/design: This is a cluster randomised feasibility trial, aiming to recruit at least 8–12 residents at each of 12 residential care homes across Yorkshire, UK. Care homes will be randomly allocated on a 1:1 basis to receive either the intervention alongside usual care, or to continue to provide usual care alone. Assessment will be undertaken with participating residents at baseline (prior to care home randomisation) and at 3, 6, and 9 months post-randomisation. Data relating to changes in physical activity, physical function, level of cognitive impairment, mood, perceived health and wellbeing, and quality of life will be collected. Data at the level of the home will also be collected and will include staff experience of care, and changes in the numbers and types of adverse events residents experience (for example, hospital admissions, falls). Details of National Health Service (NHS) usage will be collected to inform the economic analysis. An embedded process evaluation will obtain information to test out the theory of change underpinning the intervention and its acceptability to staff and residents. Discussion: This feasibility trial with embedded process evaluation and collection of health economic data will allow us to undertake detailed feasibility work to inform a future large-scale trial. It will provide valuable information to inform research procedures in this important but challenging area

Recruiting Care Homes to a Randomised Controlled Trial

Background There are over a quarter of a million individuals aged ≥65 years resident in care homes in England and Wales. Care home residents have high levels of cognitive impairment, physical disability, multimorbidity and polypharmacy. Research is needed to ensure there are robust, evidence-based interventions to improve the quality of life of this frail group. However, there is a paucity of research studies in this area. Recruiting care homes and their residents to research is challenging. A feasibility, cluster randomised controlled trial was undertaken as part of a research programme to identify ways to develop and test methods to enhance the physical activity of care home residents. This paper describes two methods of recruiting care homes to the trial and draws out learning to inform future studies. Methods Eligible care homes: were within a defined geographical area in the north of England; provided residential care for adults ≥65 years of age; had not previously been involved in the research programme; were not taking part in a conflicting study; were not recorded on the Care Quality Commission website as ‘inadequate’ or ‘requiring improvements’ in any area; had ≥10 beds. Care homes were identified by: a ‘systematic approach’ using the Care Quality Commission website database of care homes; a ‘targeted approach’ via a network of research-ready care homes. A standardised method was used to recruit care homes including: eligibility screening; invitation letters; telephone contact; visits; formal letter of agreement. Results In the systematic approach, 377 care homes were screened, 230 (61%) were initially eligible and invited to participate, 11 were recruited (recruitment rate (RR) 4.8%). In the targeted approach, 15 care homes were invited to participate, two were recruited (RR 13.3%). Overall, 245 care homes were approached and 13 recruited (RR 5.3%). A variety of care homes were recruited to the trial in terms of size, location, ownership and care provision. Conclusions Systematic recruitment of care homes to the study was time-consuming and resource-heavy but led to a variety of care homes being recruited. The targeted approach led to a higher recruitment rate. Trial registration ISRCTN registry, ISRCTN16076575. Registered 25 June 2015, http://www.isrctn.com/ISRCTN1607657

PATCH: posture and mobility training for care staff versus usual care in care homes: study protocol for a randomised controlled trial

Background: Residents of care homes have high levels of disability and poor mobility, but the promotion of health and wellbeing within care homes is poorly realised. Residents spend the majority of their time sedentary which leads to increased dependency and, coupled with poor postural management, can have many adverse outcomes including pressure sores, pain and reduced social interaction. The intervention being tested in this project (the Skilful Care Training Package) aims to increase the awareness and skills of care staff in relation to poor posture in the older, less mobile adult and highlight the benefits of activity, and how to skilfully assist activity, in this group to enable mobility and reduce falls risk. Feasibility work will be undertaken to inform the design of a definitive cluster randomised controlled trial. Methods: This is a cluster randomised controlled feasibility trial, aiming to recruit at least 12–15 residents at each of 10 care homes across Yorkshire. Care homes will be randomly allocated on a 1:1 basis to receive either the Skilful Care Training Package alongside usual care or to continue to provide usual care alone. Assessments will be undertaken by blinded researchers with participating residents at baseline (before care home randomisation) and at three and six months post randomisation. Data relating to changes in physical activity, mobility, posture, mood and quality of life will be collected. Data at the level of the home will also be collected and will include staff experience of care and changes in the numbers and types of adverse events residents experience (for example, hospital admissions, falls). Details of NHS service usage will be collected to inform the economic analysis. An embedded process evaluation will explore intervention delivery and its acceptability to staff and residents. Discussion: Participant uptake, engagement and retention are key feasibility outcomes. Exploration of barriers and facilitators to intervention delivery will inform intervention optimisation. Study results will inform progression to a definitive trial and add to the body of evidence for good practice in care home research. Trial registration: ISRCTN Registry, ISRCTN50080330. Registered on 27 March 2017

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead