Novel control of cardiac myofilament response to calcium by S-glutathionylation at specific sites of myosin binding protein C

Our previous studies demonstrated a relation between glutathionylation of cardiac myosin binding protein C (cMyBP-C) and diastolic dysfunction in a hypertensive mouse model stressed by treatment with salt, deoxycorticosterone acetate, and unilateral nephrectomy. Although these results strongly indicated an important role for S-glutathionylation of myosin binding protein C as a modifier of myofilament function, indirect effects of other post-translational modifications may have occurred. Moreover, we did not determine the sites of thiol modification by glutathionylation. To address these issues, we developed an in vitro method to mimic the in situ S-glutathionylation of myofilament proteins and determined direct functional effects and sites of oxidative modification employing Western blotting and mass spectrometry. We induced glutathionylation in vitro by treatment of isolated myofibrils and detergent extracted fiber bundles (skinned fibers) with oxidized glutathione (GSSG). Immuno-blotting results revealed increased glutathionylation with GSSG treatment of a protein band around 140 kDa. Using tandem mass spectrometry, we identified the 140 kDa band as cMyBP-C and determined the sites of glutathionylation to be at cysteines 655, 479, and 627. Determination of the relation between Ca(2+)-activation of myofibrillar acto-myosin ATPase rate demonstrated an increased Ca(2+)-sensitivity induced by the S-glutathionylation. Force generating skinned fiber bundles also showed an increase in Ca-sensitivity when treated with oxidized glutathione, which was reversed with the reducing agent, dithiothreitol (DTT). Our data demonstrate that a specific and direct effect of S-glutathionylation of myosin binding protein C is a significant increase in myofilament Ca(2+)-sensitivity. Our data also provide new insights into the functional significance of oxidative modification of myosin binding protein C and the potential role of domains not previously considered to be functionally significant as controllers of myofilament Ca(2+)-responsiveness and dynamics

Universities and community-based research in developing countries: community voice and educational provision in rural Tanzania

The main focus of recent research on the community engagement role of universities has been in developed countries, generally in towns and cities and usually conducted from the perspectives of universities rather than the communities with which they engage. The purpose of this paper is to investigate the community engagement role of universities in the rural areas of developing countries, and its potential for strengthening the voice of rural communities. The particular focus is on the provision of primary and secondary education. The paper is based on the assumption that in order for community members to have both the capacity and the confidence to engage in political discourse for improving educational capacity and quality, they need the opportunity to become involved and well-versed in the options available, beyond their own experience. Particular attention is given in the paper to community-based research (CBR). CBR is explored from the perspectives of community members and local leaders in the government-community partnerships which have responsibility for the provision of primary and secondary education in rural Tanzania. The historical and policy background of the partnerships, together with findings from two case studies, provide the context for the paper

ICDP Workshop on the Lake Tanganyika Scientific Drilling Project: A Late Miocene–Present Record of Climate, Rifting, and Ecosystem Evolution from the World\u27s Oldest Tropical Lake

The Neogene and Quaternary are characterized by enormous changes in global climate and environments, including global cooling and the establishment of northern high-latitude glaciers. These changes reshaped global ecosystems, including the emergence of tropical dry forests and savannahs that are found in Africa today, which in turn may have influenced the evolution of humans and their ancestors. However, despite decades of research we lack long, continuous, well-resolved records of tropical climate, ecosystem changes, and surface processes necessary to understand their interactions and influences on evolutionary processes. Lake Tanganyika, Africa, contains the most continuous, long continental climate record from the mid-Miocene (∼10 Ma) to the present anywhere in the tropics and has long been recognized as a top-priority site for scientific drilling. The lake is surrounded by the Miombo woodlands, part of the largest dry tropical biome on Earth. Lake Tanganyika also harbors incredibly diverse endemic biota and an entirely unexplored deep microbial biosphere, and it provides textbook examples of rift segmentation, fault behavior, and associated surface processes. To evaluate the interdisciplinary scientific opportunities that an ICDP drilling program at Lake Tanganyika could offer, more than 70 scientists representing 12 countries and a variety of scientific disciplines met in Dar es Salaam, Tanzania, in June 2019. The team developed key research objectives in basin evolution, source-to-sink sedimentology, organismal evolution, geomicrobiology, paleoclimatology, paleolimnology, terrestrial paleoecology, paleoanthropology, and geochronology to be addressed through scientific drilling on Lake Tanganyika. They also identified drilling targets and strategies, logistical challenges, and education and capacity building programs to be carried out through the project. Participants concluded that a drilling program at Lake Tanganyika would produce the first continuous Miocene–present record from the tropics, transforming our understanding of global environmental change, the environmental context of human origins in Africa, and providing a detailed window into the dynamics, tempo and mode of biological diversification and adaptive radiations

