293 research outputs found

    Comparison of fluorescence-based techniques for the quantification of particle-induced hydroxyl radicals

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    <p>Abstract</p> <p>Background</p> <p>Reactive oxygen species including hydroxyl radicals can cause oxidative stress and mutations. Inhaled particulate matter can trigger formation of hydroxyl radicals, which have been implicated as one of the causes of particulate-induced lung disease. The extreme reactivity of hydroxyl radicals presents challenges to their detection and quantification. Here, three fluorescein derivatives [aminophenyl fluorescamine (APF), amplex ultrared, and dichlorofluorescein (DCFH)] and two radical species, proxyl fluorescamine and tempo-9-ac have been compared for their usefulness to measure hydroxyl radicals generated in two different systems: a solution containing ferrous iron and a suspension of pyrite particles.</p> <p>Results</p> <p>APF, amplex ultrared, and DCFH react similarly to the presence of hydroxyl radicals. Proxyl fluorescamine and tempo-9-ac do not react with hydroxyl radicals directly, which reduces their sensitivity. Since both DCFH and amplex ultrared will react with reactive oxygen species other than hydroxyl radicals and another highly reactive species, peroxynitite, they lack specificity.</p> <p>Conclusion</p> <p>The most useful probe evaluated here for hydroxyl radicals formed from cell-free particle suspensions is APF due to its sensitivity and selectivity.</p

    Role of pyrite in formation of hydroxyl radicals in coal: possible implications for human health

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    BACKGROUND: The harmful effects from inhalation of coal dust are well-documented. The prevalence of lung disease varies by mining region and may, in part, be related to regional differences in the bioavailable iron content of the coal. Pyrite (FeS(2)), a common inorganic component in coal, has been shown to spontaneously form reactive oxygen species (ROS) (i.e., hydrogen peroxide and hydroxyl radicals) and degrade nucleic acids. This raises the question regarding the potential for similar reactivity from coal that contains pyrite. Experiments were performed to specifically evaluate the role of pyrite in coal dust reactivity. Coal samples containing various amounts of FeS(2 )were compared for differences in their generation of ROS and degradation of RNA. RESULTS: Coals that contain iron also show the presence of FeS(2), generate ROS and degrade RNA. Coal samples that do not contain pyrite do not produce ROS nor degrade RNA. The concentration of generated ROS and degradation rate of RNA both increase with greater FeS(2 )content in the coals. CONCLUSION: The prevalence of coal workers' pneumoconiosis can be correlated to the amount of FeS(2 )in the coals. Considering the harmful effects of generation of ROS by inhaled particles, the results presented here show a possible mechanism whereby coal samples may contribute to CWP. This suggests that the toxicity of coal may be explained, in part, by the presence of FeS(2)

    Transcriptional profiling of putative human epithelial stem cells

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    <p>Abstract</p> <p>Background</p> <p>Human interfollicular epidermis is sustained by the proliferation of stem cells and their progeny, transient amplifying cells. Molecular characterization of these two cell populations is essential for better understanding of self renewal, differentiation and mechanisms of skin pathogenesis. The purpose of this study was to obtain gene expression profiles of alpha 6<sup>+</sup>/MHCI<sup>+</sup>, transient amplifying cells and alpha 6<sup>+</sup>/MHCI<sup>-</sup>, putative stem cells, and to compare them with existing data bases of gene expression profiles of hair follicle stem cells. The expression of Major Histocompatibility Complex (MHC) class I, previously shown to be absent in stem cells in several tissues, and alpha 6 integrin were used to isolate MHCI positive basal cells, and MHCI low/negative basal cells.</p> <p>Results</p> <p>Transcriptional profiles of the two cell populations were determined and comparisons made with published data for hair follicle stem cell gene expression profiles. We demonstrate that presumptive interfollicular stem cells, alpha 6<sup>+</sup>/MHCI<sup>- </sup>cells, are enriched in messenger RNAs encoding surface receptors, cell adhesion molecules, extracellular matrix proteins, transcripts encoding members of IFN-alpha family proteins and components of IFN signaling, but contain lower levels of transcripts encoding proteins which take part in energy metabolism, cell cycle, ribosome biosynthesis, splicing, protein translation, degradation, DNA replication, repair, and chromosome remodeling. Furthermore, our data indicate that the cell signaling pathways Notch1 and NF-κB are downregulated/inhibited in MHC negative basal cells.</p> <p>Conclusion</p> <p>This study demonstrates that alpha 6<sup>+</sup>/MHCI<sup>- </sup>cells have additional characteristics attributed to stem cells. Moreover, the transcription profile of alpha 6<sup>+</sup>/MHCI<sup>- </sup>cells shows similarities to transcription profiles of mouse hair follicle bulge cells known to be enriched for stem cells. Collectively, our data suggests that alpha 6<sup>+</sup>/MHCI<sup>- </sup>cells may be enriched for stem cells. This study is the first comprehensive gene expression profile of putative human epithelial stem cells and their progeny that were isolated directly from neonatal foreskin tissue. Our study is important for understanding self renewal and differentiation of epidermal stem cells, and for elucidating signaling pathways involved in those processes. The generated data base may serve those working with other human epithelial tissue progenitors.</p

