1,721,040 research outputs found
Gerichte evolutie van XNA polymerase: faag display als selectietechniek
No full textArtificial or xeno nucleic acids (XNAs) present an alternative to natural genetic polymers by expanding chemical diversity and improving chemical and biological stability, with potential applications in, for example, therapeutics. XNA differs from its natural counterparts by modifications applied to the nucleobases, sugar-phosphate backbone, nucleotide leaving group, or a combination of these. In the long run, XNA could form the basis of an orthogonal genetic system, “invisible” to and replicating independently from the natural world by chemical and enzymatic decoupling. Key to the development and manipulation of these XNAs are suitable polymerases that can incorporate synthetic nucleotides into a growing XNA chain in a template-dependent way. These polymerases can be created starting from natural variants by directed evolution, an in vitro process of repeated mutagenesis and selection. In this project, we propose bacteriophage phi29 DNA polymerase as a new candidate for directed evolution towards XNA polymerase activity. The symmetrical, protein-primed replication mode of this polymerase is an important feature that could enable the establishment of a straightforward in vivo XNA episome. We showed promiscuous activity of phi29 DNA polymerase towards sugar-modified nucleotides, further justifying our choice for this enzyme. A number of mutant libraries was designed and created, guided by structural information to increase the chance of finding interesting variants. The polymerase was successfully displayed on phage and shown to be active. A novel phage display system involving co-display of polymerase and HaloTag was developed to simplify attachment of a primer-template substrate and selection based on modified nucleotide incorporation. The presented co-display system might also be used for the evolution of a wide range of other enzymes, different from polymerases. Preliminary selection experiments exemplified the importance of high-quality libraries, precise control of growth conditions and protein expression, and an optimal selection procedure to maximize enrichment of active variants. Further refinement of selection conditions is needed for the isolation of functional XNA polymerases, to be eventually applied in an orthogonal XNA episome.status: Publishe
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Moleculaire evolutie van een thermoresistent DNA polymerase voor nucleotiden met gewijzigde uittredende groep ,,
No full textMany details about the complex enzyme mechanism of polymerases are still unknown, despite their essential rol in vivo and in biochemical laboratories. In this project we aim to develop a polymerase capable of accepting and incorporating synthetic nucleotides with modified leaving groups by means of molecular evolution in order to illucidate the enzyme mechanism. Furthermore, this polymerase will represent a valuable contribution to an orthogonal genetic system as the addition of synthetic nucleotide triphosphates will likely interfere with DNA/RNA metabolism, energie storage and phosphorylation regulation of the host. Of course, modification of the leaving group alone will not result in complete orthogonality. Therefore, a polymerase will be designed, based on information gathered from literature and acquired from ongoing projects within the research group, capable of incorporating synthetic nucleotides with both modified sugar and leaving groups. These artificial nucleic acids will not be able to interact with natural systems. In short term, these enzymes are directly applicable in the development of aptamers based on xeno nucleic acids (XNA).status: Publishe
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe 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
Single-molecule studies met ClyA nanoporiën. De ontwikkeling van een nieuw platform voor biosensing and single-protein studies ,,
No full textstatus: Publishe
New proteomic initiatives to study multiple sclerosis
No full textNeurological diseases, including MS, often provoke changes in the functioning of the endothelial and epithelial brain barriers and give rise to disease associated alterations of the CSF proteome. Therefore, CSF analysis is believed to represent a valuable approach to identify disease-related proteins. The goal of this chapter is the construction of a protein database of CSF from MS patients, using gel-based and gel-free identification approaches. In a first setup, twodimensional gel electrophoresis was applied on individual CSF samples of five MS patients and high-performance liquid chromatography (HPLC) coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS) resulted in the identification of 65 different proteins. Eighteen of these proteins have not been described previously on 20 gels of CSF and their potential relation to MS is discussed. In a second setup, unseparated protein mixtures from ultrafiltered CSF of MS and non-MS patients were digested with trypsin and analyzed by offline strong cation exchange chromatography (SCX) coupled to on-line reversed phase LC-ESI-MS/MS. Alternatively, the trypsin-treated sub-proteomes were analyzed directly by LC-ESI-MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both gel-free proteomic approaches in combination with a three-step evaluation process including the search engines Sequest and Mascot, and the validation software Scaffold, resulted in the identification of 148 proteins. Sixty proteins were identified in CSF for the first time by mass spectrometry. In addition, the capacity of the gel-free approach to identify disease-related molecules is discussed. Finally, three identified proteins, cystatin A, annexin A5 and psoriasin were validated with respect to their expression in CSF and/or brain slices of MS and control patients
- …