Optimizing Livelihood and Environmental Benefits from Crop Residues in Smallholder Crop-Livestock Systems in Southern Africa. Series Paper Number 11

Sustainable forms of intensification are needed to address the low and stagnant production of farming systems in southern Africa. More efficient use of crop-livestock interactions can contribute to this; in this context the effective use of crop residues is becoming increasingly important and also contested. Crop residues left on the field for mulching are expected to bring long-term environmental benefits but when fed to livestock they provide farmers with short-term livelihood benefits. This study aims at better understanding the diversity of farming systems and uses of crop residues, in particular the trade-offs in using the residues for soil amendment versus livestock feed. It is part of a global comparison with sites along a human and livestock population density gradient across sub-Saharan Africa and South Asia. This southern Africa study represents the most extensive case of semi-arid areas with lowest biomass production. Three sites were compared, at different levels of agricultural intensification, extent of crop-livestock integration and use of crop residues. 1. Mzimba in Northern Malawi – intensified crop oriented production. 2. Nkayi in southwest Zimbabwe – integrated crop-livestock systems. 3. Changara in Tete province in Central Mozambique – extensive crop-livestock farming. Across the three sites, crop residues are clearly needed as livestock feed. In Nkayi and Changara low crop yields and low biomass production against the existing demand from livestock prevents farmers from using residues for purposes other than livestock feed. The practice of collecting and kraal feeding residues in Nkayi illustrates that the pressure on residues is at a level where farmers start privatizing residues in order to ensure their individual benefits. When feeding crop residues in the kraal, farmers also increase the amount of manure for soil fertility improvement. Even in Mzimba, with higher residue production and lower livestock ownership, very few farmers retain the residues to achieve real impact on soil health. Although farmers see soil fertility as a critical constraint, they have limited residues to spare for mulching. The trade-offs of reallocating crop residues from livestock feed to mulching for soil amendment will be high as long as alternative feed technologies and access to input and output markets are not developed. The trade-offs will be lower in areas with higher biomass production and less competition with livestock. Technical options need to increase biomass on existing croplands, addressing feed shortages and the need for soil amendment concurrently. Viable institutional structures and appropriate policies need to support this intensification processes through better access to inputs, knowledge and markets. The pathways for sustainable intensification and more efficient crop residue utilization need to be developed within the local context. We found strong growth potential for livestock-oriented agricultural development in extensive areas (Changara), strengthening crop and livestock integration to support intensification in areas like Nkayi, and enhancing crop-livestock integration for more efficient resource utilization where biomass is less limiting (Mzimba)

Natural disease history of mouse models for limb girdle muscular dystrophy types 2D and 2F

Limb-girdle muscular dystrophy types 2D and 2F (LGMD 2D and 2F) are autosomal recessive disorders caused by mutations in the alpha- and delta sarcoglycan genes, respectively, leading to severe muscle weakness and degeneration. The cause of the disease has been well characterized and a number of animal models are available for pre-clinical studies to test potential therapeutic interventions. To facilitate transition from drug discovery to clinical trials, standardized procedures and natural disease history data were collected for these mouse models. Implementing the TREAD-NMD standardized operating procedures, we here subjected LGMD2D (SGCA-null), LGMD2F (SGCD-null) and wild type (C57BL/6J) mice to five functional tests from the age of 4 to 32 weeks. To assess whether the functional test regime interfered with disease pathology, sedentary groups were taken along. Muscle physiology testing of tibialis anterior muscle was performed at the age of 34 weeks. Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Mice successfully accomplished the functional tests, which did not interfere with disease pathology. Muscle function of SGCA- and SGCD-null mice was impaired and declined over time. Interestingly, female SGCD-null mice outperformed males in the two and four limb hanging tests, which proved the most suitable non-invasive tests to assess muscle function. Muscle physiology testing of tibialis anterior muscle revealed lower specific force and higher susceptibility to eccentric-induced damage in LGMD mice. Analyzing muscle histopathology and gene expression, we identified the diaphragm as the most affected muscle in LGMD strains. Cardiac fibrosis was found in SGCD-null mice, being more severe in males than in females. Our study offers a comprehensive natural history dataset which will be useful to design standardized tests and future pre-clinical studies in LGMD2D and 2F miceFunctional Genomics of Muscle, Nerve and Brain Disorder