    Differential Regulation of Lipoprotein and Hepatitis C Virus Secretion by Rab1b

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    Secretory cells produce diverse cargoes, yet how they regulate concomitant secretory traffic remains insufficiently explored. Rab GTPases control intracellular vesicular transport. To map secretion pathways, we generated a library of lentivirus-expressed dominant-negative Rab mutants and used it in a large-scale screen to identify regulators of hepatic lipoprotein secretion. We identified several candidate pathways, including those mediated by Rab11 and Rab8. Surprisingly, inhibition of Rab1b, the major regulator of transport from the endoplasmic reticulum to the Golgi, differently affected the secretion of the very-low-density lipoprotein components ApoE and ApoB100, despite their final association on mature secreted lipoprotein particles. Since hepatitis C virus (HCV) incorporates ApoE and ApoB100 into its virus particle, we also investigated infectious HCV secretion and show that its regulation by Rab1b mirrors that of ApoB100. These observations reveal differential regulation of hepatocyte secretion by Rab1b and advance our understanding of lipoprotein assembly and lipoprotein and HCV secretion

    Absolute dimensions of eclipsing binaries. XXVI, Setting a new standard : masses, radii, and abundances for the F-type systems AD Bootis, VZ Hydrae, and WZ Ophiuchi

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    Context. Accurate mass, radius, and abundance determinations from binaries provide important information on stellar evolution, fundamental to central fields in modern astrophysics and cosmology. Aims. We aim to determine absolute dimensions and abundances for the three F-type main-sequence detached eclipsing binaries ADBoo, VZHya, and WZOph and to perform a detailed comparison with results from recent stellar evolutionary models. Methods. uvby light curves and uvbyβ standard photometry were obtained with the Strömgren Automatic Telescope at ESO, La Silla, radial velocity observations at CfA facilities, and supplementary high-resolution spectra with ESO’s FEROS spectrograph. State-ofthe-art methods were applied for the analyses: the EBOP andWilson-Devinney binary models, two-dimensional cross-correlation and disentangling, and the VWA abundance analysis tool. Results. Masses and radii that are precise to 0.5–0.7% and 0.4–0.9%, respectively, have been established for the components, which span the ranges of 1.1 to 1.4 M and 1.1 to 1.6 R. The [Fe/H] abundances are from –0.27 to +0.10, with uncertainties between 0.07 and 0.15 dex. We find indications of a slight α-element overabundance of [α/Fe] ∼ +0.1 for WZOph. The secondary component of ADBoo and both components of WZOph appear to be slightly active. Yale-Yonsai and Victoria-Regina evolutionary models fit the components of ADBoo and VZHya almost equally well, assuming coeval formation, at ages of about 1.75/1.50 Gyr (ADBoo) and 1.25/1.00 Gyr (VZHya). BaSTI models, however, predict somewhat different ages for the primary and secondary components. For WZOph, the models from all three grids are significantly hotter than observed. A low He content, decreased envelope convection coupled with surface activity, and/or higher interstellar absorption would remove the discrepancy, but its cause has not been definitively identified. Conclusions. We have demonstrated the power of testing and comparing recent stellar evolutionary models using eclipsing binaries, provided their abundances are known. The strongest limitations and challenges are set by Teff and interstellar absorption determinations, and by their effects on and correlation with abundance results

    Patients and animal models of CNGβ1-deficient retinitis pigmentosa support gene augmentation approach.

