1,354,408 research outputs found
Enhanced borohydride oxidation kinetics at gold-rare earth alloys
No full textsponsorship: The authors would like to thank the Ministry of Education, Science and Technological Development of the Republic of Serbia (contract no. 451-03-68/2020-14/200146). The authors would also like to thank the Portuguese Foundation for Science and Technology (FCT, Portugal) for a research grant within project UID/CTM/04540/2013 (G. Backovic), for contract IST-ID/156/2018 (B. Sljukic), and for a research contract in the scope of programmatic funding UIDP/04540/2020 (D.M.F. Santos). (Ministry of Education, Science and Technological Development of the Republic of Serbia|451-03-68/2020-14/200146, Portuguese Foundation for Science and Technology (FCT, Portugal)|UID/CTM/04540/2013, Portuguese Foundation for Science and Technology (FCT, Portugal)|IST-ID/156/2018, Portuguese Foundation for Science and Technology (FCT, Portugal)|UIDP/04540/2020)status: Publishe
Capsid Assembly and Single Stranded DNA Genome Formation of Adeno-Associated Virus Type2 in Yeast Cells
No full textSaccharomyces cerevisiae has provided an array of genetic tools to study unknown aspects
of viral life cycles, supporting replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses). It also provides means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus) and as such represents a useful tool for vaccine development. To extend the utility of the S. cerevisiae expression system, we expressed AAV2 structural and nonstructural proteins in yeast cells, using both authentic AAV2 and heterologous yeast promoters. For the first time, we described the assembly of AAV2 virus-like particles from yeast-expressed AAV2 structural proteins. To do this we used AAV p40 promoter, whose activity in yeast cells resembled the one of yeast glycolytic promoters, resulting in the synthesis of the most abundant capsid protein VP3 when transformed yeast cells were
grown on glucose as a carbon source. The expression of other two VPs was induced from yeast, galactose inducible pGal1 promoter. Simultaneous production of all three VPs was achieved by growing the yeast cells in the medium containing both glucose and galactose, while their relative production levels were further optimized by varying amounts of each
carbon source in the induction medium, followed by the fine tuning of the induction time.
Moreover, we investigated the ability of the yeast Saccharomyces cerevisiae to carry out
the replication of a recombinant rAAV2. When a plasmid harboring the rAAV2 genome in
which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in
yeast with a plasmid expressing Rep68 from constitutive yeast promoter pADH, a
significant number of URA3+ clones were scored (more than 30-fold over controls).
Molecular analysis of low molecular weight DNA revealed that the single stranded DNA is
formed, in Rep68 and ITR dependent manner, and that the plasmid is entirely replicated.
The ss DNA contained the ITRs, URA3 gene and also vector sequences suggesting that ss
rAAV genomes were not obtained by the canonical AAV replication mechanism.
These results could open new prospects for using yeast cell in two ways: (i) as a model
system for studying viral and cellular factors involved in AAV2 capsid assembly and
packaging of rAAV ss genomes; and (ii) as a novel cell factory for developing superior
recombinant rAAV production technologies
Capsid protein expression and adeno-associated virus like particles assembly in Saccharomyces cerevisiae.
No full textThe budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses) and has provided means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus). We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast.To do this, at least two out of three AAV structural proteins, VP1 and VP3, have to be simultaneously expressed in yeast cells and their intracellular stoichiometry has to resemble the one found in the particles derived from mammalian or insect cells. This was achieved by stable co-transformation of yeast cells with two plasmids, one expressing VP3 from its natural p40 promoter and the other one primarily expressing VP1 from a modified AAV2 Cap gene under the control of the inducible yeast promoter Gal1. Among various induction strategies we tested, the best one to yield the appropriate VP1:VP3 ratio was 4.5 hour induction in the medium containing 0.5\% glucose and 5\% galactose. Following such induction, AAV virus like particles (VLPs) were isolated from yeast by two step ultracentrifugation procedure. The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells.Taken together, the results show for the first time that yeast can be used to assemble AAV capsid and, therefore, as a genetic system to identify novel cellular factors involved in AAV biology
Capsid protein expression and adeno-associated virus like particles assembly in <it>Saccharomyces cerevisiae</it>
No full textAbstract Background The budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses) and has provided means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus). We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast. Results To do this, at least two out of three AAV structural proteins, VP1 and VP3, have to be simultaneously expressed in yeast cells and their intracellular stoichiometry has to resemble the one found in the particles derived from mammalian or insect cells. This was achieved by stable co-transformation of yeast cells with two plasmids, one expressing VP3 from its natural p40 promoter and the other one primarily expressing VP1 from a modified AAV2 Cap gene under the control of the inducible yeast promoter Gal1. Among various induction strategies we tested, the best one to yield the appropriate VP1:VP3 ratio was 4.5 hour induction in the medium containing 0.5% glucose and 5% galactose. Following such induction, AAV virus like particles (VLPs) were isolated from yeast by two step ultracentrifugation procedure. The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells. Conclusions Taken together, the results show for the first time that yeast can be used to assemble AAV capsid and, therefore, as a genetic system to identify novel cellular factors involved in AAV biology.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
No full textThe 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
Solar γ rays as a complementary probe of dark matter
No full textWe show that observations of solar γ rays offer a novel probe of dark matter in scenarios where interactions with the visible sector proceed via a long-lived mediator. As a proof of principle, we demonstrate that there exists a class of models which yield solar γ-ray fluxes observable with the next generation of γ-ray telescopes, while being allowed by a variety of current experimental constraints. The parameter space allowed by big bang nucleosynthesis and beam dump experiments naturally leads to mediator lifetimes sufficient to produce observable solar γ-ray signals. The model allows for solar γ-ray fluxes up to orders of magnitude larger compared to dwarf spheroidal galaxies, without reaching equilibrium between dark matter annihilation and capture rate. Our results suggest that solar γ-ray observations are complementary, and in some cases superior, to existing and future dark matter detection efforts
Variations on the Author
No full text“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
No full textWe 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
No full textWe 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
Author, publisher and bookseller : a tripartite synergy in Nigerian book industry
No full textThis work is about the roles of Author, Publisher and Bookseller in Book development in
Nigeria. The paper started by delving into the history of Book Publishing in Nigeria after
which it proceeded by defining who an author, a publisher, and a bookseller is and
expatiated on the indispensable roles of these key actors in Nigerian Book Industry and in
the emerging Information Society. Furthermore, the various constraints to book
development were identified while the paper advised on how the Book Industry can be
further promoted in Nigeria. However, the paper concluded and made recommendations
on how the Book sector can help in enhancing scholarship in the country
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