Simvastatin Treatment Does Not Ameliorate Muscle Pathophysiology in a Mouse Model for Duchenne Muscular Dystrophy

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 suggested that simvastatin, a cholesterol lowering drug used for cardiovascular diseases, has beneficial effects on several parameters in mdx mice. To validate properly the effectiveness of simvastatin, two independent labs tested the effects of 12-week simvastatin treatment in either young (starting at 4 weeks of age) or adult (starting at 12 weeks of age) mdx mice. In neither study were benefits of simvastatin treatment observed on muscle function, histology or expression of genes involved in fibrosis, regeneration, oxidative stress and autophagy. Unexpectedly, although the treatment protocol was similar, simvastatin plasma levels were found be much lower than observed in a previous study. In conclusion, in two laboratories, simvastatin did not ameliorate disease pathology in mdx mice, which could either be due to the ineffectiveness of simvastatin itself or due to the low simvastatin plasma levels following oral administration via the food

Peripheral blood transcriptome profiling enables monitoring disease progression in dystrophic mice and patients

DMD is a rare disorder characterized by progressive muscle degeneration and premature death. Therapy development is delayed by difficulties to monitor efficacy non-invasively in clinical trials. In this study, we used RNA-sequencing to describe the pathophysiological changes in skeletal muscle of 3 dystrophic mouse models. We show how dystrophic changes in muscle are reflected in blood by analyzing paired muscle and blood samples. Analysis of repeated blood measurements followed the dystrophic signature at five equally spaced time points over a period of seven months. Treatment with two antisense drugs harboring different levels of dystrophin recovery identified genes associated with safety and efficacy. Evaluation of the blood gene expression in a cohort of DMD patients enabled the comparison between preclinical models and patients, and the identification of genes associated with physical performance, treatment with corticosteroids and body measures. The presented results provide evidence that blood RNA-sequencing can serve as a tool to evaluate disease progression in dystrophic mice and patients, as well as to monitor response to (dystrophin-restoring) therapies in preclinical drug development and in clinical trials.Development and application of statistical models for medical scientific researc

Influence of production systems and sex on nutritional value and meat quality of native Malawian Muscovy ducks

This study was conducted to assess effects of production systems and sex on nutritional value and meat quality of native Malawian Muscovy ducks. One hundred twenty ducks were randomly assigned to either an intensive (IS), duck-rice integration (DR) or free-range (FR) production system. A starter ration containing 20% crude protein and a finisher containing 17% crude protein were fed to ducks in IS (1 to 4 weeks), and provided as a supplement to ducks in DR and FR (5 to 10 weeks). Feed and water were offered ad libitum. At 10 weeks of age, 16 ducks per treatment were selected randomly, slaughtered and chilled at 4 °C for 24 hours. Carcass temperature, pH and meat colour were measured at 45 min, and at 3, 6, 12 and 24 hours post mortem. Tenderness, cooking loss, proximate and mineral composition were determined 24 hours post mortem. Production system and sex had no effect on carcass temperature, pH and proximate composition of duck breast meat. However, production system affected tenderness and mineral composition of the meat and sex influenced moisture and tenderness. Males were moister and had less tender meat than females. Carcasses from ducks in the FR system contained more zinc, copper, manganese, and potassium, but less iron while those in IS had the lowest mineral content of the three production systems. Thus, DR can be adopted to improve the current FR system of native Malawian Muscovy duck production with supplementation to produce duck with acceptable mineral composition and better meat quality. Keywords: cooking loss, meat colour, mineral, proximate compositio

