709 research outputs found

    Intra-amniotic delivery of CFTR-expressing adenovirus does not reverse cystic fibrosis phenotype in inbred CFTR-knockout mice

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    This article is available open access through the publisher’s website at the link below. Copyright © 2008 The American Society of Gene Therapy.Due to its early onset and severe prognosis, cystic fibrosis (CF) has been suggested as a candidate disease for in utero gene therapy. In 1997, a study was published claiming that to how transient prenatal expression of CF transmembrane conductance regulator (CFTR) from an in utero –injected adenovirus vector could achieve permanent reversal of the CF intestinal pathology in adult CF knockout mice, despite the loss of CFTR transgene expression by birth. This would imply that the underlying cause of CF is a prenatal defect for which lifelong cure can be achieved by transient prenatal expression of CFTR. Despite criticism at the time of publication, no independent verification of this contentious finding has been published so far. This is vital for the development of future therapeutic strategies as it may determine whether CF gene therapy should be performed prenatally or postnatally. We therefore reinvestigated this finding with an identical adenoviral vector and a knockout CF mouse line (CftrtmlCam) with a completely inbred genetic background to eliminate any effects due to genetic variation. After delivery of the CFTR-expressing adenovirus to the fetal mouse, both vector DNA and transgenic CFTR expression were detected in treated animals postpartum but statistically no significant difference in survival was observed between the Cftr–/– mice treated with the CFTR-adenovirus and those treated with the control vector.Sport Aiding Medical Research for Kids, the Cystic Fibrosis Trust, and the Katharine Dormandy Trust

    A Mechanistic Model of PCR for Accurate Quantification of Quantitative PCR Data

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    Background: Quantitative PCR (qPCR) is a workhorse laboratory technique for measuring the concentration of a target DNA sequence with high accuracy over a wide dynamic range. The gold standard method for estimating DNA concentrations via qPCR is quantification cycle (Cq) standard curve quantification, which requires the time- and labor-intensive construction of a Cq standard curve. In theory, the shape of a qPCR data curve can be used to directly quantify DNA concentration by fitting a model to data; however, current empirical model-based quantification methods are not as reliable as Cq standard curve quantification. Principal Findings: We have developed a two-parameter mass action kinetic model of PCR (MAK2) that can be fitted to qPCR data in order to quantify target concentration from a single qPCR assay. To compare the accuracy of MAK2-fitting to other qPCR quantification methods, we have applied quantification methods to qPCR dilution series data generated in three independent laboratories using different target sequences. Quantification accuracy was assessed by analyzing the reliability of concentration predictions for targets at known concentrations. Our results indicate that quantification by MAK2-fitting is as reliable as Cq standard curve quantification for a variety of DNA targets and a wide range of concentrations. Significance: We anticipate that MAK2 quantification will have a profound effect on the way qPCR experiments are designed and analyzed. In particular, MAK2 enables accurate quantification of portable qPCR assays with limited sampl

    Small Hairy Black Holes in Global AdS Spacetime

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    We study small charged black holes in global AdS spacetime in the presence of a charged massless minimally coupled scalar field. In a certain parameter range these black holes suffer from well known superradiant instabilities. We demonstrate that the end point of the resultant tachyon condensation process is a hairy black hole which we construct analytically in a perturbative expansion in the black hole radius. At leading order our solution is a small undeformed RNAdS black hole immersed into a charged scalar condensate that fills the AdS `box'. These hairy black hole solutions appear in a two parameter family labelled by their mass and charge. Their mass is bounded from below by a function of their charge; at the lower bound a hairy black hole reduces to a regular horizon free soliton which can also be thought of as a nonlinear Bose condensate. We compute the microcanonical phase diagram of our system at small mass, and demonstrate that it exhibits a second order `phase transition' between the RNAdS black hole and the hairy black hole phases.Comment: 68+1 pages, 18 figures, JHEP format. v2 : small typos corrected and a reference adde

    The secreted triose phosphate isomerase of Brugia malayi is required to sustain microfilaria production in vivo

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    Human lymphatic filariasis is a major tropical disease transmitted through mosquito vectors which take up microfilarial larvae from the blood of infected subjects. Microfilariae are produced by long-lived adult parasites, which also release a suite of excretory-secretory products that have recently been subject to in-depth proteomic analysis. Surprisingly, the most abundant secreted protein of adult Brugia malayi is triose phosphate isomerase (TPI), a glycolytic enzyme usually associated with the cytosol. We now show that while TPI is a prominent target of the antibody response to infection, there is little antibody-mediated inhibition of catalytic activity by polyclonal sera. We generated a panel of twenty-three anti-TPI monoclonal antibodies and found only two were able to block TPI enzymatic activity. Immunisation of jirds with B. malayi TPI, or mice with the homologous protein from the rodent filaria Litomosoides sigmodontis, failed to induce neutralising antibodies or protective immunity. In contrast, passive transfer of neutralising monoclonal antibody to mice prior to implantation with adult B. malayi resulted in 60–70% reductions in microfilarial levels in vivo and both oocyte and microfilarial production by individual adult females. The loss of fecundity was accompanied by reduced IFNγ expression by CD4+ T cells and a higher proportion of macrophages at the site of infection. Thus, enzymatically active TPI plays an important role in the transmission cycle of B. malayi filarial parasites and is identified as a potential target for immunological and pharmacological intervention against filarial infections
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