1,721,003 research outputs found
Engineering stable cytoplasmic intrabodies with designed specificity
No full textMany attempts have been made to develop antibody fragments that can be expressed in the cytoplasm ("intrabodies") in a stable and functional form. The recombinant antibody fragment scFv(F8) is characterised by peculiarly high in vitro stability and functional folding in both prokaryotic and eukaryotic cytoplasm. To dissect the relative contribution of different scFv(F8) regions to cytoplasmic stability and specificity we designed and constructed five chimeric molecules (scFv-P1 to P5) in which several groups of residues important for antigen binding in the poorly stable anti-hen egg lysozyme (HEL) scFv(D1.3) were progressively grafted onto the scFv(F8) scaffold. All five chimeric scFvs were expressed in a soluble form in the peri-plasm and cytoplasm of Escherichia coli. All the periplasmic oxidised forms and the scFv(P3) extracted from the cytoplasm in reducing conditions had HEL binding affinities essentially identical (K-d = 15 nM) to that of the cognate scFv(D1.3) fragment (K-d = 16 nM). The successful grafting of the antigen binding properties of D1.3 onto the scFv(F8) opens the road to the exploitation of this molecule as a scaffold for the reshaping of intrabodies with desired specificities to be targeted to the cytoplasm. (C) 2003 Elsevier Science Ltd. All rights reserved
Humanization of a highly stable single-chain antibody by structure-based antigen-binding site grafting
No full textThe murine single-chain variable fragment F8 (scFv(F8)) is endowed with high intrinsic thermodynamic stability and can be functionally expressed in the reducing environment of both prokaryotic and eukaryotic cytoplasm. The stability and intracellular functionality of this molecule can be ascribed mostly to its framework regions and are essentially independent of the specific sequence and structure of the supported antigen-binding site. Therefore, the scFv(F8) represents a suitable scaffold to construct stable scFv chimeric molecules against different antigens by in vitro evolution or antigen-binding site grafting. Thanks to the favourable pharmacokinetic properties associated to a high thermodynamic stability of antibody fragments, such scFv(F8) variants may be exploited for a wide range of biomedical applications, from in vivo diagnosis to therapy, as well as to interfere with the function of intracellular proteins and pathogens, and for functional genomics studies. However, the potential immunogenicity of the murine framework regions represents a limitation for their exploitation in therapeutic applications. To overcome this limitation, we humanized a derivative of the scFv(FS), the anti-lysozyme scFv(11E), which is endowed with even higher thermodynamic stability than the parent antibody. The humanization was carried out by substituting the framework residues differing from closely related V-H and V-L domains of human origin with their human counterparts. Site-directed mutagenesis generated the fully humanized product and four intermediate scFvs, which were analyzed for protein expression and antigen binding. We found that the substitution Tyr 90 -> Phe in the V-H domain dramatically reduced the bacterial expression of all mutants. The back-mutation of Phe H90 to Tyr led to the final humanized variant named scFv(H5)H90Tyr. This molecule comprises humanized V-H and V-L framework regions and is endowed with HEL-binding affinity, stability in human serum and functionality under reducing conditions comparable to the murine cognate antibody. Consequently, the humanized scFv(H5)H90Tyr represents a suitable scaffold onto which new specificities towards antigens of therapeutic interest can be engineered for biomedical applications. (C) 2008 Elsevier Ltd. All rights reserved
A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold
No full textWe report the design, construction and use of an antibody bacteriophage display library built on the scaffold of a single-chain variable fragment (scFv) previously proven to be functionally expressed in the reducing environment of both bacterial and plant cytoplasm and endowed with intrinsic high thermodynamic stability. Four amino acid residues of the third hypervariable loop (CDR3) of both V-H and V-L were combinatorially mutated, generating a repertoire of approximately 5 x 10(7) independent scFvs, cloned in a phagemid vector. The ability of the antibody phage library to yield specific binders was tested by biopanning against several antigens. Successful selection of fully active scFvs was obtained, confirming the notion that combinatorial mutagenesis of few amino acid residues centrally located in the antigen-binding site is sufficient to provide binding specificities against virtually any target. High yields of both soluble and phage antibodies were obtained in Escherichia coil. Maintenance of the cognate scFv antibody stability in the newly selected scFv fragments was demonstrated by guanidinium chloride denaturation/renaturation studies and by soluble antibody expression in the bacterial cytoplasm. The antibody library described here allows the isolation of new stable binding specificities, potentially exploitable as immunochemical reagents for intracellular applications. (C) 2001 Academic Press
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
Variations 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
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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