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    ABSTRACT: RNA folding pathways can be complex and even include kinetic traps or misfolded intermediates that can be slow to resolve. Characterizing these pathways is critical to understanding how RNA molecules acquire their biological function. We have previously developed a novel approach to help characterize such misfolded intermediates. Laser-assisted single-molecule refolding (LASR) is a powerful technique that combines temperature-jump (T-jump) kinetics with single-molecule detection. In a typical LASR experiment, the temperature is rapidly increased and conformational dynamics are characterized, in real-time, at the single-molecule level using single-molecule fluorescence resonance energy transfer (smFRET). Here, we provide detailed protocols for performing LASR experiments including sample preparation, temperature calibration, and data analysis.
    Full-text · Article · Jan 2014 · Methods in molecular biology (Clifton, N.J.)
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    ABSTRACT: Viruses that establish a persistent infection, involving intracellular latency, commonly stimulate cellular DNA synthesis and sometimes cell division early after infection. However, most cells of metazoans have evolved "fail-safe" responses that normally monitor unscheduled DNA synthesis and prevent cell proliferation when, for instance, cell proto-oncogenes are "activated" by mutation, amplification, or chromosomal rearrangements. These cell intrinsic defense mechanisms that reduce the risk of neoplasia and cancer are collectively called oncogenic stress responses (OSRs). Mechanisms include the activation of tumor suppressor genes and the so-called DNA damage response that together trigger pathways leading to cell cycle arrest (e.g., cell senescence) or complete elimination of cells (e.g., apoptosis). It is not surprising that viruses that can induce cellular DNA synthesis and cell division have the capacity to trigger OSR, nor is it surprising that these viruses have evolved countermeasures for inactivating or bypassing OSR. The main focus of this review is how the human tumor-associated Epstein-Barr virus manipulates the host polycomb group protein system to control - by epigenetic repression of transcription - key components of the OSR during the transformation of normal human B cells into permanent cell lines.
    Full-text · Article · Oct 2013 · Frontiers in Genetics
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    ABSTRACT: Herpes simplex virus 1 (HSV1) is an enveloped virus that uses undefined transport carriers for trafficking of its glycoproteins to envelopment sites. Screening of an siRNA library against 60 Rab GTPases revealed Rab6 as the principal Rab involved in HSV1 infection, with its depletion preventing Golgi-to-plasma membrane transport of HSV1 glycoproteins in a pathway used by several integral membrane proteins but not the luminal secreted protein Gaussia luciferase. Knockdown of Rab6 reduced virus yield to 1% and inhibited capsid envelopment, revealing glycoprotein exocytosis as a prerequisite for morphogenesis. Rab6-dependent virus production did not require the effectors myosin-II, bicaudal-D, dynactin-1 or rabkinesin-6, but was facilitated by ERC1, a factor involved in linking microtubules to the cell cortex. Tubulation and exocytosis of Rab6-positive, glycoprotein-containing membranes from the Golgi was substantially augmented by infection, resulting in enhanced and targeted delivery to cell tips. This reveals HSV1 morphogenesis as one of the first biological processes shown to be dependent on the exocytic activity of Rab6.
    Full-text · Article · Oct 2013 · Traffic
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Journal of Virological Methods 07/2007; 143(1):81-5. DOI:10.1016/j.jviromet.2007.02.009
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