Methods in molecular biology
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Before there was access to space, all experiments on plant tropisms were conducted upon the background of gravity. The gravity vector could be disrupted, such as with clinorotation and random positioning machines, and by manipulating incident angles of root growth with respect to gravity, such as with Darwin's plants on slanted plates, but gravity could not be removed from the experimental equation. Access to microgravity through spaceflight has opened new doors to plant research. Here we provide an overview of some of the methodologies of conducting plant research in the unique spaceflight environment.
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Reprogramming of somatic cells, such as skin fibroblasts, to pluripotency was first achieved by forced expression of four transcription factors using integrating retroviral or lentiviral vectors, which result in integration of exogenous DNA into cellular genome and present a formidable barrier to therapeutic application of induced pluripotent stem cells (iPSCs). To facilitate the translation of iPSC technology to clinical practice, mRNA reprogramming method that generates transgene-free iPSCs is a safe and efficient method, eliminating bio-containment concerns associated with viral vectors, as well as the need for weeks of screening of cells to confirm that viral material has been completely eliminated during cell passaging.
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Visualization of protein-protein interactions in vivo offers a powerful tool to resolve spatial and temporal aspects of cellular functions. The bimolecular fluorescence complementation (BiFC) makes use of nonfluorescent fragments of green fluorescent protein or its variants that are added as "tags" to target proteins under study. Only upon target protein interaction is a fluorescent protein complex assembled, and the site of interaction can be monitored by microscopy. In this chapter, we describe the method and tools for the use of BiFC in the yeast Saccharomyces cerevisiae and in mammalian cells.
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Retinal cell apoptosis occurs in many eye conditions, including glaucoma, diabetic retinopathy and Alzheimer's disease. Real-time detection of retinal cell apoptosis has potential clinical value in early disease detection, as well as evaluating disease progression and treatment efficacy. Here, we describe our novel imaging technology DARC (Detection of Apoptosing Retinal Cells), which can be used to visualize single retinal neurons undergoing apoptosis in real time, by using fluorescently labeled Annexin A5 and confocal scanning laser ophthalmoscopy (cSLO ). Clinical trials of DARC in glaucoma patients are due to start shortly, but in this chapter, we describe this technique in experimental animal models.
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Tremor is the most common movement disorder. However; characterizing it in large populations is not easily accomplished since current methodologies are not adapted to large-scale field studies. To overcome this challenge, a smartphone application was developed as a stand-alone platform to assess tremor. ⋯ This allowed for the identification of the tremor properties that could reliably be characterized with the smartphone as well as the limits of the hardware. It also allowed for the identification of tasks that could be performed with the smartphone when tremor was being assessed. Finally, we confirmed the clinical relevance of the results provided by the smartphone application.