3,318 research outputs found

    Analysis of replication factories in human cells by super-resolution light microscopy.

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    BACKGROUND: DNA replication in human cells is performed in discrete sub-nuclear locations known as replication foci or factories. These factories form in the nucleus during S phase and are sites of DNA synthesis and high local concentrations of enzymes required for chromatin replication. Why these structures are required, and how they are organised internally has yet to be identified. It has been difficult to analyse the structure of these factories as they are small in size and thus below the resolution limit of the standard confocal microscope. We have used stimulated emission depletion (STED) microscopy, which improves on the resolving power of the confocal microscope, to probe the structure of these factories at sub-diffraction limit resolution. RESULTS: Using immunofluorescent imaging of PCNA (proliferating cell nuclear antigen) and RPA (replication protein A) we show that factories are smaller in size (approximately 150 nm diameter), and greater in number (up to 1400 in an early S- phase nucleus), than is determined by confocal imaging. The replication inhibitor hydroxyurea caused an approximately 40% reduction in number and a 30% increase in diameter of replication factories, changes that were not clearly identified by standard confocal imaging. CONCLUSIONS: These measurements for replication factory size now approach the dimensions suggested by electron microscopy. This agreement between these two methods, that use very different sample preparation and imaging conditions, suggests that we have arrived at a true measurement for the size of these structures. The number of individual factories present in a single nucleus that we measure using this system is greater than has been previously reported. This analysis therefore suggests that each replication factory contains fewer active replication forks than previously envisaged.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

    Continuing and community education as engaged learning

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    CPE providers focus on understanding learning needs, designing and providing educational offerings, and evaluating programs. Cervero and Daly believe these decisions are made within the contested spaces of the social, professional, institutional, and educational systems; however, CPE continues to focus on the needs of the workplace and economic demands. We argue for a re-framing of how CPE is conceptualized and situated within the realm of adult learning and development specific to higher education institutions which engage in the endeavor, proposing the Community Transformation Model for Continuing and Extended Education to address neo-liberalism bias

    The Carina Nebula and Gum 31 molecular complex: II. The distribution of the atomic gas revealed in unprecedented detail

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    We report high spatial resolution observations of the HI 21cm line in the Carina Nebula and the Gum 31 region obtained with the Australia Telescope Compact Array. The observations covered \sim 12 deg2^2 centred on l=287.5deg,b=1degl= 287.5\deg,b = -1\deg, achieving an angular resolution of \sim 35 arcseconds. The HI map revealed complex filamentary structures across a wide range of velocities. Several "bubbles" are clearly identified in the Carina Nebula Complex, produced by the impact of the massive star clusters located in this region. An HI absorption profile obtained towards the strong extragalactic radio source PMN J1032--5917 showed the distribution of the cold component of the atomic gas along the Galactic disk, with the Sagittarius-Carina and Perseus spiral arms clearly distinguishable. Preliminary calculations of the optical depth and spin temperatures of the cold atomic gas show that the HI line is opaque (τ\tau \gtrsim 2) at several velocities in the Sagittarius-Carina spiral arm. The spin temperature is 100\sim100 K in the regions with the highest optical depth, although this value might be lower for the saturated components. The atomic mass budget of Gum 31 is 35%\sim35 \% of the total gas mass. HI self absorption features have molecular counterparts and good spatial correlation with the regions of cold dust as traced by the infrared maps. We suggest that in Gum 31 regions of cold temperature and high density are where the atomic to molecular gas phase transition is likely to be occurring.Comment: 20 pages, 1 table, 16 Figures, Accepted for Publication in the Monthly Notices of the Royal Astronomical Society Journa

    Highly neurotic never-depressed students have negative biases in information processing

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    BACKGROUND: Cognitive theories associate depression with negative biases in information processing. Although negatively biased cognitions are well documented in depressed patients and to some extent in recovered patients, it remains unclear whether these abnormalities are present before the first depressive episode. METHOD: High neuroticism (N) is a well-recognized risk factor for depression. The current study therefore compared different aspects of emotional processing in 33 high-N never-depressed and 32 low-N matched volunteers. Awakening salivary cortisol, which is often elevated in severely depressed patients, was measured to explore the neurobiological substrate of neuroticism. RESULTS: High-N volunteers showed increased processing of negative and/or decreased processing of positive information in emotional categorization and memory, facial expression recognition and emotion-potentiated startle (EPS), in the absence of global memory or executive deficits. By contrast, there was no evidence for effects of neuroticism on attentional bias (as measured with the dot-probe task), over-general autobiographical memory, or awakening cortisol levels. CONCLUSIONS: These results suggest that certain negative processing biases precede depression rather than arising as a result of depressive experience per se and as such could in part mediate the vulnerability of high-N subjects to depression. Longitudinal studies are required to confirm that such cognitive vulnerabilities predict subsequent depression in individual subjects

    One ring to rule them all? Another cellular responsibility for PCNA

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    To prevent duplication or loss of genomic regions during DNA replication, it is essential that the entire genome is copied precisely once every S phase. Cells achieve this by mutually exclusive regulation of origin firing and licensing. A crucial protein that is involved in origin licensing is chromatin licensing and DNA replication factor 1 (CDT1) and, therefore, activity of this protein must be strictly controlled. Four recent articles have demonstrated that proliferating cell nuclear antigen (PCNA), an essential sliding clamp used in replication and DNA repair, has a crucial role in this process by mediating the proteasomal degradation of CDT1

    Optimization Of Detergent-Mediated Reconstitution Of Influenza A M2 Protein Into Proteoliposomes

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    We report the optimization of detergent-mediated reconstitution of an integral membrane-bound protein, full-length influenza M2 protein, by direct insertion into detergent-saturated liposomes. Detergent-mediated reconstitution is an important method for preparing proteoliposomes for studying membrane proteins, and must be optimized for each combination of protein and membrane constituents used. The purpose of the reconstitution was to prepare samples for site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) studies. Our goals in optimizing the protocol were to minimize the amount of detergent used, reduce overall proteoliposome preparation time, and confirm the removal of all detergent. The liposomes were comprised of (1-palmitoyl-2-oleyl-sn-glycero-phosphocholine (POPC) and 1-palmitoyl-2-oleyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), and the detergent octylglucoside (OG) was used for reconstitution. Rigorous physical characterization was applied to optimize each step of the reconstitution process. We used dynamic light scattering (DLS) to determine the amount of OG needed to saturate the preformed liposomes. During detergent removal by absorption with Bio-Beads, we quantified the detergent concentration by means of a colorimetric assay, thereby determining the number of Bio-Bead additions needed to remove all detergent from the final proteoliposomes. We found that the overnight Bio-Bead incubation used in previously published protocols can be omitted, reducing the time needed for reconstitution. We also monitored the size distribution of the proteoliposomes with DLS, confirming that the size distribution remains essentially constant throughout the reconstitution process

    Serious Bacterial Infections Acquired During Treatment of Patients Given a Diagnosis of Chronic Lyme Disease - United States.

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    The term "chronic Lyme disease" is used by some health care providers as a diagnosis for various constitutional, musculoskeletal, and neuropsychiatric symptoms (1,2). Patients with a diagnosis of chronic Lyme disease have been provided a wide range of medications as treatment, including long courses of intravenous (IV) antibiotics (3,4). Studies have not shown that such treatments lead to substantial long-term improvement for patients, and they can be harmful (1,5). This report describes cases of septic shock, osteomyelitis, Clostridium difficile colitis, and paraspinal abscess resulting from treatments for chronic Lyme disease. Patients, clinicians, and public health practitioners should be aware that treatments for chronic Lyme disease can carry serious risks
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