Mitochondrial Allotopic Gene Therapy Approaches Using a Drosophila Model with an Endogenous ATP6 Mutation

Mitochondrial Encephalomyopathies are a group of disorders with common symptoms such as neurological, cardiac, and muscular dysfunctions. Mutations in ATP6, a protein-coding gene in the mitochondria genome, can lead to NARP, MILS, or FBSN diseases. ATP6 encodes a protein subunit of the ATP synthase, also known as complex V. Currently, there is no cure for patients with ATP6 mutations, and pharmacotherapies provide limited benefits. Because manipulation of mitochondrial genome is extremely difficult, allotopic expression of ATP6 – specifically, expressing the mitochondrially-encoded ATP6 gene in the nucleus – has been championed as a potential gene therapy strategy. Efficacies of allotopic rescue in in vitro systems have been controversial, with some studies showing the restoration of ATP synthase activity and some showing the lack of any rescue effects. We have isolated a Drosophila strain with a missense mutation in ATP6. The phenotypes of this mutant ATP6 strain have been characterized and are very similar to those of human patients, making it an excellent model for diseases caused by ATP6 mutation. The overarching goal of this dissertation is to import nucleus-encoded ATP6 protein into the mitochondria. This work examines the efficacies of multiple strategies in enhancing the functional outcomes of the first animal model with a stable and endogenous ATP6 mutation and shows that algal ATP6 protein provides the most promising rescue results

Modes of Metabolic Compensation during Mitochondrial Disease Using the Drosophila Model of ATP6 Dysfunction

Numerous mitochondrial DNA mutations cause mitochondrial encephalomyopathy: a collection of related diseases for which there exists no effective treatment. Mitochondrial encephalomyopathies are complex multisystem diseases that exhibit a relentless progression of severity, making them both difficult to treat and study. The pathogenic and compensatory metabolic changes that are associated with chronic mitochondrial dysfunction are not well understood. The Drosophila ATP61 mutant models human mitochondrial encephalomyopathy and allows the study of metabolic changes and compensation that occur throughout the lifetime of an affected animal. ATP61animals have a nearly complete loss of ATP synthase activity and an acute bioenergetic deficit when they are asymptomatic, but surprisingly we discovered no chronic bioenergetic deficit in these animals during their symptomatic period. Our data demonstrate dynamic metabolic compensatory mechanisms that sustain normal energy availability and activity despite chronic mitochondrial complex V dysfunction resulting from an endogenous mutation in the mitochondrial DNA. ATP61animals compensate for their loss of oxidative phosphorylation through increases in glycolytic flux, ketogenesis and Kreb's cycle activity early during pathogenesis. However, succinate dehydrogenase activity is reduced and mitochondrial supercomplex formation is severely disrupted contributing to the pathogenesis seen in ATP61 animals. These studies demonstrate the dynamic nature of metabolic compensatory mechanisms and emphasize the need for time course studies in tractable animal systems to elucidate disease pathogenesis and novel therapeutic avenues

M (2011) Modes of metabolic compensation during mitochondrial disease using the Drosophila model of ATP6 dysfunction

Abstract Numerous mitochondrial DNA mutations cause mitochondrial encephalomyopathy: a collection of related diseases for which there exists no effective treatment. Mitochondrial encephalomyopathies are complex multisystem diseases that exhibit a relentless progression of severity, making them both difficult to treat and study. The pathogenic and compensatory metabolic changes that are associated with chronic mitochondrial dysfunction are not well understood. The Drosophila ATP6 1 mutant models human mitochondrial encephalomyopathy and allows the study of metabolic changes and compensation that occur throughout the lifetime of an affected animal. ATP6 1 animals have a nearly complete loss of ATP synthase activity and an acute bioenergetic deficit when they are asymptomatic, but surprisingly we discovered no chronic bioenergetic deficit in these animals during their symptomatic period. Our data demonstrate dynamic metabolic compensatory mechanisms that sustain normal energy availability and activity despite chronic mitochondrial complex V dysfunction resulting from an endogenous mutation in the mitochondrial DNA. ATP6 1 animals compensate for their loss of oxidative phosphorylation through increases in glycolytic flux, ketogenesis and Kreb's cycle activity early during pathogenesis. However, succinate dehydrogenase activity is reduced and mitochondrial supercomplex formation is severely disrupted contributing to the pathogenesis seen in ATP6 1 animals. These studies demonstrate the dynamic nature of metabolic compensatory mechanisms and emphasize the need for time course studies in tractable animal systems to elucidate disease pathogenesis and novel therapeutic avenues

Recording Natural Head Position Using Cone Beam Computerized Tomography

The purpose of this study was to develop a technique to record the natural head position (NHP) of a subject using the scout images of cone beam computerized tomography (CBCT) scans. The first step was to align a hanging mirror with the vertical (XY) plane of the CBCT field-of-view (FOV) volume. Then, two scout CBCT images, at frontal and at sagittal planes, were taken when the subject exhibited a NHP. A normal CBCT scan on the subject was then taken separately. These scout images were used to correct the orientation of the normal CBCT scan. A phantom head was used for validation and performance analysis of the proposed method. It was found that the orientation detection error was within 0.88°. This enables easy and economic NHP recording for CBCT without additional hardware

Alkyl Polyglucoside (APG) Nonionic Surfactant-Based Reverse Micellar Dyeing of Cotton Fabric – A Study of Reactive Dyes with Different Functional Groups

Reverse micellar dyeing of cotton woven fabrics with natural and biodegradable alkyl polyglucoside-based (APG-based) nonionic surfactant in decamethylcyclopentasiloxane (D5) non-aqueous medium with reactive dyes of different functional groups was investigated and compared with conventional aqueous water-based dyeing in terms of reflectance, color yield, levelness, CIE L*a*b* values, washing fastness and color fading properties. Experimental results have revealed that APG reverse micellar method can provide a higher color yield (K/Ssum value) and lower color reflectance (better dye uptake) than conventional water-based method, along with comparable washing fastness, leveling and color fading properties. Hetero-bifunctional reactive dyes can attain the highest color yield, followed by homo-bifunctional reactive dyes and mono-functional reactive dyes. Mono-functional reactive dyes and homo-bifunctional reactive dyes produce the best levelling properties with smallest levelness variation when compared with that of hetero-bifunctional reactive dyes in non-aqueous D5 dyeing medium

Spontaneous and Explicit Estimation of Time Delays in the Absence/Presence of Multipath Propagation

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