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    Retinitis pigmentosa (RP) is a major cause of blindness that affects 1.5 million people worldwide. Mutations in cyclic nucleotide-gated channel β 1 (CNGB1) cause approximately 4% of autosomal recessive RP. Gene augmentation therapy shows promise for treating inherited retinal degenerations; however, relevant animal models and biomarkers of progression in patients with RP are needed to assess therapeutic outcomes. Here, we evaluated RP patients with CNGB1 mutations for potential biomarkers of progression and compared human phenotypes with those of mouse and dog models of the disease. Additionally, we used gene augmentation therapy in a CNGβ1-deficient dog model to evaluate potential translation to patients. CNGB1-deficient RP patients and mouse and dog models had a similar phenotype characterized by early loss of rod function and slow rod photoreceptor loss with a secondary decline in cone function. Advanced imaging showed promise for evaluating RP progression in human patients, and gene augmentation using adeno-associated virus vectors robustly sustained the rescue of rod function and preserved retinal structure in the dog model. Together, our results reveal an early loss of rod function in CNGB1-deficient patients and a wide window for therapeutic intervention. Moreover, the identification of potential biomarkers of outcome measures, availability of relevant animal models, and robust functional rescue from gene augmentation therapy support future work to move CNGB1-RP therapies toward clinical trials

    Cascading signaling pathways improve the fidelity of a stochastically and deterministically simulated molecular RS latch

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    <p>Abstract</p> <p>Background</p> <p>While biological systems have often been compared with digital systems, they differ by the strong effect of crosstalk between signals due to diffusivity in the medium, reaction kinetics and geometry. Memory elements have allowed the creation of autonomous digital systems and although biological systems have similar properties of autonomy, equivalent memory mechanisms remain elusive. Any such equivalent memory system, however, must silence the effect of crosstalk to maintain memory fidelity.</p> <p>Results</p> <p>Here, we present a system of enzymatic reactions that behaves like an RS latch (a simple memory element in digital systems). Using both a stochastic molecular simulator and ordinary differential equation simulator, we showed that crosstalk between two latches operating in the same spatial localization disrupts the memory fidelity of both latches. Crosstalk was reduced or silenced when simple reaction loops were replaced with multiple step or cascading reactions, showing that cascading signaling pathways are less susceptible to crosstalk.</p> <p>Conclusion</p> <p>Thus, the common biological theme of cascading signaling pathways is advantageous for maintaining the fidelity of a memory latch in the presence of crosstalk. The experimental implementation of such a latch system will lead to novel approaches to cell control using synthetic proteins and will contribute to our understanding of why cells behave differently even when given the same stimulus.</p

    Towards the clinical implementation of pharmacogenetics in bipolar disorder.

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    BackgroundBipolar disorder (BD) is a psychiatric illness defined by pathological alterations between the mood states of mania and depression, causing disability, imposing healthcare costs and elevating the risk of suicide. Although effective treatments for BD exist, variability in outcomes leads to a large number of treatment failures, typically followed by a trial and error process of medication switches that can take years. Pharmacogenetic testing (PGT), by tailoring drug choice to an individual, may personalize and expedite treatment so as to identify more rapidly medications well suited to individual BD patients.DiscussionA number of associations have been made in BD between medication response phenotypes and specific genetic markers. However, to date clinical adoption of PGT has been limited, often citing questions that must be answered before it can be widely utilized. These include: What are the requirements of supporting evidence? How large is a clinically relevant effect? What degree of specificity and sensitivity are required? Does a given marker influence decision making and have clinical utility? In many cases, the answers to these questions remain unknown, and ultimately, the question of whether PGT is valid and useful must be determined empirically. Towards this aim, we have reviewed the literature and selected drug-genotype associations with the strongest evidence for utility in BD.SummaryBased upon these findings, we propose a preliminary panel for use in PGT, and a method by which the results of a PGT panel can be integrated for clinical interpretation. Finally, we argue that based on the sufficiency of accumulated evidence, PGT implementation studies are now warranted. We propose and discuss the design for a randomized clinical trial to test the use of PGT in the treatment of BD
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