1,720,962 research outputs found
Intracellular antibodies for proteomics
No full textThe intracellular antibody technology has many applications for proteomics studies. The potential of intracellular antibodies for the systematic study of the proteome has been made possible by the development of new experimental strategies that allow the selection of antibodies under conditions of intracellular expression. The Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid-based method originally developed for the selection of antibodies readily folded for ectopic expression. IACT has been used for the rapid and effective identification of novel antigen-antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by selected intracellular antibodies. IACT opens the way to the use of intracellular antibody technology for large-scale applications in proteomics. In its present format, its use is however somewhat limited by the need of a preselection of the input phage antibody libraries on protein antigens or by the construction of an antibody library from mice immunized against the target protein(s), to provide an enriched input library to compensate for the suboptimal efficiency of transformation of the yeast cells. These enrichment steps require expressing the corresponding proteins, which represents a severe bottleneck for the scaling up of the technology. We describe here the construction of a single pot library of intracellular antibodies (SPLINT), a naïve library of scFv fragments expressed directly in the yeast cytoplasm in a format such that antigen-specific intrabodies can be isolated directly from gene sequences, with no manipulation whatsoever of the corresponding proteins. We describe also the isolation from SPLINT of a panel of intrabodies against a number of different proteins. The application of SPLINT on a genome-wide scale should help the systematic study of the functional organization of cell proteome
Direct in vivo intracellular selection of conformation-sensitive antibody domains targeting Alzheimer's amyloid-beta oligomers. J Mol Biol. 2009 Apr 3;387(3):584-606
No full textThe development of conformation-sensitive antibody domains targeting the misfolding beta amyloid (Abeta) peptide is of great interest for research into Alzheimer's disease (AD). We describe the direct selection, by the Intracellular Antibody Capture Technology (IACT), of a panel of anti-Abeta single chain Fv antibody fragments (scFvs), targeting pathologically relevant conformations of Abeta. A LexA-Abeta1-42 fusion protein was expressed in yeast cells, as the "intracellular antigen". Two different scFv antibody libraries (Single Pot Libraries of Intracellular Antibodies, SPLINT) were used for the intracellular selections: (i) a naïve library, derived from a natural, non-immune, source of mouse antibody variable region (V) genes; and (ii) an immune library constructed from the repertoire of antibody V genes of Abeta-immunized mice. This led to the isolation of 18 different anti-Abeta scFvs, which bind Abeta both in the yeast cell, as well as in vitro, if used as purified recombinant proteins. Surprisingly, all the anti-Abeta scFvs isolated are conformation-sensitive, showing a high degree of specificity towards Abeta oligomers with respect to monomeric Abeta, while also displaying some degree of sequence-specificity, recognizing either the N-terminal or the C-terminal part of Abeta1-42; in particular, the scFvs selected from Abeta-immune SPLINT library show a relevant N-terminal epitope bias. Representative candidates from this panel of the anti-Abeta scFvs were shown to recognize in vivo-produced Abeta "deposits" in histological sections from human AD brains and to display good neutralization properties, significantly inhibiting Abeta oligomer-induced toxicity and synaptic binding of Abeta oligomers in neuronal cultured cells. The properties of these anti-Abeta antibody domains, as well as their direct availability for intra- or extra-cellular "genetic delivery" make them ideally suited for new experimental approaches to study and image the intracellular processing and trafficking of Abeta oligomers
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Novel generation of recombinant antibodies against oligomeric form of human ß amyloid 1-42 peptide.
No full textVariations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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