On how to reconcile flexibility with rigidity during evolution: the caudal system in seahorses

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Engineered flock house virus for targeted gene suppression through RNAi in fruit flies (Drosophila melanogaster) in vitro and in vivo

RNA interference (RNAi) is a powerful tool to study functional genomics in insects and the potential of using RNAi to suppress crop pests has made outstanding progress. However, the delivery of dsRNA is a challenging step in the development of RNAi bioassays. In this study, we investigated the ability of engineered Flock House virus (FHV) to induce targeted gene suppression through RNAi under in vitro and in vivo condition. As proxy for fruit flies of agricultural importance, we worked with S2 cells as derived from Drosophila melanogaster embryos, and with adult stages of D. melanogaster. We found that the expression level for all of the targeted genes were reduced by more than 70% in both the in vitro and in vivo bioassays. Furthermore, the cell viability and median survival time bioassays demonstrated that the recombinant FHV expressing target gene sequences caused a significantly higher mortality (60-73% and 100%) than the wild type virus (24 and 71%), in both S2 cells and adult insects, respectively. This is the first report showing that a single stranded RNA insect virus such as FHV, can be engineered as an effective in vitro and in vivo RNAi delivery system. Since FHV infects many insect species, the described method could be exploited to improve the efficiency of dsRNA delivery for RNAi-related studies in both FHV susceptible insect cell lines and live insects that are recalcitrant to the uptake of naked dsRNA

Automated method for the determination of a new matrix metalloproteinase inhibitor in ovine plasma and serum by coupling of restricted access material for on-line sample clean-up to liquid chromatography

A fully automated liquid chromatographic method was developed for the determination of Ro 28-2653, a new synthetic inhibitor of matrix metalloproteinases (MMPs), in ovine serum and plasma. The method was based on the coupling of a pre-column packed with restricted access material, namely LiChrospher RP-8 ADS (alkyl diol silica), for sample clean-up to an analytical column containing octyl silica stationary phase. One hundred mul of biological sample, to which 2-propanol was automatically added, were injected onto the ADS pre-column, which was then washed with a washing liquid consisting of a mixture of 25 mM phosphate buffer (pH 7.0) and acetonitrile (90: 10; v/v) for 10 min. By rotation of the switching valve, the analyte was then eluted in the back-flush mode with the LC mobile phase composed of a mixture of acetonitrile and 25 mM phosphate buffer (pH 7.0) (57:43; v/v). The UV detection was performed at 395 nm. The main parameters likely to influence the sample preparation technique were investigated. The method was then validated over a concentration range from 17.5 to 1950 ng/mI, the first concentration level corresponding to the lower limit of quantitation. At this concentration level, the mean bias and the R.S.D. value for intermediate precision were -2.4% and 4.2%, respectively. (C) 2004 Elsevier B.V. All rights reserved

Generation of virus- and dsRNA-derived siRNAs with species-dependent length in insects

Double-stranded RNA (dsRNA) molecules of viral origin trigger a post-transcriptional gene-silencing mechanism called RNA interference (RNAi). Specifically, virally derived dsRNA is recognized and cleaved by the enzyme Dicer2 into short interfering RNAs (siRNAs), which further direct sequence-specific RNA silencing, ultimately silencing replication of the virus. Notably, RNAi can also be artificially triggered by the delivery of gene-specific dsRNA, thereby leading to endogenous gene silencing. This is a widely used technology that holds great potential to contribute to novel pest control strategies. In this regard, research efforts have been set to find methods to efficiently trigger RNAi in the field. In this article, we demonstrate the generation of dsRNA- and/or virus-derived siRNAs-the main RNAi effectors-in six insect species belonging to five economically important orders (Lepidoptera, Orthoptera, Hymenoptera, Coleoptera, and Diptera). In addition, we describe that the siRNA length distribution is species-dependent. Taken together, our results reveal interspecies variability in the (antiviral) RNAi mechanism in insects and show promise to contribute to future research on (viral-based) RNAi-triggering mechanisms in this class of animals

Engineered Flock House Virus for Targeted Gene Suppression Through RNAi in Fruit Flies (Drosophila melanogaster) in Vitro and in Vivo

RNA interference (RNAi) is a powerful tool to study functional genomics in insects and the potential of using RNAi to suppress crop pests has made outstanding progress. However, the delivery of dsRNA is a challenging step in the development of RNAi bioassays. In this study, we investigated the ability of engineered Flock House virus (FHV) to induce targeted gene suppression through RNAi under in vitro and in vivo condition. As proxy for fruit flies of agricultural importance, we worked with S2 cells as derived from Drosophila melanogaster embryos, and with adult stages of D. melanogaster. We found that the expression level for all of the targeted genes were reduced by more than 70% in both the in vitro and in vivo bioassays. Furthermore, the cell viability and median survival time bioassays demonstrated that the recombinant FHV expressing target gene sequences caused a significantly higher mortality (60–73% and 100%) than the wild type virus (24 and 71%), in both S2 cells and adult insects, respectively. This is the first report showing that a single stranded RNA insect virus such as FHV, can be engineered as an effective in vitro and in vivo RNAi delivery system. Since FHV infects many insect species, the described method could be exploited to improve the efficiency of dsRNA delivery for RNAi-related studies in both FHV susceptible insect cell lines and live insects that are recalcitrant to the uptake of naked dsRNA

DNA Sensors with Diamond as a Promising Alternative Transducer Material

Bio-electronics is a scientific field coupling the achievements in biology with electronics to obtain higher sensitivity, specificity and speed. Biosensors have played a pivotal role, and many have become established in the clinical and scientific world. They need to be sensitive, specific, fast and cheap. Electrochemical biosensors are most frequently cited in literature, often in the context of DNA sensing and mutation analysis. However, many popular electrochemical transduction materials, such as silicon, are susceptible to hydrolysis, leading to loss of bioreceptor molecules from the surface. Hence, increased attention has been shifted towards diamond, which surpasses silicon on many levels

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

EurOP2E – the European Open Platform for Prescribing Education, a consensus study among clinical pharmacology and therapeutics teachers

Purpose Sharing and developing digital educational resources and open educational resources has been proposed as a way to harmonize and improve clinical pharmacology and therapeutics (CPT) education in European medical schools. Previous research, however, has shown that there are barriers to the adoption and implementation of open educational resources. The aim of this study was to determine perceived opportunities and barriers to the use and creation of open educational resources among European CPT teachers and possible solutions for these barriers. Methods CPT teachers of British and EU medical schools completed an online survey. Opportunities and challenges were identified by thematic analyses and subsequently discussed in an international consensus meeting. Results Data from 99 CPT teachers from 95 medical schools were analysed. Thirty teachers (30.3%) shared or collaboratively produced digital educational resources. All teachers foresaw opportunities in the more active use of open educational resources, including improving the quality of their teaching. The challenges reported were language barriers, local differences, lack of time, technological issues, difficulties with quality management, and copyright restrictions. Practical solutions for these challenges were discussed and include a peer review system, clear indexing, and use of copyright licenses that permit adaptation of resources. Conclusion Key challenges to making greater use of CPT open educational resources are a limited applicability of such resources due to language and local differences and quality concerns. These challenges may be resolved by relatively simple measures, such as allowing adaptation and translation of resources and a peer review system

Harmonizing and improving European education in prescribing: An overview of digital educational resources used in clinical pharmacology and therapeutics

Aim: Improvement and harmonization of European clinical pharmacology and therapeutics (CPT) education is urgently required. Because digital educational resources can be easily shared, adapted to local situations and re-used widely across a variety of educational systems, they may be ideally suited for this purpose. Methods: With a cross-sectional survey among principal CPT teachers in 279 out of 304 European medical schools, an overview and classification of digital resources was compiled. Results: Teachers from 95 (34%) medical schools in 26 of 28 EU countries responded, 66 (70%) of whom used digital educational resources in their CPT curriculum. A total of 89 of such resources were described in detail, including e-learning (24%), simulators to teach pharmacokinetics and/or pharmacodynamics (10%), virtual patients (8%), and serious games (5%). Together, these resources covered 235 knowledge-based learning objectives, 88 skills, and 13 attitudes. Only one third (27) of the resources were in-part or totally free and only two were licensed open educational resources (free to use, distribute and adapt). A narrative overview of the largest, free and most novel resources is given. Conclusion: Digital educational resources, ranging from e-learning to virtual patients and games, are widely used for CPT education in EU medical schools. Learning objectives are based largely on knowledge rather than skills or attitudes. This may be improved by including more real-life clinical case scenarios. Moreover, the majority of resources are neither free nor open. Therefore, with a view to harmonizing international CPT education, more needs to be learned about why CPT teachers are not currently sharing their educational materials