Mutations in PTEN-induced kinase 1 (pink1) or parkin cause autosomal-recessive and some sporadic forms of Parkinson's disease. pink1 acts upstream of parkin in a common genetic pathway to regulate mitochondrial integrity in Drosophila. Mitochondrial morphology is maintained by a dynamic balance between the opposing actions of mitochondrial fusion, controlled by Mitofusin (mfn) and Optic atrophy 1 (opa1), and mitochondrial fission, controlled by drp1. Here, we explore interactions between pink1/parkin and the mitochondrial fusion/fission machinery. Muscle-specific knockdown of the fly homologue of Mfn (Marf ) or opa1, or overexpression of drp1, results in significant mitochondrial fragmentation. Mfn-knockdown flies also display altered cristae morphology. Interestingly, knockdown of Mfn or opa1 or overexpression of drp1, rescues the phenotypes of muscle degeneration, cell death, and mitochondrial abnormalities in pink1 or parkin mutants. In the male germline, we also observe genetic interactions between pink1 and the testes-specific mfn homologue fuzzy onion, and between pink1 and drp1. Our data suggest that the pink1/parkin pathway promotes mitochondrial fission and/or inhibits fusion by negatively regulating mfn and opa1 function, and/or positively regulating drp1. However, pink1 and parkin mutant flies show distinct mitochondrial phenotypes from drp1 mutant flies, and flies carrying a heterozygous mutation in drp1 enhance the pink1-null phenotype, resulting in lethality. These results suggest that pink1 and parkin are likely not core components of the drp1-mediated mitochondrial fission machinery. Modification of fusion and fission may represent a novel therapeutic strategy for Parkinson's disease.arkinson's disease (PD), the second most common neurodegenerative disorder, is characterized by degeneration of dopaminergic neurons in the midbrain (1). Although the exact cause of PD is unclear, mitochondrial toxins such as 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) can selectively destroy dopaminergic neurons and cause clinical features similar to PD (2, 3). Moreover, mitochondrial respiratory dysfunction also occurs in sporadic PD (4). The most compelling evidence for a mitochondrial etiology of PD, however, derives from the study of genes mediating familial forms of the disease (4, 5). Mutations in PTEN-induced kinase 1 (Pink1; PARK6), which encodes a serine-threonine kinase localized to mitochondria, and parkin (PARK2), which encodes a RING finger-containing E3 ubiquitin ligase, have been found in recessively inherited and sporadic PD cases (6-9). Previously, we and others have reported that Drosophila pink1 and parkin function in the same genetic pathway, with pink1 acting upstream of parkin, to regulate mitochondrial integrity in testes, muscle, and dopaminergic neurons (10-12). Flies lacking pink1 or parkin function are viable and show muscle degeneration and TUNEL staining, indicative of cell death (10-13). Subsequent studies have shown that parkin can suppress mitochondrial defects caused by pink1...

Replacement of wild insect populations with genetically modified individuals unable to transmit disease provides a self-perpetuating method of disease prevention but requires a gene drive mechanism to spread these traits to high frequency. Drive mechanisms requiring that transgenes exceed a threshold frequency in order to spread are attractive because they bring about local but not global replacement, and transgenes can be eliminated through dilution of the population with wild-type individuals. These features are likely to be important in many social and regulatory contexts. Here we describe the first creation of a synthetic threshold-dependent gene drive system, designated maternal-effect lethal underdominance (UD(MEL)), in which two maternally expressed toxins, located on separate chromosomes, are each linked with a zygotic antidote able to rescue maternal-effect lethality of the other toxin. We demonstrate threshold-dependent replacement in single- and two-locus configurations in Drosophila. Models suggest that transgene spread can often be limited to local environments. They also show that in a population in which single-locus UD(MEL) has been carried out, repeated release of wild-type males can result in population suppression, a novel method of genetic population manipulation.

Caspase family proteases play important roles in the regulation of apoptotic cell death. Initiator caspases are activated in response to death stimuli, and they transduce and amplify these signals by cleaving and thereby activating effector caspases. In Drosophila, the initiator caspase Nc (previously Dronc) cleaves and activates two short-prodomain caspases, Dcp-1 and Ice (previously Drice), suggesting these as candidate effectors of Nc killing activity. dcp-1-null mutants are healthy and possess few defects in normally occurring cell death. To explore roles for Ice in cell death, we generated and characterized an Ice null mutant. Animals lacking Ice show a number of defects in cell death, including those that occur during embryonic development, as well as during formation of adult eyes, arista and wings. Ice mutants exhibit subtle defects in the destruction of larval tissues, and do not prevent destruction of salivary glands during metamorphosis. Cells from Ice animals are also markedly resistant to several stresses, including X-irradiation and inhibition of protein synthesis. Mutations in Ice also suppress cell death that is induced by expression of Rpr, Wrinkled (previously Hid) and Grim. These observations demonstrate that Ice plays an important non-redundant role as a cell death effector. Finally, we demonstrate that Ice participates in, but is not absolutely required for, the non-apoptotic process of spermatid differentiation.

