A new method to customize protein expression vectors for fast, efficient and background free parallel cloning

Background: Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any selected vector. Results: Here we describe a method to tailor selected expression vectors for parallel Sequence and Ligation Independent Cloning. SLIC cloning enables precise and sequence independent engineering and is based on joining vector and insert with 15-25 bp homologies on both DNA ends by homologous recombination. We modified expression vectors based on pET, pFastBac and pTT backbones for parallel PCR-based cloning and screening in E. coli, insect cells and HEK293E cells, respectively. We introduced the toxic ccdB gene under control of a strong constitutive promoter for counterselection of insert less vector. In contrast to DpnI treatment commonly used to reduce vector background, ccdB used in our vector series is 100% efficient in killing parental vector carrying cells and reduces vector background to zero. In addition, the 3' end of ccdB functions as a primer binding site common to all vectors. The second shared primer binding site is provided by a HRV 3C protease cleavage site located downstream of purification and solubility enhancing tags for tag removal. We have so far generated more than 30 different parallel expression vectors, and successfully cloned and expressed more than 250 genes with this vector series. There is no size restriction for gene insertion, clone efficiency is > 95% with clone numbers up to 200. The procedure is simple, fast, efficient and cost-effective. All expression vectors showed efficient expression of eGFP and different target proteins requested to be produced and purified at our Core Facility services. Conclusion: This new expression vector series allows efficient and cost-effective parallel cloning and thus screening of different protein constructs, tags and expression hosts

Inflammatory bowel disease-specific autoantibodies in HLA-B27-associated spondyloarthropathies: Increased prevalence of ASCA and pANCA

Aims: An association between inflammatory bowel disease (IBD) and spondyloarthropathies (SpA) has repeatedly been reported. The aim of the present study was to investigate whether serologic markers of IBD, e. g. antibodies against Saccharomyces cerevisiae (ASCA), antibodies against exocrine pancreas (PAB) and perinuclear antineutrophil cytoplasmic antibodies (pANCA) are present in HLA-B27-associated SpA. Methods: 87 patients with HLA-B27-positive SpA and 145 controls were tested for ASCA, PAB and pANCA employing ELISA or indirect immunofluorescence, respectively. Antibody-positive patients were interviewed regarding IBD-related symptoms using a standardized questionnaire. Results/Conclusion: When compared to the controls, ASCA IgA but not ASCA IgG levels were significantly increased in patients with SpA, in particular in ankylosing spondylitis (AS) and undifferentiated SpA (uSpA). pANCA were found in increased frequency in patients with SpA whereas PAB were not detected. The existence of autoantibodies was not associated with gastrointestinal symptoms but sustains the presence of a pathophysiological link between bowel inflammation and SpA. Copyright (C) 2004 S. Karger AG, Basel

Rfx6 Maintains the Functional Identity of Adult Pancreatic β Cells.

SummaryIncreasing evidence suggests that loss of β cell characteristics may cause insulin secretory deficiency in diabetes, but the underlying mechanisms remain unclear. Here, we show that Rfx6, whose mutation leads to neonatal diabetes in humans, is essential to maintain key features of functionally mature β cells in mice. Rfx6 loss in adult β cells leads to glucose intolerance, impaired β cell glucose sensing, and defective insulin secretion. This is associated with reduced expression of core components of the insulin secretion pathway, including glucokinase, the Abcc8/SUR1 subunit of KATP channels and voltage-gated Ca2+ channels, which are direct targets of Rfx6. Moreover, Rfx6 contributes to the silencing of the vast majority of “disallowed” genes, a group usually specifically repressed in adult β cells, and thus to the maintenance of β cell maturity. These findings raise the possibility that changes in Rfx6 expression or activity may contribute to β cell failure in humans

Valence band photoemission from the GaN(0001) surface

A detailed investigation by one-step photoemission calculations of the GaN(0001)-(1x1) surface in comparison with recent experiments is presented in order to clarify its structural properties and electronic structure. The discussion of normal and off-normal spectra reveals through the identified surface states clear fingerprints for the applicability of a surface model proposed by Smith et al. Especially the predicted metallic bonds are confirmed. In the context of direct transitions the calculated spectra allow to determine the valence band width and to argue in favor of one of two theoretical bulk band structures. Furthermore a commonly used experimental method to fix the valence band maximum is critically tested.Comment: 8 pages, 11 eps files, submitted to PR

Historical-institutionalist perspectives on the development of the EU budget system

The EU budget has only recently started to feature in theories of European integration. Studies typically adopt a historical-institutionalist framework, exploring notions such as path dependency. They have, however, generally been rather aggregated, or coarse-grained, in their approach. The EU budget has thus been treated as a single entity rather than a series of inter-linked institutions. This paper seeks to address these lacunae by adopting a fine-grained approach. This enables us to emphasize the connections that exist between EU budgetary institutions, in both time and space. We show that the initial set of budgetary institutions was unable, over time, to achieve consistently their treaty-based objectives. In response, rather than reform these institutions at potentially high political cost, additional institutions were layered on top of the extant structures. We thus demonstrate how some EU budgetary institutions have remained unchanged, whilst others have been added or changed over time

Measuring the Discernability of Virtual Objects in Conventional and Stylized Augmented Reality

In augmented reality, virtual graphical objects are overlaid over the real environment of the observer. Conventional augmented reality systems normally use standard real-time rendering methods for generating the graphical representations of virtual objects. These renderings contain the typical artifacts of computer generated graphics, e.g., aliasing caused by the rasterization process and unrealistic, manually configured illumination models. Due to these artifacts, virtual objects look artifical and can easily be distinguished from the real environment. A different approach to generating augmented reality images is the basis of stylized augmented reality [FBS05c]. Here, similar types of artistic or illustrative stylization are applied to the virtual objects and the camera image of the real enviroment. Therefore, real and virtual image elements look significantly more similar and are less distinguishable from each other. In this paper, we present the results of a psychophysical study on the effectiveness of stylized augmented reality. In this study, a number of participants were asked to decide whether objects shown in images of augmented reality scenes are virtual or real. Conventionally rendered as well as stylized augmented reality images and short video clips were presented to the participants. The correctness of the participants' responses and their reaction times were recorded. The results of our study show that an equalized level of realism is achieved by using stylized augmented reality, i.e., that it is significantly more difficult to distinguish virtual objects from real objects

Terahertz dynamics of a topologically protected state: quantum Hall effect plateaus near cyclotron resonance in a GaAs/AlGaAs heterojunction

We measure the Hall conductivity of a two-dimensional electron gas formed at a GaAs/AlGaAs heterojunction in the terahertz regime close to the cyclotron resonance frequency by employing a highly sensitive Faraday rotation method coupled with electrical gating of the sample to change the electron density. We observe clear plateau-and step-like features in the Faraday rotation angle vs. electron density and magnetic field (Landau-level filling factor), which are the high frequency manifestation of quantum Hall plateaus - a signature of topologically protected edge states. The results are compared to a recent dynamical scaling theory.Comment: 18 pages, 3 figure