1,720,955 research outputs found
An advanced method for the small-scale production of high-quality minicircle DNA
No full textMinicircle DNA is a promising tool in the field of gene therapy, whose products are increasingly gaining market access. Greater transfection efficiency and longer expression time as well as lower immunogenicity contrast with cost-intensive production, which also stands in the way of a broader use of the advantages of this technology in research. Starting from a commercial minicircle production kit a simple protocol for the cost-effective small-scale production of high-quality minicircle DNA to be used at a research scale has been developed by combining and improving procedures of various publications. An optimized size-exclusion chromatography method led to almost pure minicircle DNA with a superior proportion of the desired supercoiled plasmid conformation. The pharmaceutical potential of the produced minicircle DNA was investigated in vitro by real-time impedance assays in a tumor cell model in case of coded suicide genes as well as by ELISA of the translation product in case of coded human coagulation factor IX
Investigation of saponins as novel transfection enhancer
No full textThe ability of specific triterpenoid saponins to act as a novel mediator of the endosomal release was investigated thoroughly in the past. By interacting with the endosomal membrane, these sec-ondary plant metabolites from Saponaria- or Gypsophila-specimen are able to release the endo-somal or lysosomal content before being degraded by lysosomal enzymes. Special attention hereby was paid to the use of ribosome-inactivating proteins, which can induce the apoptotic cell death by cleaving an adenine out of the 28S-Subunit of the ribosomes. A synergy between triterpenoid saponins and RIPs achieved an amplified cell toxicity in several studies, which was even tested successfully in many in-vivo-studies. Resulting out of a less specific release from the endosomes, a logic consequence was to test the ability of the triterpenoid saponins in other scien-tific fields. Therefore, the quest of this work happened to be the investigation of the saponin me-diated enhanced gene delivery (transfection). For this purpose, a number of different nanocarriers were formulated and analyzed for their suitability as stable nanoplexes in terms of size, zetapotential, uniformity, stability and DNA-complexation efficiency. After the establishment of a robust transfection method with peptide-based nanoplexes, a number of different saponins could be tested on their transfection enhancing properties, among them SO1861, isolated from Saponaria officinalis L., and GE1741, isolated from Gypsophila elegans M.Bieb. In addition, a further breakdown of the structure-activity relationship regarding the endosomal release could be achieved. After the identification of the highly active saponins SO1861 and GE1741, not only the transfection efficiency, but also the universal applicability was investigated. A flexible as-signment could be demonstrated in different cell lines, transfection methods as well as with dif-ferent nucleic acids. The fact of a not elucidated mechanism of the modulated endosomal escape was a further topic of this work. By applying microscopic and fluorescence-based methods, val-uable insights regarding the intracellular uptake and release could be gathered. With the transfec-tion of a RIP-gene the opportunity of a concrete therapeutic application was investigated. Initial in-vitro-tests confirmed a non-toxic reporter gene delivery as well as an effective and highly toxic suicide gene delivery. On neuroblastoma bearing NMRI nu/nu mice, finally an in-vivo-efficacy was demonstrated, after the combined application of SO1861 and targeted YD-nanocarrier (suicide gene) led to a 50% decrease of tumor growth.
