Insects, as a group, have been remarkably successful in adapting to a great range of physical and biological environments, in large part because of their ability to fly. The evolution of flight in small insects was accompanied by striking adaptations of the thoracic musculature that enabled very high wing beat frequencies. At the cellular and protein filament level, a stretch activation mechanism evolved that allowed high-oscillatory work to be achieved at very high frequencies as contraction and nerve stimulus became asynchronous. At the molecular level, critical adaptations occurred within the motor protein myosin II, because its elementary interactions with actin set the speed of sarcomere contraction. Here, we show that the key myosin enzymatic adaptations required for powering the very fast flight muscles in the fruit fly Drosophila melanogaster include the highest measured detachment rate of myosin from actin (forward rate constant, 3,698 s ؊1 ), an exceptionally weak affinity of MgATP for myosin (association constant, 0.2 mM ؊1 ), and a unique rate-limiting step in the cross-bridge cycle at the point of inorganic phosphate release. The latter adaptations are constraints imposed by the overriding requirement for exceptionally fast release of the hydrolytic product MgADP. Otherwise, as in Drosophila embryonic muscle and other slow muscle types, a step associated with MgADP release limits muscle contraction speed by delaying the detachment of myosin from actin.cross-bridge cycle ͉ Drosophila ͉ kinetics ͉ myosin I n Drosophila melanogaster two sets of antagonistic, asynchronous flight muscles oscillate at Ϸ200 beats per second, powering the wings indirectly by deforming the thoracic cuticle into which the muscles and wings insert. Although the myofibrillar basis of oscillatory work and power production is known (1-5), the molecular adaptations that allow the indirect flight muscles (IFM) to operate at very high frequencies are less well understood. In muscles of slow-to-moderate speed, muscle velocity is thought to be limited by prolonging the time myosin spends strongly bound to actin before detachment (6, 7). The prolongation is essential for coupling enzyme chemical kinetics, which normally occur rapidly, to the slower movements of the sarcomere during normal muscle function. In most vertebrate striated muscle types, the comparatively slow release of MgADP (one of the products of MgATP hydrolysis) is thought to be the ratelimiting step (8-13). However, recent studies suggest MgADP release may not be rate limiting for faster muscle types (14-16). If so, we reasoned that a shift in rate-limiting step to another part of the cross-bridge cycle should be most readily apparent in working indirect insect flight muscle, the fastest known muscle type.We had directly shown that myosin isoforms determine Drosophila IFM speed by using genetic engineering methods to substitute a relatively slow embryonic myosin (EMB) for the native fast myosin (IFI) in the IFM (Fig. 1A Inset) (17,18). This substitution transformed the ...

Objective Congenital muscular dystrophy type 1A (MDC1A) is an autosomal recessive disease that is caused by loss-of-function mutations in the laminin-α2 gene and results in motor nerve and skeletal muscle dysfunction. In a previous study, we used genetic modifications to show that inappropriate induction of apoptosis was a significant contributor to pathogenesis in a laminin-α2-deficient mouse model of MDC1A. To identify a possible pharmacological therapy for laminin-α2-deficency, this study was designed to determine if treatment with minocycline or doxycycline, which are tetracycline derivatives reported to have anti-apoptotic effects in mammals, would significantly increase lifespan and improve neuromuscular function in laminin-α2-deficient mice. Methods Mice that were homozygous for a targeted, inactivating mutation of the laminin-α2-gene were placed into control, minocycline-treated, or doxycycline-treated groups. Drug treatment began within two weeks of birth and the progression of disease was followed over time using behavioral, growth, histological, and molecular assays. Results We found that treatment with either minocycline or doxycycline increased the median lifespan of laminin-α2-null mice from ∼32 days to ∼70 days. Furthermore, doxycycline improved postnatal growth rate and delayed the onset of hindlimb paralysis. Doxycycline-treated laminin-α2-deficient muscles had increased Akt phosphorylation, decreased inflammation, and decreased levels of Bax protein, TUNEL-positive myonuclei, and activated caspase-3. Interpretation Doxycycline or other drugs with similar functional profiles may be a possible route to improving neuromuscular dysfunction due to laminin-α2-deficiency.