Tropomyosin Phosphorylation and Oxidative Stress in Hypertrophic Cardiomyopathy

The work described within this dissertation investigates the general hypothesis that post-translational modifications of α-Tm elicit a progression of a pathophysiological phenotype through a mechanism involving altered redox signals. Hypertrophic cardiomyopathy (HCM) is a genetic disease of the sarcomere that ultimately results in left ventricular hypertrophy and diastolic dysfunction. This disease affects 1 in 500 and is the leading cause of sudden cardiac death in young, especially athletes. The exact mechanism by which these mutations elicit hypertrophy is unknown. In order to gain a better understanding of this disorder and how to devise effective treatments, a mouse model has been generated that mimics human HCM where cardiac specific α-tropomyosin (α-Tm) glutamic acid (E) at position 180 was exchanged with uncharged glycine (G) (α-cMyBP-C). This well characterized model displays severely enlarged left atria, fibrosis, myocyte disarray, hypertrophy, and diastolic dysfunction associated with an increased Ca2+-sensitivity of myofilaments. Human HCM hearts have been shown to exhibit increases in oxidation that inappropriately activate kinases and phosphatases. When we examined the HCM myofibrils, we detected an increase in oxidative markers that target myofilament proteins. Moreover, in the adult stages of this model, the 30-60% reduction in the phosphorylation status of α-Tm was not evident compared to non-transgenic (NTG) littermates. Collectively these data led us to hypothesize that decreasing phosphorylation of α-Tm at S283 would facilitate the rescue of the phenotype, including preventing the oxidative modifications associated with cMyBP-C. We further hypothesized that antioxidant treatment using N-acetylcysteine (NAC) would reverse the HCM phenotype. Experiments reported in chapter one uses a double mutant transgenic (DMTG) mouse model incorporating α-cMyBP-C with dephosphorylated α-Tm (S283A) to understand the role phosphorylation plays in disease progression. Dephosphorylation prevented the hypertrophic remodeling, decreased the Ca2+ sensitivity, improved relaxation, and reduced oxidative glutathionylation of myosin binding protein C (cMyBP-C) and carbonylation of myofibrillar proteins. These data suggest that altered charge status and post-translational modification of α-Tm causes a strain on the heart that elicits oxidative modifications. Chapter two examines the direct functional effect and specific sites of glutathionylation of myofilament proteins. Employing Western blotting and mass spectrometry, we induced glutathionylation in vitro by treatment of isolated myofibrils and detergent extracted fiber bundles (skinned fibers) with oxidized glutathione (GSSG). Results indicate that there are at least three cysteines on cMyBP-C that are susceptible to oxidative modification. Force generating skinned fiber bundles showed an increase in Ca2+-sensitivity when treated with oxidized glutathione, which was reversed with the reducing agent, dithiothreitol. These results indicated that antioxidants would be beneficial to reduce Ca2+-sensitivity in HCM. The object of experiments reported in chapter three aimed to determine whether antioxidant treatment could reverse HCM. In our approach, we treated NTG and cMyBP-C mice with N-Acetylcysteine (NAC) for one month. NAC is a precursor to the major intracellular antioxidant glutathione. Glutathione is found in every mammalian cell at a concentration between 1-10 mM. Oxidative stress has been demonstrated to decrease the cellular antioxidant capacity and alter the redox potential. We found that oral administration of NAC reverses redox mediated hypertrophic signaling, remodeling, and improves the relaxation kinetics by a mechanism that involves modifying key sarcomeric and Ca2+ handling proteins that mediate contraction and relaxation. Our findings provide a new appreciation of the role of tropomyosin phosphorylation in control of hypertrophic and oxidative signaling. Results of this thesis work show the complexity of the relation between myofilament modifications, oxidative stress, and the hypertrophic phenotype in HCM. HCM mutations inducing oxidative myofilament modifications could be resolved by treatment with antioxidants that relieve the constraint placed on the molecular motors that drive the cardiac cycle