Insects act as vectors for diseases of plants, animals and humans. Replacement of wild insect populations with genetically modified individuals unable to transmit disease provides a potentially self-perpetuating method of disease prevention. Population replacement requires a gene drive mechanism in order to spread linked genes mediating disease refractoriness through wild populations. We previously reported the creation of synthetic Medea selfish genetic elements able to drive population replacement in Drosophila. These elements use microRNA-mediated silencing of myd88, a maternally expressed gene required for embryonic dorso-ventral pattern formation, coupled with early zygotic expression of a rescuing transgene, to bring about gene drive. Medea elements that work through additional mechanisms are needed in order to be able to carry out cycles of population replacement and/or remove existing transgenes from the population, using second-generation elements that spread while driving first-generation elements out of the population. Here we report the synthesis and population genetic behavior of two new synthetic Medea elements that drive population replacement through manipulation of signaling pathways involved in cellular blastoderm formation or Notch signaling, demonstrating that in Drosophila Medea elements can be generated through manipulation of diverse signaling pathways. We also describe the mRNA and small RNA changes in ovaries and early embryos associated from Medea-bearing females. Finally, we use modeling to illustrate how Medea elements carrying genes that result in diapause-dependent female lethality could be used to bring about population suppression.

One proposed strategy for controlling the transmission of insect-borne pathogens uses a drive mechanism to ensure the rapid spread of transgenes conferring disease refractoriness throughout wild populations. Here, we report the creation of maternal-effect selfish genetic elements in Drosophila that drive population replacement and are resistant to recombination-mediated dissociation of drive and disease refractoriness functions. These selfish elements use microRNA-mediated silencing of a maternally expressed gene essential for embryogenesis, which is coupled with early zygotic expression of a rescuing transgene.

BackgroundThe inter-patient classification schema and the Association for the Advancement of Medical Instrumentation (AAMI) standards are important to the construction and evaluation of automated heartbeat classification systems. The majority of previously proposed methods that take the above two aspects into consideration use the same features and classification method to classify different classes of heartbeats. The performance of the classification system is often unsatisfactory with respect to the ventricular ectopic beat (VEB) and supraventricular ectopic beat (SVEB).MethodsBased on the different characteristics of VEB and SVEB, a novel hierarchical heartbeat classification system was constructed. This was done in order to improve the classification performance of these two classes of heartbeats by using different features and classification methods. First, random projection and support vector machine (SVM) ensemble were used to detect VEB. Then, the ratio of the RR interval was compared to a predetermined threshold to detect SVEB. The optimal parameters for the classification models were selected on the training set and used in the independent testing set to assess the final performance of the classification system. Meanwhile, the effect of different lead configurations on the classification results was evaluated.ResultsResults showed that the performance of this classification system was notably superior to that of other methods. The VEB detection sensitivity was 93.9% with a positive predictive value of 90.9%, and the SVEB detection sensitivity was 91.1% with a positive predictive value of 42.2%. In addition, this classification process was relatively fast.ConclusionsA hierarchical heartbeat classification system was proposed based on the inter-patient data division to detect VEB and SVEB. It demonstrated better classification performance than existing methods. It can be regarded as a promising system for detecting VEB and SVEB of unknown patients in clinical practice.

Advances in insect transgenesis and our knowledge of insect physiology and genomics are making it possible to create transgenic populations of beneficial or pest insects that express novel traits. There are contexts in which we may want the transgenes responsible for these traits to spread so that all individuals within a wild population carry them, a process known as population replacement. Transgenes of interest are unlikely to confer an overall fitness benefit on those who carry them. Therefore, an essential component of any population replacement strategy is the presence of a drive mechanism that will ensure the spread of linked transgenes. We discuss contexts in which population replacement might be desirable and the requirements a drive system must satisfy to be both effective and safe. We then describe the creation of synthetic Medea elements, the first selfish genetic elements synthesized de novo, with the capability of driving population replacement, in this case in Drosophila. The strategy used to create Drosophila Medea is applicable to a number of other insect species and the Medea system satisfies key requirements for scientific and social acceptance. Finally, we highlight several challenges to implementing population replacement in the wild.

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