In this work, for the first time the big potential of specific triterpenoid saponins as potent trans-fection enhancer could be described. Despite detailed investigations and connected with that, an establishment of a sound basis, a further breakdown of the mechanistic actions and precise opti-mizations are highly necessary in order to manifest a big value of saponins in future gene and cancer therapies.Die Fähigkeit bestimmter Triterpensaponine als Mediator des endosomalen Release zu agieren, wurde in der Vergangenheit intensiv beforscht. Durch Interaktion mit der Endosomenmembran sind diese Sekundärstoffe aus u.a. Saponaria- oder Gypsophila-Arten in der Lage, den endoso-malen Inhalt vor dem lysosomalen Abbau aus den späten Endosomen oder Lysosomen zu befrei-en. Ein besonderes Interesse galt dabei dem Einsatz von Ribosomen-inaktivierenden Proteinen, die durch enzymatische Abspaltung eines Adenins aus der 28S-Unterheit der Ribosomen den apoptotischen Zelltod induzieren. Ein Synergismus zwischen den Triterpensaponine und RIPs verzeichnete in zahlreichen Experimenten eine vervielfachte Zytotoxizität, die gar in vielen in-vivo-Studien erfolgreich getestet wurde. Aus der weniger spezifischen Freigabe aus den Endo-somen resultierend, war es lediglich eine logische Konsequenz, die Fähigkeit der Triterpensa-ponine an weiteren Gebieten zu testen. So wurde es in dieser Arbeit zur Aufgabe, die Möglich-keiten eines durch Saponine verstärkten Gentransfers (Transfektion) zu testen.
Für diesen Zweck wurden zunächst eine Reihe verschiedener Nanocarrier formuliert und durch ihre Größe, Zetapotential, Uniformität, Stabilität sowie DNA-Komplexierungseffizienz hinsicht-lich ihrer Eignung als stabile Nanoplexe analysiert. Nach der Etablierung einer robusten Trans-fektionsmethode mit peptidbasierten Nanoplexen konnte eine Vielzahl verschiedener Saponine auf ihre transfektionsverstärkenden Eigenschaften getestet werden. Darunter gehörten unter an-derem das aus Saponaria officinalis L. isolierte SO1861 und aus Gypsophila elegans M.Bieb. isolierte GE1741. Mithilfe der Screenings konnte zudem eine weitere Aufschlüsselung der bisher beschriebenen Struktur-Wirkungs-Beziehung bezüglich des endosomalen Release erreicht wer-den. Mit der Identifizierung der hochpotenten Saponine SO1861 und GE1741 wurden nicht nur die Transfektionseffizienz, sondern gleichermaßen die universellen Anwendungsmöglichkeiten untersucht. Dabei wurde ein flexibler Einsatz der Saponine hinsichtlich verschiedener Zelllinien, verschiedener Transfektionsmethoden oder gar verschiedener Nukleinsäuren demonstriert. Die Tatsache eines bisher nicht aufgeklärten Mechanismus des modulierten endosomalen Release war ferner Gegenstand dieser Arbeit. Anhand diverser mikroskopischen oder fluoreszenzbasierten Methoden konnten wertvolle Erkenntnisse hinsichtlich der intrazellulären Aufnahme sowie Freigabe gewonnen werden.
Mit der Transfektion eines RIP-Gens wurde eine neue Methode der nicht viralen Tumor-Gentherapie demonstriert, sowie die Möglichkeit einer konkreten therapeutischen Anwendung erstmalig ergründet. Initiale in-vitro-Tests bestätigten hierbei einen nicht toxischen Reporter-Gentransfer sowie einen effektiven und hochtoxischen Suizid-Gentransfer. An NMRI nu/nu-Mäusen mit beimpftem Neuroblastoma wurde schließlich zum ersten Mal durch eine saponin-vermittelte in-vivo-Gentherapie eine Wirksamkeit demonstriert, nachdem durch eine kombinierte Gabe von SO1861 und gerichteten YD-Nanocarriern (Suizidgen) eine 50%ige Tumorwachs-tumshemmung beobachtet worden war.
In dieser Arbeit konnte somit erstmals das große Potential spezifischer Triterpensaponine als potente Transfektionsverstärker beschrieben werden. Trotz der umfassenden Untersuchungen und der damit verbundenen Erstellung einer fundierten Wissensgrundlage ist eine weitere Erfor-schung der beschriebenen Anwendung jedoch dringend notwendig. Eine weitere Entschlüsselung der genauen mechanistischen Vorgänge sowie präzise Optimierungsschritte sind von ungemeiner Wichtigkeit, um einen hohen Wert für die zukünftige Gen- und Krebstherapie zu manifestieren
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
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
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe 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
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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