Comparison of static immersion and intravenous injection systems for exposure of zebrafish embryos to the natural pathogen Edwardsiella tarda

<p>Abstract</p> <p>Background</p> <p>The zebrafish embryo is an important <it>in vivo </it>model to study the host innate immune response towards microbial infection. In most zebrafish infectious disease models, infection is achieved by micro-injection of bacteria into the embryo. Alternatively, <it>Edwardsiella tarda</it>, a natural fish pathogen, has been used to treat embryos by static immersion. In this study we used transcriptome profiling and quantitative RT-PCR to analyze the immune response induced by <it>E. tarda </it>immersion and injection.</p> <p>Results</p> <p>Mortality rates after static immersion of embryos in <it>E. tarda </it>suspension varied between 25-75%, while intravenous injection of bacteria resulted in 100% mortality. Quantitative RT-PCR analysis on the level of single embryos showed that expression of the proinflammatory marker genes <it>il1b </it>and <it>mmp9 </it>was induced only in some embryos that were exposed to <it>E. tarda </it>in the immersion system, whereas intravenous injection of <it>E. tarda </it>led to <it>il1b </it>and <it>mmp9 </it>induction in all embryos. In addition, microarray expression profiles of embryos subjected to immersion or injection showed little overlap. <it>E. tarda</it>-injected embryos displayed strong induction of inflammatory and defense genes and of regulatory genes of the immune response. <it>E. tarda</it>-immersed embryos showed transient induction of the cytochrome P450 gene <it>cyp1a</it>. This gene was also induced after immersion in <it>Escherichia coli </it>and <it>Pseudomonas aeruginosa </it>suspensions, but, in contrast, was not induced upon intravenous <it>E. tarda </it>injection. One of the rare common responses in the immersion and injection systems was induction of <it>irg1l</it>, a homolog of a murine immunoresponsive gene of unknown function.</p> <p>Conclusions</p> <p>Based on the differences in mortality rates between experiments and gene expression profiles of individual embryos we conclude that zebrafish embryos cannot be reproducibly infected by exposure to <it>E. tarda </it>in the immersion system. Induction of <it>il1b </it>and <it>mmp9 </it>was consistently observed in embryos that had been systemically infected by intravenous injection, while the early transcriptional induction of <it>cyp1a </it>and <it>irg1l </it>in the immersion system may reflect an epithelial or other tissue response towards cell membrane or other molecules that are shed or released by bacteria. Our microarray expression data provide a useful reference for future analysis of signal transduction pathways underlying the systemic innate immune response versus those underlying responses to external bacteria and secreted virulence factors and toxins.</p

Improving the drug discovery process by using multiple classifier systems

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.Machine learning methods have become an indispensable tool for utilizing large knowledge and data repositories in science and technology. In the context of the pharmaceutical domain, the amount of acquired knowledge about the design and synthesis of pharmaceutical agents and bioactive molecules (drugs) is enormous. The primary challenge for automatically discovering new drugs from molecular screening information is related to the high dimensionality of datasets, where a wide range of features is included for each candidate drug. Thus, the implementation of improved techniques to ensure an adequate manipulation and interpretation of data becomes mandatory. To mitigate this problem, our tool (called D2-MCS) can split homogeneously the dataset into several groups (the subset of features) and subsequently, determine the most suitable classifier for each group. Finally, the tool allows determining the biological activity of each molecule by a voting scheme. The application of the D2-MCS tool was tested on a standardized, high quality dataset gathered from ChEMBL and have shown outperformance of our tool when compare to well-known single classification models

Analysis of RNAseq datasets from a comparative infectious disease zebrafish model using GeneTiles bioinformatics.

Animal science

Identification of TNF-alpha-Responsive Promoters and Enhancers in the Intestinal Epithelial Cell Model Caco-2

The Caco-2 cell line is one of the most important in vitro models for enterocytes, and is used to study drug absorption and disease, including inflammatory bowel disease and cancer. In order to use the model optimally, it is necessary to map its functional entities. In this study, we have generated genome-wide maps of active transcription start sites (TSSs), and active enhancers in Caco-2 cells with or without tumour necrosis factor (TNF)-α stimulation to mimic an inflammatory state. We found 520 promoters that significantly changed their usage level upon TNF-α stimulation; of these, 52% are not annotated. A subset of these has the potential to confer change in protein function due to protein domain exclusion. Moreover, we locate 890 transcribed enhancer candidates, where ∼50% are changing in usage after TNF-α stimulation. These enhancers share motif enrichments with similarly responding gene promoters. As a case example, we characterize an enhancer regulating the laminin-5 γ2-chain (LAMC2) gene by nuclear factor (NF)-κB binding. This report is the first to present comprehensive TSS and enhancer maps over Caco-2 cells, and highlights many novel inflammation-specific promoters and enhancers

Differences in the Cognitive Skills of Bonobos and Chimpanzees

While bonobos and chimpanzees are both genetically and behaviorally very similar, they also differ in significant ways. Bonobos are more cautious and socially tolerant while chimpanzees are more dependent on extractive foraging, which requires tools. The similarities suggest the two species should be cognitively similar while the behavioral differences predict where the two species should differ cognitively. We compared both species on a wide range of cognitive problems testing their understanding of the physical and social world. Bonobos were more skilled at solving tasks related to theory of mind or an understanding of social causality, while chimpanzees were more skilled at tasks requiring the use of tools and an understanding of physical causality. These species differences support the role of ecological and socio-ecological pressures in shaping cognitive skills over relatively short periods of evolutionary time

Robotic injection of zebrafish embryos for high-throughput screening in disease models

The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines