117 research outputs found
The application of scientific research by professionals: an empirical research
The gap between scientific research and its application in practice seems hard to bridge. Through scientific mechanisms, scientific knowledge can reach practitioners, such as accountants and controllers. In this study, an exploratory survey has been used to examine to what extent the mechanisms identified from the literature also apply to the way in which Dutch professionals in accountancy and controlling use scientific knowledge. The results show, among other things, that these mechanisms work differently for different professionals, and that accountants in particular tend to attach less importance to the lessons from science
Communicated by Michael Meyer
In diesem Artikel wird ein mathematisches Modell entwickelt für die Ausbreitung des Wollschafs unter Hirten im Nahen Osten und in Südosteuropa zwischen 6200 und 4200 v. Chr. In unserem Modell werden Hirten als Agenten betrachtet, deren Bewegungen durch Zufallsprozesse gesteuert werden, sodass sich die Agenten mit größerer Wahrscheinlichkeit in Regionen aufhalten, die attraktiv für die Schafhaltung sind. Das Modell berücksichtigt außerdem soziale Interaktionen zwischen Agenten und erlaubt die Weitergabe der Innovation zwischen Agenten mit einer bestimmten Wahrscheinlichkeit. Die Parameter des agentenbasierten Modells werden an die verfügbaren archäologischen Daten angepasst. Ein Simulationsverfahren für die räumliche und zeitliche Entwicklung des Ausbreitungsprozesses soll es ermöglichen, qualitative Effekte von verschiedenen Aspekten zu studieren, die den Ausbreitungsprozess beeinflussen
A Helitron transposon reconstructed from bats reveals a novel mechanism of genome shuffling in eukaryotes
Helitron transposons capture and mobilize gene fragments in eukaryotes, but experimental evidence for their transposition is lacking in the absence of an isolated active element. Here we reconstruct Helraiser, an ancient element from the bat genome, and use this transposon as an experimental tool to unravel the mechanism of Helitron transposition. A hairpin close to the 3'-end of the transposon functions as a transposition terminator. However, the 3'-end can be bypassed by the transposase, resulting in transduction of flanking sequences to new genomic locations. Helraiser transposition generates covalently closed circular intermediates, suggestive of a replicative transposition mechanism, which provides a powerful means to disseminate captured transcriptional regulatory signals across the genome. Indeed, we document the generation of novel transcripts by Helitron promoter capture both experimentally and by transcriptome analysis in bats. Our results provide mechanistic insight into Helitron transposition, and its impact on diversification of gene function by genome shuffling
Diversification of the Caenorhabditis heat shock response by Helitron transposable elements.
Heat Shock Factor 1 (HSF-1) is a key regulator of the heat shock response (HSR). Upon heat shock, HSF-1 binds well-conserved motifs, called Heat Shock Elements (HSEs), and drives expression of genes important for cellular protection during this stress. Remarkably, we found that substantial numbers of HSEs in multiple Caenorhabditis species reside within Helitrons, a type of DNA transposon. Consistent with Helitron-embedded HSEs being functional, upon heat shock they display increased HSF-1 and RNA polymerase II occupancy and up-regulation of nearby genes in C. elegans. Interestingly, we found that different genes appear to be incorporated into the HSR by species-specific Helitron insertions in C. elegans and C. briggsae and by strain-specific insertions among different wild isolates of C. elegans. Our studies uncover previously unidentified targets of HSF-1 and show that Helitron insertions are responsible for rewiring and diversifying the Caenorhabditis HSR
Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells
The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing the non-viral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4 and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed putative iPS cells showed long term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5 and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives of the three germ layers. Upon injection of putative iPS cells under the skin of immunodeficient mice, we observed teratomas in 3 of 6 cases. These results form the basis for in-depth studies towards the derivation of porcine iPS cells, which hold great promise for preclinical testing of novel cell therapies in the pig model
Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells.
The discovery of direct cell reprogramming and induced
pluripotent stem (iPS) cell technology opened up new avenues for
the application of non-viral, transposon-based gene delivery
systems. The Sleeping Beauty (SB) transposon is highly advanced
for versatile genetic manipulations in mammalian cells. We
established iPS cell reprogramming of mouse embryonic
fibroblasts and human foreskin fibroblasts by transposition of
OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28)
expression cassettes mobilized by the SB100X hyperactive
transposase. The efficiency of iPS cell derivation with SB
transposon system was in the range of that obtained with
retroviral vectors. Co-expression of the miRNA302/367 cluster
together with OSKM significantly improved reprogramming
efficiency and accelerated the temporal kinetics of
reprogramming. The iPS cells displayed a stable karyotype, and
hallmarks of pluripotency including expression of stem cell
markers and the ability to differentiate into embryoid bodies in
vitro. We demonstrate Cre recombinase-mediated exchange allowing
simultaneous removal of the reprogramming cassette and targeted
knock-in of an expression cassette of interest into the
transposon-tagged locus in mouse iPS cells. This strategy would
allow correction of a genetic defect by site-specific insertion
of a therapeutic gene construct into 'safe harbor' sites in the
genomes of autologous, patient-derived iPS cells
Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling
In drug discovery, prediction of selectivity and toxicity
require the evaluation of cellular calcium homeostasis. The rat
is a preferred laboratory animal for pharmacology and
toxicology studies, while currently no calcium indicator
protein expressing rat model is available. We established a
transgenic rat strain stably expressing the GCaMP2
fluorescent calcium sensor by a transposon-based methodology.
Zygotes were co-injected with mRNA of transposase and a CAG-
GCaMP2 expressing construct, and animals with one
transgene copy were pre-selected by measuring fluorescence in
blood cells. A homozygous rat strain was generated with high
sensor protein expression in the heart, kidney, liver, and
blood cells. No pathological alterations were found in these
animals, and fluorescence measurements in cardiac tissue slices
and primary cultures demonstrated the applicability of this
system for studying calcium signaling. We show here that the
GCaMP2 expressing rat cardiomyocytes allow the
prediction of cardiotoxic drug side-effects, and provide
evidence for the role of Na+/Ca2+ exchanger and its beneficial
pharmacological modulation in cardiac reperfusion. Our data
indicate that drug-induced alterations and pathological
processes can be followed by using this rat model, suggesting
that transgenic rats expressing a calcium-sensitive protein
provide a valuable system for pharmacological and toxicological
studies
Structural determinants of Sleeping Beauty transposase activity
Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called "sectors", which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold.Molecular Therapy (2016); doi:10.1038/mt.2016.110
Germline Transgenic Pigs by Sleeping Beauty Transposition in Porcine Zygotes and Targeted Integration in the Pig Genome
Genetic engineering can expand the utility of pigs for modeling human diseases, and for developing advanced therapeutic approaches. However, the inefficient production of transgenic pigs represents a technological bottleneck. Here, we assessed the hyperactive Sleeping Beauty (SB100X) transposon system for enzyme-catalyzed transgene integration into the embryonic porcine genome. The components of the transposon vector system were microinjected as circular plasmids into the cytoplasm of porcine zygotes, resulting in high frequencies of transgenic fetuses and piglets. The transgenic animals showed normal development and persistent reporter gene expression for >12 months. Molecular hallmarks of transposition were confirmed by analysis of 25 genomic insertion sites. We demonstrate germ-line transmission, segregation of individual transposons, and continued, copy number-dependent transgene expression in F1-offspring. In addition, we demonstrate target-selected gene insertion into transposon-tagged genomic loci by Cre-loxP-based cassette exchange in somatic cells followed by nuclear transfer. Transposase-catalyzed transgenesis in a large mammalian species expands the arsenal of transgenic technologies for use in domestic animals and will facilitate the development of large animal models for human diseases
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