33,653 research outputs found

    Double-stranded break can be repaired by single-stranded oligonucleotides via the ATM/ATR pathway in mammalian cells

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    Single-stranded oligonucleotide (SSO)-mediated gene modification is a newly developed tool for site-specific gene repair in mammalian cells; however, the corrected cells always show G2/M arrest and cannot divide to form colonies. This phenomenon and the unclear mechanism seriously challenge the future application of this technique. In this study, we developed an efficient SSO-mediated DNA repair system based on double-stranded break (DSB) induction. We generated a mutant EGFP gene with insertions of 24 bp to 1.6 kb in length as a reporter integrated in mammalian cell lines. SSOs were successfully used to delete the insertion fragments upon DSB induction at a site near the insertion. We demonstrated that this process is dependent on the ATM/ATR pathway. Importantly, repaired cell clones were viable. Effects of deletion length, SSO length, strand bias, and SSO modification on gene repair frequency were also investigated. © 2008 Mary Ann Liebert, Inc.published_or_final_versio

    Cell death caused by single-stranded oligodeoxynucleotide-mediated targeted genomic sequence modification

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    Targeted gene repair directed by single-stranded oligodeoxynucleotides (ssODNs) offers a promising tool for biotechnology and gene therapy. However, the methodology is currently limited by its low frequency of repair events, variability, and low viability of "corrected" cells. In this study, we showed that during ssODN-mediated gene repair reaction, a significant population of corrected cells failed to divide, and were much more prone to undergo apoptosis, as marked by processing of caspases and PARP-1. In addition, we found that apoptotic cell death triggered by ssODN-mediated gene repair was largely independent of the ATM/ATR kinase. Furthermore, we examined the potential involvement of the mismatch repair (MMR) proteins in this "correction reaction-induced" cell death. Result showed that while defective MMR greatly enhanced the efficiency of gene correction, compromising the MMR system did not yield any viable corrected clone, indicating that the MMR machinery, although plays a critical role in determining ssODN-directed repair, was not involved in the observed cellular genotoxic responses. © 2009. Mary Ann Liebert, Inc.published_or_final_versio

    How is rising obesity tackled in China and Europe?

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    Overweight and obesity are a growing problem in China but their causes may be differently viewed as a consequence of globalisation and the development of an obesogenic environment or as a consequence of an individual’s wellbeing. This paper explores these different explanations and the ways that the problem is addressed in Sino and European policy and TCM practice. The diagnosis of obesity and its treatment by herbs, acupuncture and auricular acupuncture are described but there is a lack of evidence for their efficacy. This paper argues that at an individual level, obesity can be linked to an unhealthy diet and eating practices and a lack of activity. Behavioural changes as well as TCM treatments that address the gastrointestinal digestive function may be effective. Obesity is, however, a complex and global issue that also needs to be recognised as a social problem where the environments in which people live and work will influence their ability to adopt healthier lifestyles

    Analysis of the characteristics of DC nozzle arcs in air and guidance for the search of SF6 replacement gas

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    It is shown that the arc model based on laminar flow cannot predict satisfactorily the voltage of an air arc burning in a supersonic nozzle. The Prandtl mixing length model (PML) and a modified k-epsilon turbulence model (MKE) are used to introduce turbulence enhanced momentum and energy transport. Arc voltages predicted by these two turbulence models are in good agreement with experiments at the stagnation pressure (P 0) of 10 bar. The predicted arc voltages by MKE for P 0  =  13 bar and 7 bar are in better agreement with experiments than those predicted by PML. MKE is therefore a preferred turbulence model for an air nozzle arc. There are two peaks in ρC P of air at 4000 K and 7000 K due, respectively, to the dissociation of oxygen and that of nitrogen. These peaks produce corresponding peaks in turbulent thermal conductivity, which results in very broad radial temperature profile and a large arc radius. Thus, turbulence indirectly enhances axial enthalpy transport, which becomes the dominant energy transport process for the overall energy balance of the arc column at high currents. When the current reduces, turbulent thermal conduction gradually becomes dominant. The temperature dependence of ρC P has a decisive influence on the radial temperature profile of a turbulent arc, thus the thermal interruption capability of a gas. Comparison between ρC P for air and SF6 shows that ρC P for SF6 has peaks below 4000 K. This renders a distinctive arc core and a small arc radius for turbulent SF6, thus superior arc quenching capability. It is suggested, for the first time, that ρC P provides guidance for the search of a replacement switching gas for SF6