BackgroundReal-time and dynamic assessment of an individual’s lipid homeostatic state in blood is complicated due to the need to collect samples in a clinical environment. In the context of precision medicine and population health, tools that facilitate sample collection and empower the individual to participate in the process are necessary to complement advanced bioanalytical analysis. The dried blood spot (DBS) methodology via finger prick or heel prick is a minimally invasive sample collection method that allows the relative ease and low cost of sample collection as well as transport. However, it has yet to be integrated into broad scale personalized lipidomic analysis. Therefore, in this study we report the development of a novel DBS high resolution MS/MSALL lipidomics workflow.MethodsIn this report we compared lipidomic analysis of four types of blood sample collection methods (DBS, venous whole blood, serum, and plasma) across several parameters, which include lipidomics coverage of each matrix and the effects of temperature and time on the coverage and stability of different lipid classes and molecular species. The novel DBS-MS/MSALL lipidomics platform developed in this report was then applied to examine postprandial effects on the blood lipidome and further to explore the temporal fluctuation of the lipidome across hours and days.ResultsMore than 1,200 lipid molecular species from a single DBS sample were identified and quantified. The lipidomics profile of the DBS samples is comparable to whole blood matrix. DBS-MS/MSALL lipidomic analysis in postprandial experiments revealed significant alterations in triacylglyceride species. Temporal analysis of the lipidome at various times in the day and across days identified several lipid species that fluctuate as a function of time, and a subset of lipid species were identified to be significantly altered across hours within a day and within successive days of the week.ConclusionsA novel DBS-MS/MSALL lipidomics method has been established for human blood. The feasibility and application of this method demonstrate the potential utility for lipidomics analysis in both healthy and diverse diseases states. This DBS MS-based lipidomics analysis represents a formidable approach for empowering patients and individuals in the era of precision medicine to uncover novel biomarkers and to monitor lipid homeostasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12986-017-0182-6) contains supplementary material, which is available to authorized users.

(1) Background: Interest in the application of metabolomics toward clinical diagnostics development and population health monitoring has grown significantly in recent years. In spite of several advances in analytical and computational tools, obtaining a sufficient number of samples from patients remains an obstacle. The dried blood spot (DBS) and dried urine strip (DUS) methodologies are a minimally invasive sample collection method allowing for the relative simplicity of sample collection and minimal cost. (2) Methods: In the current report, we compared results of targeted metabolomics analyses of four types of human blood sample collection methods (with and without DBS) and two types of urine sample collection (DUS and urine) across several parameters including the metabolite coverage of each matrix and the sample stability for DBS/DUS using commercially available Whatman 903TM paper. The DBS/DUS metabolomics protocols were further applied to examine the temporal metabolite level fluctuations within hours and days of sample collection. (3) Results: Several hundred polar metabolites were monitored using DBS/DUS. Temporal analysis of the polar metabolites at various times of the day and across days identified several species that fluctuate as a function of day and time. In addition, a subset of metabolites were identified to be significantly altered across hours within a day and within successive days of the week. (4) Conclusion: A comprehensive DBS/DUS metabolomics protocol was developed for human blood and urine analyses. The described methodology demonstrates the potential for enabling patients to contribute to the expanding bioanalytical demands of precision medicine and population health studies.

The severely debilitating disease Congenital Muscular Dystrophy Type 1A (MDC1A) is caused by mutations in the gene encoding laminin-alpha2. Bax-mediated muscle cell death is a significant contributor to the severe neuromuscular pathology seen in the Lama2-null mouse model of MDC1A. To extend our understanding of pathogenesis due to laminin-alpha2-deficiency, we have now analyzed molecular mechanisms of Bax regulation in normal and laminin-alpha2-deficient muscles and cells, including myogenic cells obtained from patients with a clinical diagnosis of MDC1A. In mouse myogenic cells, we found that, as in non-muscle cells, Bax co-immunoprecipitated with the multifunctional protein Ku70. In addition, cell permeable pentapeptides designed from Ku70, termed Bax-inhibiting peptides (BIPs), inhibited staurosporine-induced Bax translocation and cell death in mouse myogenic cells. We also found that acetylation of Ku70, which can inhibit binding to Bax and can be an indicator of increased susceptibility to cell death, was more abundant in Lama2-null than in normal mouse muscles. Furthermore, myotubes formed in culture from human laminin-alpha2-deficient patient myoblasts produced high levels of activated caspase-3 when grown on poly-L-lysine, but not when grown on a laminin-alpha2-containing substrate or when treated with BIPs. Finally, cytoplasmic Ku70 in human laminin-alpha2-deficient myotubes was both reduced in amount and more highly acetylated than in normal myotubes. Increased susceptibility to cell death thus appears to be an intrinsic property of human laminin-alpha2-deficient myotubes. These results identify Ku70 as a regulator of Bax-mediated pathogenesis and a therapeutic target in laminin-alpha2-deficiency.