    Glucagon-like peptide-2 modulates neurally evoked mucosal chloride secretion in guinea pig small intestine in vitro

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    Glucagon-like peptide-2 (GLP-2) is an important neuroendocrine peptide in intestinal physiology. It influences digestion, absorption, epithelial growth, motility, and blood flow. We studied involvement of GLP-2 in intestinal mucosal secretory behavior. Submucosal-mucosal preparations from guinea pig ileum were mounted in Ussing chambers for measurement of shortcircuit current (Isc) as a surrogate for chloride secretion. GLP-2 action on neuronal release of acetylcholine was determined with ELISA. Enteric neuronal expression of the GLP-2 receptor (GLP-2R) was studied with immunohistochemical methods. Application of GLP-2 (0.1–100 nM) to the serosal or mucosal side of the preparations evoked no change in baseline Isc and did not alter transepithelial ionic conductance. Transmural electrical field stimulation (EFS) evoked characteristic biphasic increases in Isc, with an initially rapid rising phase followed by a sustained phase. Application of GLP-2 reduced the EFS-evoked biphasic responses in a concentration-dependent manner. The GLP-2R antagonist GLP-2-(3-33) significantly reversed suppression of the EFS-evoked responses by GLP-2. Tetrodotoxin, scopolamine, and hexamethonium, but not vasoactive intestinal peptide type 1 receptor (VPAC1) antagonist abolished or reduced to near zero the EFS-evoked responses. GLP-2 suppressed EFS-evoked acetylcholine release as measured by ELISA. Pretreatment with GLP-2- (3-33) offset this action of GLP-2. In the submucosal plexus, GLP-2R immunoreactivity (-IR) was expressed in choline acetyltransferase-IR neurons, somatostatin-IR neurons, neuropeptide Y-IR neurons, and vasoactive intestinal peptide-IR neurons. We conclude that submucosal neurons in the guinea pig ileum express GLP-2R. Activation of GLP-2R decreases neuronally evoked epithelial chloride secretion by suppressing acetylcholine release from secretomotor neurons

    Regulation of pituitary MT1 melatonin receptor expression by gonadotrophin-releasing hormone (GnRH) and early growth response factor-1 (Egr-1) : in vivo and in vitro studies

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    Copyright: © 2014 Bae et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the UK Biotechnology and Biological Sciences Research Council (BBSRC; grant BB/F020309/1; http://www.bbsrc.ac.uk/home/home.aspx). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

    Altered distribution of mucosal NK cells during HIV infection.

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    The human gut mucosa is a major site of human immunodeficiency virus (HIV) infection and infection-associated pathogenesis. Increasing evidence shows that natural killer (NK) cells have an important role in control of HIV infection, but the mechanism(s) by which they mediate antiviral activity in the gut is unclear. Here, we show that two distinct subsets of NK cells exist in the gut, one localized to intraepithelial spaces (intraepithelial lymphocytes, IELs) and the other to the lamina propria (LP). The frequency of both subsets of NK cells was reduced in chronic infection, whereas IEL NK cells remained stable in spontaneous controllers with protective killer immunoglobulin-like receptor/human leukocyte antigen genotypes. Both IEL and LP NK cells were significantly expanded in immunological non-responsive patients, who incompletely recovered CD4+ T cells on highly active antiretroviral therapy (HAART). These data suggest that both IEL and LP NK cells may expand in the gut in an effort to compensate for compromised CD4+ T-cell recovery, but that only IEL NK cells may be involved in providing durable control of HIV in the gut
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